Solid phase peptide synthesis method and device
阅读说明：本技术 固相肽合成方法及装置 (Solid phase peptide synthesis method and device ) 是由 奥利弗·约翰内斯·克鲁泽 于 2020-02-04 设计创作，主要内容包括：本发明涉及进行固相肽合成的方法、自动并行的固相肽合成以及设计用于实施这种方法的装置。根据本发明,在进行固相肽合成的反应介质上的所述方法期间,频率超过25kHz的超声至少间歇地作用。(The present invention relates to a method for performing solid phase peptide synthesis, automated parallel solid phase peptide synthesis and apparatus designed for carrying out such method. According to the invention, ultrasound with a frequency exceeding 25kHz is applied at least intermittently during the process on a reaction medium for carrying out solid-phase peptide synthesis.)
1. A method for performing solid phase peptide synthesis comprising the steps of:
a) the amino acid protected by a protecting group at the N-terminal is bonded to a solid support material through the C-terminal of the amino acid,
b) the cleavage of the protecting group is carried out,
c) carrying out at least one peptide multiplication, and
d) the reaction is terminated by cleaving the peptide from the support material,
wherein steps a) to d) take place in a liquid reaction medium and ultrasound with a frequency in the range of 25 to 2000kHz acts at least intermittently on the reaction medium at least during one of the steps.
2. The method according to claim 1, characterized in that the ultrasound acts on the reaction medium at a frequency in the range of more than 40kHz, in particular more than 75kHz, preferably more than 100kHz, particularly preferably more than 110 kHz.
3. Method according to claim 1 or 2, characterized in that the ultrasonic waves are applied to the reaction medium at a frequency in the range of not more than 1000kHz, preferably not more than 500 kHz.
4. The method according to any one of the preceding claims, wherein the ultrasound is transmitted to the reaction medium via an external liquid bath.
5. The method according to any of the preceding claims, further comprising a washing step W occurring after step b)b) A washing step W which takes place after step c)c) And/or a washing step W which takes place after step d)d) WhereinUltrasound is also applied to the reaction medium during at least one of these steps.
6. Method according to any one of the preceding claims, characterized in that the amino acid is protected at the N-terminus by a base-labile protecting group, in particular a protecting group cleavable by a secondary amine, in particular fluorenylmethyloxycarbonyl (Fmoc).
7. The method according to any one of the preceding claims, characterized in that the amino acid comprises a protecting group for protecting the side chain, in particular S-2,4, 6-trimethoxybenzyl (Tmob), trityl (Trt), tert-butyl (tBu), tert-butoxycarbonyl (Boc), 2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf).
8. The method according to any one of the preceding claims, characterized in that ultrasound is applied to the reaction medium in only one step, in particular in step c).
9. The method according to any of the preceding claims, characterized in that in all steps a) to d) and/or Wb)、Wc) And Wd) The ultrasound acting on the reaction medium is not interrupted and/or acts at the same frequency.
10. Method according to any of the preceding claims, characterized in that when the ultrasound acts on the reaction medium in several steps, the ultrasound frequency is varied between the steps, in particular in the reaction steps a) to d) and the washing step Wb-d) To change between.
11. The method according to any of the preceding claims, characterized in that during at least one washing step the frequency is in the range of 25 to 2000kHz, preferably in the range of more than 40kHz, in particular more than 75kHz, preferably more than 100kHz, more preferably more than 110 kHz.
12. The method according to any of the preceding claims, characterized in that the ultrasonic bath is controlled in a temperature range of 20 to 100 ℃, preferably 20 to 70 ℃, more preferably 40 to 60 ℃.
13. Method according to any of the preceding claims, characterized in that the method is performed semi-automatically/automatically and/or in parallel.
14. The method according to any one of the preceding claims, wherein step d) comprises a dosing step, a washing step and a filtering step, and in case of semi-automatic execution, the dosing step is performed manually and the other steps are performed in an automated manner.
15. An automated parallel solid phase peptide synthesis method comprising a method according to any one of the preceding claims.
16. Device (1) for carrying out solid phase peptide synthesis, designed for carrying out the method according to any one of the preceding claims.
17. The apparatus (1) according to claim 16, comprising means (4) for receiving a synthesis vessel (5), in particular a synthesis plate, a plurality of synthesis cartridges, a reaction flask or a reactor having at least one opening for filling with a reaction medium; and an ultrasonic bath (50) containing a liquid, wherein the synthesis vessel (5) can be arranged in the ultrasonic bath (50) in such a way that the synthesis vessel (5) is wetted to a minimum height of the liquid of the ultrasonic bath (50).
18. Device according to claim 16 or 17, characterized in that the ultrasonic bath is arranged height and/or temperature adjustable.
19. A peptide prepared according to the method of any one of claims 1-14.
Technical Field
The present invention relates to a method for performing solid phase peptide synthesis, automated parallel solid phase peptide synthesis and apparatus suitable for performing such method.
Background
Solid phase peptide synthesis (SPPS or also known as Merrifield synthesis) is a method of peptide synthesis introduced in 1962 by Robert Bruce Merrifield, a prize-earier under the nobel prize, using an insoluble polymeric support. Linear peptides are constructed by stepwise ligation of sequence-specific, temporarily protected amino acids, in which the C-terminus of the growing polypeptide chain is covalently linked to a synthetic resin support. To ensure a controlled reaction and to avoid side reactions, the reactive functional side chains of amino acids must be blocked with suitable protecting groups. Although the alpha-amino group of the amino acid to be attached only needs to be protected during the actual coupling reaction, the permanent side chain protecting group is cleaved from the peptide only after the synthesis is complete. In contrast to ribosomal protein biosynthesis, extension of the peptide chain occurs from the C-terminus to the N-terminus. Copolymers of polystyrene and 1-2% of 1, 4-divinylbenzene have proven to be suitable polymeric supports. Resin beads having a diameter of between 20 and 100 microns obtained by bead polymerization swell in the solvent used for the synthesis and become permeable to the agent. The tert-butyloxycarbonyl (Boc) and fluorenyl-9-methoxycarbonyl (Fmoc) groups were mainly used as intermediate alpha-amino protecting groups. The Boc group is stable in catalytic hydrogenation and alkaline hydrolysis and can be decomposed by mild acid hydrolysis, e.g. with 50% trifluoroacetic acid (TFA). Repeated acid deblocking after each coupling step results in partial deblocking of the side chain protecting groups and slight hydrolysis of the anchor bond to the polymeric support. The advantage of the Fmoc group is that it can be cleaved by treatment with a suitable base (e.g.morpholine, 2-aminoethanol or piperidine). If acid-labile, alkali-resistant groups are used as anchoring groups on the polymer support, and in order to protect the third function of the corresponding amino acid building blocks, the intermediate protecting groups and the permanent protecting groups can advantageously be separated independently of one another.
The coupling reaction (also called condensation or peptide propagation) is an extremely important step in the synthesis, since complete conversion is an essential requirement for the homogeneity of the final product. usually, reagents are used in excess, preferably anhydrides, active esters or so-called in situ activators, in which intermediate activated ester derivatives are formed, the reaction steps of decomposing the alpha-amino protecting group and linking the next N alpha-protected amino acid (coupling reaction, condensation) are repeated over and over to allow extensive automation of the synthesis steps and the construction of the peptide synthesizer, most of which works according to the flow-through principle. Depending on the chosen protecting group scheme, the anchor bond between the C-terminal amino acid and the support is selectively cleaved, or simultaneously results in partial or complete deblocking of the synthetic peptide. Various peptide synthesis methods have evolved from solid phase peptide synthesis. [ E.Atherton and R.C.Sheppard Solid-Phase Synthesis-A Practical Approach, Oxford University Press, 1989; H. jakubbek Peptides Chemistry and Biology, published by Spektrum Akademiescher Verlag Heidelberg, 1996)
One of the major challenges in automated peptide synthesis is to avoid cross-contamination, since in known procedures and automated devices the reagents are passed through the same tubing and cannula. In order to prevent cross-contamination, in known arrangements of the apparatus, the entire system is flushed with a large quantity of flushing agent.
DE 10131088B 4 was initiated and provided a device capable of automated, simultaneous, multiple and parallel synthesis, in which cross-contamination can be excluded. This results in a significant reduction in synthesis time.
Another method of reducing synthesis time in peptide synthesis is to expose the reagents to microwave radiation during synthesis. This makes it possible to reduce the synthesis time to one tenth. However, microwave-assisted reactions have to be carried out in special protected spaces. Thus, the process is limited to smaller reactors and thus the throughput is lower. Furthermore, it has been found that not all commonly used protecting groups are stable to microwave radiation, and thus yield losses and further impurities may occur.
In 1977, CA 1019324 published attempts to assist synthesis by ultrasound. However, in the following years, it is clear that this method, at least in the form shown, does not produce the expected results. Reproducible acceleration of the synthesis time cannot be observed with the usual methods.
It is now an object of the present invention to further accelerate the synthesis time of solid phase peptide synthesis while maintaining or improving yield and purity. The method is particularly suitable for automated parallel methods.
Disclosure of Invention
This object is achieved by a method for performing automated parallel solid phase peptide synthesis and by an apparatus for performing said method having the features of the independent claims.
Accordingly, the first aspect of the present invention relates to a method for performing solid phase peptide synthesis (hereinafter also referred to as synthesis or peptide synthesis). The method according to the invention comprises the following steps:
a) the amino acid protected by a protecting group at the N-terminal is bonded to a solid support material through the C-terminal of the amino acid,
b) the cleavage of the protecting group is carried out,
c) carrying out at least one peptide multiplication, and
d) the reaction is terminated by cleaving the peptide from the support material,
wherein steps a) to d) occur in a liquid reaction medium and ultrasound having a frequency in the range >25 to 2000kHz is at least intermittently applied to the reaction medium at least during one of the steps.
It has been found that ultrasound only accelerates the reaction in question in solid phase peptide synthesis from frequencies above 40 kHz. Thus, the method according to the invention enables a reproducible reduction of the synthesis time of solid phase peptide synthesis, at least over the synthesis time range in microwave-assisted peptide synthesis. Advantageously, however, no special safety precautions have to be taken. In addition, the purchase and maintenance costs of the required equipment are low. This means that the method can be used in almost any synthesis apparatus, in particular a parallel and/or automated synthesis apparatus.
Step a) is herein understood to mean the direct or indirect binding of functional groups to a suitable support material, such as a pre-loaded or non-pre-loaded resin or an amide resin for solid phase peptide synthesis. The functional group may in particular be protected by an Fmoc protecting group. Here, preloaded or not preloaded refers to the fact that at least one first and optionally at least one subsequent amino acid, i.e.the primary amino acid in the amino acid sequence to be synthesized, has been bound directly to the carrier material.
Frequencies above 40kHz, preferably above 50kHz, in particular above 75kHz, particularly preferably above 100kHz have proven to be particularly suitable, since a more significant reduction in synthesis time can be achieved with higher frequencies. It has been found that the formation of cavitation has a significant positive effect on peptide synthesis, in particular an improvement in quality. The associated cavitation starts at a frequency of 40kHz and increases with increasing frequency. In the frequency range of 20 to 40kHz, only vibrational excitation occurs.
Preferably, the ultrasound frequency of the method according to the invention does not exceed 2MHz, in particular 1 MHz. Further explanation of preferred frequencies follows.
The ultrasound-assisted solid phase peptide synthesis (USPS) described herein falls within the category of sonochemistry in chemical synthesis.
The chemical effect of ultrasound cannot be a direct effect of the acoustic field, since the typical frequency is too low, several orders of magnitude lower, and cannot be excited even with simple rotational motion.
We assume that the positive effect is directly related to cavitation triggered by ultrasound and the resulting pressure pulses. Cavitation occurs in the frequency range of 40kHz to 2 MHz.
Three types of sonochemical reactions are assumed.
1. In a homogeneous system consisting of free radicals or free radical ionic intermediates. In cavitation bubbles, extreme pressures and high temperatures can produce, for example, OH in the aqueous phase.Radicals, which, among other effects, lead to H formation in the bubbles2O2。
2. In heterogeneous systems by ionic reactions. These are mainly assisted by the mechanical effect of solvent hollowing. Asymmetric bubbles are formed on the solid particles. The collapse of asymmetric bubbles on a particle produces a jet of liquid directed at a single side collapsing the bubble. This helps to absorb the solvent and dissolved substance into the porous material. On the other hand, in the other liquid phase, mixing of the phases occurs.
3. In heterogeneous systems, free radical reactions can also occur. The free radical pathway may produce different products than the ionic pathway, for example in the Kornblum-Russell reaction.
Cavitation bubbles are more likely to form in the lower frequency range and then also become larger and asymmetric. This results in stronger but less uniform mixing. On the other hand, at higher frequencies, more smaller, symmetrical bubbles are produced, and there is more exchange of radicals between the cavitation bubbles and the environment.
Cavitation is the "formation, growth and implosion and collapse of bubbles in a liquid. Cavitation collapse can result in localized high temperatures (-5000K), high pressures (-1000 atm), large heating and cooling rates (>109K/s) "and liquid jets (-400 km/h). The cavitation bubbles are vacuum bubbles (Suslick 1998). The vacuum is created by a fast moving surface and an inert liquid. The resulting pressure differential overcomes the cohesive and adhesive forces within the liquid.
From frequencies of 110kHz and higher, in particular from 125kHz, preferably at frequencies of 130kHz and higher, accelerated reaction processes and the associated shortened reaction times and increased yields are observed. It has been found that it is not necessary to use a 40-fold excess of amino acids compared to the standard system, and the same results can already be obtained with a 4-fold excess. This in turn results in a significant reduction in the number of reactants and thus in significant cost savings. Furthermore, no racemization was observed in the frequency range of 110 to 500kHz, which gave high yields.
In a preferred embodiment of the process according to the invention, it is provided that the ultrasound is transmitted to the reaction medium via an external liquid bath. This is in marked contrast to devices that transmit ultrasound directly or exclusively through a solid delivery device to a reaction medium. It has been found that the transfer through at least one liquid medium provides more consistent, repeatable and gentle synthetic results. The necessary synthesis time shows a lower dispersion when using a liquid-containing transport medium compared to, for example, the synthesis time when using a probe immersed in the reaction medium. Furthermore, the test equipment is much simpler than when probes are used. Immersion of the probe will inevitably lead to contamination of the probe and require periodic cleaning of the probe, which will equal or at least significantly reduce the gain in synthesis time.
The energy converted to cavitation depends on several factors that indicate the motion transferred from the cavitation-producing device to the liquid. The intensity of acceleration is one of the most important factors affecting the efficient conversion of energy into cavitation. Higher acceleration produces higher pressure differentials. This increases the likelihood of vacuum bubbles being created in the liquid rather than waves. This means that the higher the acceleration, the higher the proportion of energy converted into cavitation. In the case of (ultrasonic) transducers, the strength of the acceleration is determined by the amplitude of the vibration. In addition to the intensity of the ultrasonic waves, it is also important that the liquid is accelerated in such a way that the losses of turbulence, friction and wave generation are as low as possible. The path of the unilateral motion direction is best suited to this situation.
Therefore, the choice of the transport medium is crucial for the effectiveness of peptide synthesis. In addition to the choice of state, the material of the transmission medium must also be optimized. In addition to water as a transport medium, organic solvents, in particular lower and medium alcohols, such as ethanol, propanol and butanol, are also preferably used as transport media.
It is also advantageous to choose different transmission media to combine. The test equipment is understood to be a bath in a bath. In other words, the reaction takes place in a reaction medium arranged in the (reaction) vessel. The reaction vessel is in turn arranged in a vessel with a first transport medium, which in turn is arranged in a further transport medium. The ultrasound is thus transmitted via the further transmission medium to the first transmission medium and from there to the reaction medium.
It is particularly advantageous if the first transport medium consists of a lower and a middle alcohol, in particular of the type mentioned above, and/or the further transport medium consists of water.
With high ultrasonic frequencies, the temperature in the liquid bath will rise as expected. However, at frequencies up to 500kHz, this effect can be well controlled, since the temperature increase which occurs can easily be compensated for by cooling devices, for example by means of a continuous cooler (cryostat) cooling a water bath or a Peltier element, so that racemization which may also occur here does not reduce the yield.
When frequencies significantly above 500kHz to 1000kHz or higher are used, it is clear that counteracting the temperature increase is meaningful for quality assurance purposes, for example by cooling the bath.
The ultrasonic bath is preferably temperature-controlled, more particularly in the temperature range from 20 to 100 ℃, preferably from 20 to 70 ℃, particularly preferably from 40 to 50 ℃.
In another preferred embodiment of the present invention, there is provided protection of the amino acid at the N-terminus by a base labile group, in particular a temporary (primary) protecting group cleavable by a secondary amine, in particular fluorenylmethyloxycarbonyl (Fmoc). These protective groups prove to be particularly stable to ultrasound at the frequencies according to the invention compared with the protective groups used in the boc method, so that particularly high yields and high purities can be achieved. Deprotection is preferably carried out using a suitable base, as described previously or in the test results. Preferably, 20% piperidine in DMF (dimethylformamide) is used.
To date, the advantages of ultrasound-assisted peptide synthesis have only been demonstrated in BocOC synthesis. Even ultrasound-assisted synthesis has been found to be unsuitable for Fmoc-based synthesis, since the resins used are comminuted under ultrasound. Due to the different reaction media and side chain protecting groups used, it is assumed that only coupling to the resin can be assisted by ultrasound. Surprisingly, however, Fmoc-based peptide synthesis by ultrasound-assisted synthesis during the individual synthesis steps in the frequency range using the method of the invention can also show advantages in terms of reaction time and yield.
The reactive side chains of the peptides synthesized by the method according to the invention are preferably also protected by (secondary) protecting groups. Depending on the functional group to be protected, acid-stable protecting groups, in particular groups selected from the group consisting of S-2,4, 6-trimethoxybenzyl (Tmob), trityl (Trt), tert-butyl (tBu), tert-butoxycarbonyl (Boc), 2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), have proven particularly stable in the synthesis of peptides according to the invention using ultrasound in the frequency range mentioned.
Referring to the method according to the invention, for example, Fmoc amino acids selected from the group consisting of the amino acids listed in the one-letter code or the three-letter code 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, Fmoc-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 the L and D forms of the amino acid.
In addition, special side chain protected post-translationally modified amino acids can be used, such as:
for 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,
for sulfation of Tyr:
Fmoc-Tyr(SO3nP)-OH、Fmoc-Tyr(SO3DCV)-OH,
for methylation of Arg:
Fmoc-Arg(Me,Pbf)-OH、Fmoc-ADMA(Pbf)-OH、Fmoc-SDMA(Boc2)-ONa,
for methylation of Lys:
Fmoc-Lys(Me,Boc)-OH、Fmoc-Lys(Me2)-OH、Fmoc-Lys(Me3Cl)-OH，
for citrullination:
Fmoc-citrulline-OH (Fmoc-citrulline-OH),
for glycosylation of Asn:
Fmoc-Asn(β-DGlcNAc(Ac)3)-OH、Fmoc-Asn(β-DGlcNAc(Ac)3-(1-4)-β-DGlcNAc(Ac)2)-OH，
for 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(Ac)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。
likewise, synthetic building blocks developed for the synthesis of complex peptide sequences can be used, such as the FmocPseudoProline (Fmoc pseudoproline) dipeptide, the so-called Dmb building block: for example Fmoc- (Dmb) Gly-OH, or the dipeptide FmocXaaDmbGly, the Hmb building block: fmochhmxaa, and Hmsb building blocks, Hnb building blocks, Mmsb building blocks, unnatural amino acids such as:
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 amino polyglycol acids.
Specifically derivatized amino acids, such as:
Fmoc-Lys (biotin) -OH, FMOC-Lys (Cy)5)-OH。
In general, all building blocks with temporarily protected amine functionality (preferably Fmoc protected) and carboxylic acid functionality convertible to an active ester or amine reactive group can be used in the USPS.
Advantageously, the ultrasound acts on the reaction medium in only one step, in particular in step c). Alternatively or additionally, ultrasound is applied to the reaction medium in at least one further step, preferably in steps a), b) and/or d). The reaction acceleration characteristics of the ultrasound according to the invention can be observed in each of the steps mentioned.
It is particularly preferred here that the action of the ultrasound is not interrupted between the individual steps or in the individual steps, since interruptions lead to a reduction in the yield.
To date, the greatest reduction in synthesis time and high yields have been achieved when ultrasound waves are applied to the reaction medium throughout the synthesis, i.e. also during washing, deprotection, condensation and coupling. In contrast, ultrasound is not necessarily advantageous during pre-swelling as a preparatory step or during final washing.
Solid phase peptide synthesis involves several washing steps that can be distinguished from each other. The individual types of washing steps can be distinguished by their respective upstream reactions. Thus, mention may be made of at least one wash (initial wash) after the coupling of the first amino acid to the resin, the uncoupling of the protective group in step b) (hereinafter referred to as step W)b) In-line with the aboveAfter washing, coupling with amino acids for extending the peptide chain (hereinafter, step W)c) Subsequent washing and cleavage of the last temporary protecting group of the final peptide from the support material in step d) (hereinafter step W)d) Followed by a final wash.
The ultrasound also preferably has an effect on the reaction during the individual washing steps and leads to a significant reduction in the necessary cleaning agents and cleaning times. Thus, washing using only one cleaning step with ultrasound has achieved the same result as the four typical washes in standard synthesis. Washing step WcI.e. washing after step c), is particularly important for improving yield and quality. Studies have shown that the washing step W can even be omitted completelyb. Preferably, however, all washing steps are carried out in the process according to the invention.
Preferably, in particular for this washing step, the ultrasonic frequency is in the range of 100 to 500kHz, preferably in the range of 100 to 200kHz, in particular in the range of 120 to 140 kHz. Within these ranges, the amount of solvent required for washing or rinsing, e.g. DMF, in the individual rinsing cycles of the washing steps may be reduced in such a way that each washing step WbOnly a single cleaning step is required.
It is particularly advantageous to include a washing step W during all steps of peptide synthesis a) to d)b、WcAnd WdDuring this time, the ultrasound acts on the reaction medium in the frequency range described above, in particular without being completely interrupted.
It has been found that, depending on the peptide to be synthesized, different frequencies are optimal for the individual steps a) to d), in particular for step Wb. c and dI.e. they show a more beneficial effect in terms of improved quality and reduced reaction time. Therefore, it is preferable that the ultrasonic waves act on the reaction medium at different frequencies in the respective steps. In particular, it is preferred that the frequencies between the steps are changed and/or that ultrasound waves with different frequencies are superimposed on each other.
The support materials are materials which are basically known to the person skilled in the art of peptide synthesis. These are synthetic resins/synthetic resins, of which resins from the following group are particularly advantageous:
knorr Amid resin LS 1% DVB, Wang resin, chlorotrityl resin, PRG resin, Tentagel resin, Chemcatarix resin.
Pre-loaded or non-pre-loaded and/or functionalized resins typically used for solid phase synthesis.
Non-paramagnetic synthetic resins are preferred because the separation of synthetic peptides by magnetic means is much more complicated than separation by filtration and it has been shown that paramagnetic resins are crushed to form very fine particles in low frequency ultrasound (up to 40kHz) and in the process clog the filter material.
In the context of the present invention, preference is given to using solvents such as DMF (N, N-dimethylformamide), NMP (N-methyl-2-pyrrolidone) or DMA (N, N-dimethylacetamide).
The base used to catalyze the condensation reaction is preferably NMP, 4-methylmorpholine or DIPEA, diisopropylethylamine in DMF or another solvent.
The solution used to cleave the temporary Fmoc protecting group is preferably 20% piperidine in DMF. Other cutting methods are known in principle to the person skilled in the art.
Preferably, the coupling agents used are HBTU (2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate), HCTU (2- (6-chloro-1H-benzotriazol-1-yl) -1,1,3, 3-tetramethylhexafluorophosphate), PyBOP (benzotriazol-1-yl-oxytriazolidinium hexafluorophosphate), DCC, dicyclohexylcarbodiimide; DIC, diisopropylcarbodiimide; or EDC, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.
All amino acids/reagents are dissolved in the solvent used, resulting in a reaction medium/reactant medium/reagent solution. When referring to reagents in the context of methods and devices, this is also understood to include solutions of the reagents.
The concentration of amino acid used always depends on the scale of the synthesis and its solubility in the solvent used (e.g. DMF, NMP or DMA).
In order to obtain good synthesis results, the Amino Acid (AA) to be coupled and the reagent for forming the active ester (activator) are preferably each used at least equimolar to the scale of synthesis. However, it is common to use AA and the activator together in equimolar amounts and to use them both beyond the scale of synthesis. The excess ranges from 4-fold to 100-fold. In order to obtain a quality comparable to the present method of solid phase peptide synthesis of the prior art, at least a 40-fold excess is required, depending on the peptide sequence. The reason is that a high excess of reactants favours the formation of the product, i.e. the extended peptide chain. The concentration of amino acid here depends mainly on the solubility of the protected AA in the solvent used, preferably between 0.2M and 0.6M.
In the experiments described in this application, the amino acid was used at a concentration of 0.4M.
Also, advantageously, the ultrasound is never completely interrupted between successive (ultrasound-assisted) steps a) -d), but only the frequency is changed. Advantageously, it is envisaged that the method is performed semi-automatically/automatically and/or in parallel. In a combination of a parallel automated method with ultrasound assistance according to the invention, it is possible to be able to produce individual peptides, i.e. peptides tailored to a specific individual, at a scale and production capacity that would make a specific form of cancer treatment with a new antigen profitable for a large number of patients. Neoantigens are mutation-induced changes in tumor cell proteins. They can be identified by Next Generation Sequencing (NGS) techniques. Typically, the number of different neoantigens is between 100 and 200. A corresponding number of synthetic peptides can mimic these neo-antigens in vitro and be used for tumor-specific immunization of patients. The number, composition and amino acid sequence of the neoantigens are individualized for each patient. Therefore, the replication of the corresponding peptide must also be different from person to person, in other words personalized. In order for this promising therapy to be fully medically available, it must be possible to study a large number of patients under the same conditions in a reasonable time. This requires the provision of large amounts of personalized antigens in a very short time. Thus, the described personalized cancer treatment modalities place high demands on the speed, parallelism and quality of peptide synthesis. The method according to the invention can meet these requirements.
The process according to the invention is particularly suitable for large-scale applications. The term "large scale application" refers to a batch size of 1 liter to 50 liters, especially 100 to 500 liters.
Thus, another aspect of the invention is automated parallel solid phase peptide synthesis, including the method according to the invention in one of the embodiments.
The process according to the invention can be carried out in a particularly advantageous manner at room temperature or at moderate temperature increases. Particular preference is given to reaction temperatures in the range from 20 to 100 ℃, preferably in the range from 20 to 70 ℃ and in particular in the range from 40 to 60 ℃. The temperature control is preferably carried out at least in steps a), b) and/or d).
The process according to the invention has been found to be particularly suitable for the large-scale production of liraglutide (litriglutid) and semaglutide (Semaglutid), in particular on a scale of over 100g product.
Another aspect of the invention relates to a device for performing solid phase peptide synthesis, designed to carry out the method according to the invention in one of the described embodiments. To this end, the device according to the invention comprises an ultrasonic transducer which preferably transmits ultrasonic waves with a frequency of 25kHz to 2MHz, in particular in the range of 40kHz to 1MHz, preferably in the range of 100 to 500kHz, to the reaction medium via the liquid transmission medium.
The device preferably has a device 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 with 96, 384, 1536 or 3456 reaction chambers, a synthesis cylinder or flask or synthesis reactor, and an ultrasonic bath containing a transport liquid, wherein the synthesis vessels can be arranged in the ultrasonic bath in such a way that they are wetted to a minimum height by the transport liquid of the ultrasonic bath.
Advantageously, the minimum height is understood to be the height which ensures the transmission of the ultrasonic waves from the ultrasonic transducer of the ultrasonic device to the reaction medium in such a way that sound (Schall) occurs almost exclusively through the liquid transmission medium. For this purpose, the height of the surface of the transport medium on the outer surface of the synthesis vessel corresponds to at least half the height of the meniscus (Meniskus) of the reaction medium in the synthesis vessel, preferably a height in the range from half the height to the full height, preferably a height of three quarters to the full height.
The synthesis vessel is preferably designed as a synthesis plate, for example a microtiter plate, in particular a synthesis cylinder parallel and juxtaposed, for example in the form of a beaker or syringe, or as a synthesis flask, for example a round-bottomed flask or a reactor (for example according toOr syringee). In particular, designing the synthesis vessel as a round-bottomed flask or reactor is more suitable for large-scale semi-automated/automated solid phase peptide synthesis, since a scale of 1-50 liters in a flask or 100-500 liters in a reactor can be handled.
On the other hand, the use of microtiter plates or parallel synthesis cylinders is particularly preferred for parallel, automated solid phase peptide synthesis, whereas the above mentioned are more commonly used for large scale application of individual peptides, such as liraglutide and semaglutide. The invention proposes a device which completely does not require diluents and a pipe system for the supply and metered (dosierte) delivery of synthetic building blocks. Individual synthesis pens (synthesizers) are provided for each synthetic building block, which are placed in a holder of the synthesis apparatus for synthesis and are gripped and picked up from the holder by gripper arms of the apparatus in order to transfer metered doses of building blocks onto a support material located in a reaction chamber of a synthesis vessel, in particular a synthesis plate.
The reagent vessel and the dosing means thus form one single unit. This eliminates all of the washing procedures and associated disadvantages that were previously required when changing the synthetic building blocks and reagent distribution.
A region is defined for parallel synthesis (fig. 2). The working area is dimensioned such that the synthetic pen moved by the gripper arm can reach each point of the working area. Preferably, up to 10 synthesis stations are arranged, in particular symmetrically, on the working area. The dimensions of the synthesis station are preferably based on standard microtiter plates. The synthesis station may be of modular design and may include a base member with connections for extracting solvent and an exposed frame for holding the synthesis plate. Depending on the subdivision of the composite boards used, for example 6, 12, 24, 48, 96, 384, 1536 or 3456 individual syntheses may be performed in parallel in one composite board. For larger synthesis scales, synthetic cylinders or syringe bodies may be used in special containers.
According to a further feature of the present invention, the reaction chambers of the composite sheet are closed at the open sides with a permeable material, such as with a frit. A sample plate for receiving the peptides dissolved after the cleavage reaction may be arranged below the frame. Using the proposed solid phase synthesis apparatus, the synthesis and cleavage of the obtained compound from the support material, synthetic resin can be performed without manual intervention.
The sample plate is equipped with individual holding chambers, which are arranged and designed to correspond to the grid of reaction chambers in the synthesis plate. In this way, an error-free and easy dispensing of the specific compound after cutting from the support material or synthetic resin is ensured. It is easy to transfer the reaction products directly to a high throughput screening line.
The synthetic pen (fig. 3 and 4) has a hollow cylindrical body (reagent container) which can be closed by a screw cap and a suction nozzle fitted to the free opening of the reaction chamber at the bottom end, while being provided with an outlet opening. The outlet opening is closed by a valve needle with a shut-off valve guided by a piston rod and a piston in the body and releasably secured in its closed position by a compression spring acting on the piston. The cylinder space below the piston is used to accommodate the individual building blocks and inert gas, and the quantitative delivery of the reagents can be achieved by simply placing the suction nozzle on the permeable material covering the open side of the reaction chamber. At the same time, by pressing in the valve needle, the shut-off valve is released from the valve seat and the outlet opening is released. The amount of reagent dispensed is determined by the length of time the mouthpiece is placed on the permeable material. When the suction nozzle is lifted, the outlet opening automatically closes again.
In a further design of the synthetic pen (fig. 6), the screw cap of the reagent container is replaced by a removable cap with a bayonet cap. The piston rod is preferably arranged in the center of the cover, in particular a pressed-in piston rod, which penetrates the entire combination pen into the dosing cylinder. The dosing cylinder is closed at the bottom, for example using a check valve. By pressing the cap, a defined amount of reagent is dispensed by means of the piston. A return means, such as a spring, mounted in the synthetic pen returns the piston. At the same time or thereafter, the dosing cylinder is filled again. A suitable actuator (e.g. a check valve) in the foot-side nozzle ensures that the solution can only be dispensed by active delivery. This design of the synthetic pen allows for contactless dispensing into the reaction chamber. The closed design of the synthetic pen with closed reagent vessel ensures a high reagent stability.
Advantageously, the ultrasonic bath of the device according to the invention can be lowered or raised. This enables the reaction vessel to be lowered in the ultrasonic bath, preferably in steps or continuously, to a predetermined height.
Furthermore, the ultrasonic bath can be used for different test devices, in particular different synthesis vessels.
The ultrasonic bath is also advantageously temperature-controllable, in particular designed to achieve a controlled temperature range of 20 to 100 ℃, preferably a range of 20 to 70 ℃, in particular a range of 40 to 60 ℃. In particular, the device has a cooling system for reducing the temperature of the bath (for example as a result of heating by high ultrasound frequencies). This is particularly advantageous when frequencies from 500kHz, especially from 1000kHz, are used.
Furthermore, the ultrasonic bath of the device according to the invention or its ultrasonic generator is designed to generate and transmit variable frequencies, in particular at least one in the low frequency range (40-75kHz) and one in the high frequency range (100 to 2000kHz, preferably 100 to 500kHz), into the liquid bath. For this purpose, it is advantageous if the different frequencies can be switched alternately or additionally to each other.
In a preferred embodiment, the ultrasonic bath, or the ultrasonic generator of the ultrasonic bath, is equipped with the necessary power in the range of 40 to 100W, in particular in the range of 50 to 70W nominal power, and can achieve a peak in the range of 100 to 300W, preferably in the range of 170 to 280W, and for large-scale applications in the range of 250 to 700W, in particular in the range of 500 to 600W.
By using a separate synthesis pen for each synthesis building block and covering the reaction chambers in the synthesis plate at the open side, the risk of contamination is significantly reduced and cross-contamination is almost eliminated. Such as the residue of synthetic building blocks that often occurs during an insufficient washing process, is no longer possible.
As the washing process is omitted, not only the consumption of the organic solvent is greatly reduced, but also the synthesis speed is accelerated by many times.
The described embodiments can be combined with one another advantageously, unless stated otherwise in individual cases. Embodiments of the invention are otherwise equally applicable to the methods and apparatus.
Drawings
Hereinafter, the present invention will be explained in more detail by practical examples and results for illustrative purposes only. Shown in the drawings are:
FIG. 1 is a schematic view of an apparatus for synthesizing a peptide according to the present invention;
FIG. 2 is a plan view of the working area of the device of FIG. 1;
FIG. 3 is a schematic illustration of a synthetic pen for separate feeding, dosing and reagent storage in a preferred embodiment of the invention;
FIG. 4 is a longitudinal cross-sectional view of the composite pen of FIG. 3;
FIG. 5 is an AA cross-sectional view of FIG. 2 through a composite plate having reaction chambers formed in accordance with the present invention;
FIG. 6 is a schematic longitudinal cross-sectional view of a composite pen according to another preferred embodiment of the present invention;
FIG. 7 is a schematic diagram of a sequence of solid phase peptide synthesis processes according to a preferred embodiment of the present invention;
FIG. 8 is a graphical representation of tryptophan stability for a method of the invention based on the synthesis of endomorphins;
FIG. 9 is a graphical representation of the stability of Acyl Carrier Protein (ACP) using a method according to the present invention;
FIG. 10 is a graphical representation of the stability of Acyl Carrier Protein (ACP) using a prior art comparative method;
FIG. 11 is a graph comparing the average synthetic quality of ACP peptides in view of synthetic strategies;
FIG. 12 is a graphical representation of the resulting mass from FIG. 11;
FIG. 13 is a graphical representation of a stock solution test for amino acids of different solution durations;
FIG. 14 is a comparison of single and double couplings;
FIG. 15 is a graph comparing single and double couplings and amino acid excess; and
FIG. 16 is a graph comparing the average synthetic mass of peptides using different ultrasound frequencies.
Detailed Description
The synthesis apparatus 1 shown schematically in fig. 1 is based on a laboratory pipetting robot and has a gripper arm 2 which can be moved in the x, y and z axes. In the work area 3 there are synthesis vessels, in particular synthesis plates 5, which originate from microtiter plates known per se with regard to the grid and arrangement of reaction chambers 9 and have 6, 12, 24, 48, 96, 384, 1536 or 3456 grids of reaction chambers 9, whereby a high degree of parallelization of the synthesis is achieved. The synthetic plate 5 is placed on the valve block 6 and has a membrane 28 of porous material on the bottom side for sucking out used reagents and rinsing liquid from the reaction chamber 9 and into the waste through the valve block 6 connected to a suction pump.
The synthetic plate 5 is arranged in an ultrasonic bath 50, in particular an ultrasonic bath which is height-adjustable and can be opened and closed in a controllable manner, together with the valve block 6 and the sample plate 27. The ultrasonic bath 50 has a container with a liquid transmission medium, in which the synthesis vessel 5 is arranged. Depending on the position of the ultrasonic bath 50, the meniscus of the synthesis vessel 5 for the reaction medium is at least as high as half the filling level of the ultrasonic transmission medium of the ultrasonic bath 50, preferably at least as high as three quarters of the filling level of the ultrasonic transmission medium of the ultrasonic bath 50, in particular completely below the filling level of the ultrasonic transmission medium of the ultrasonic bath 50. The ultrasonic bath 50 is designed to transmit ultrasonic waves through the transmission medium at a frequency in the range of at least 25kHz to 2 MHz.
The synthesis device 1 is also equipped with one or more flushing combs 8, which are connected to respective flushing agent reservoirs by means of a flushing agent supply line 10. In order to rinse the sample, to which the building blocks have been coupled, located in the reaction chamber 9, after the reaction time has elapsed and the used reaction solution has been discharged, a rinse comb 8, which is picked up with the desired rinse, is moved up by the gripper arms 2 and over the reaction chamber 9 of the synthesis plate 5 to deliver the rinse quantitatively. After washing, another washing comb 8 is used to provide the solution required to cleave the temporary protecting group of the coupled synthetic building block, as described above. After a period of incubation time, the cutting solution is drained through the valve block 6 with the aid of the suction pump 7 and the sample is washed. After washing, a new synthesis cycle is started, coupling with another synthetic building block.
According to the invention, a separate synthesis pen 11 is provided for each synthetic building block, in each of which pens reagents 20 are placed in a closed space and may be surrounded by an inert gas 21. A single synthesis pen 11 with the corresponding synthesis building block is arranged in the holder 4 of the synthesis device 1 and brought by the gripper arm 2 to the reaction chamber 9 of the synthesis plate 5, the gripper arm 2 gripping the synthesis pen 11 at the gripper arm fixing part 30 for the dosed delivery of the reagents.
The synthetic pen 11 used according to the invention consists of a hollow cylindrical body 12 with a suction mouth 14 at the foot end and a screw cap 13 tightly closing the cylindrical space. In the suction nozzle 14 there is an outlet opening which is closed by the valve needle 15 and the shut-off valve 16, which in the closed position abuts against the seal 29. The valve needle 15 and the shut-off valve 16 are guided by a piston 18 via a piston rod 17. The required closing pressure of the shut-off valve 16 is generated by a compression spring 19 which is located on the piston 18 and bears against the inner end face of the screw cap 13. The free space below the piston 18 is used to display the relevant synthetic building block 20, which is advantageously surrounded by an inert gas 21. In this way, the highly reactive reagent can be kept stable for a long time under an inert gas atmosphere, thereby significantly improving the quality of the synthesized product.
In order to reliably exclude cross-contamination in the case of direct contact of the mouthpiece 14 with the sample, according to the invention the reaction chamber 9 is covered on the open side with a permeable material 25, for example a frit, wherein the sample or a solid phase 26, for example a synthetic resin, is located in the reaction chamber 9. To couple the synthetic building block 20 to the sample or synthetic resin, the suction nozzle 14 of the synthetic pen 11 is placed on the permeable material 25 to close the reaction chamber, whereby the valve needle 15 moves the compression spring 19 inwards against the closing pressure and the shut-off valve 16 is released. After this, the reagent solution can flow freely, the dose of solution flowing out being determined by the time period during which the suction nozzle 14 is placed on the material 25.
With the final cleavage of the temporary protecting group and washing of the sample, cleavage of the synthetic building block 20 coupled to the solid phase 26 occurs. To this end, the cleavage solution is added to the sample by means of the rinsing comb 8 and the cleavage 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 cutting solution enter the receiving chamber of the sample plate 27, according to another feature of the invention the sample plate 27 is arranged below the valve block 6 and connected to the extraction system. The sample plate 27 corresponds in its construction and design to the composite plate 5. The synthesis is complete as the compound dissolved from the solid phase 26 is transferred to the sample plate 27.
Fig. 6 shows another preferred embodiment of the combination pen 11, wherein like reference numerals correspond to each other. In this design, the screw cap of the reagent vessel is replaced by a removable lid 13a with a bayonet lid. The piston rod 17 is preferably arranged in the center of the cap, in particular a pressed-in piston rod, which enters the dose cylinder 31 through the entire synthetic pen. The dose cylinder 31 is closed downwards, for example by means of a check valve 32. By pressing the cap 13a, a predetermined amount of the reagent is dispensed by the piston. A return means, such as springs 33, 33a, 33b, mounted in the combination pen 11, returns the piston. At the same time or downstream, the dosing cylinder is filled 34 again. Suitable regulating means, for example check valves, in the foot-side suction nozzle 14 ensure that the solution can only be dosed by active delivery. This design of the synthetic pen 11 allows for contactless dispensing into the reaction chamber. The closed design of the synthetic pen 11 with closed reagent container ensures a high reagent stability.
Fig. 7 shows a schematic view of a device according to the invention.
The method according to the invention is part of solid phase peptide synthesis, as performed or can be performed by the device according to the invention. For this purpose, the N-terminus of the amino acid is protected from undesired reactions by a protecting group. The amino acids protected in this way are bound via their C-terminus (I) to a solid support material. Subsequently, the N-terminus is deprotected (II) so that another amino acid protected at the N-terminus is bound to the N-terminus of the previous amino acid by peptide propagation (III). Repeating steps II to III until the desired amino acid chain length is reached. When the chain length is reached, the reaction is terminated in step IV by cleaving the peptide from the support material. At least at the end, the peptide is washed with a suitable solvent (step V). Optionally, a pre-swelling of the solid support material (usually a resin) is carried out at the start of the process (step O). According to the invention, at least one of the steps is at least temporarily ultrasound-assisted (X). This is to be understood as meaning the application, at least temporarily, of ultrasound (X) with a frequency of at least more than 25kHz to the reaction medium in which the synthesis takes place. It has been found that an ultrasonic bath into which the reaction medium is introduced via the vessel is particularly suitable for transport. In terms of preparation and synthesis time, it was further found to be advantageous if the ultrasound (X) acts on the reaction medium in several steps, preferably without being switched off between the steps. With particular regard to steps I to IV, ultrasound carried out in accordance with the present invention can reduce the synthesis time by about one order of magnitude.
Table 1 compares the synthesis times of the individual steps of the prior art method without sonication and the repeating unit according to the invention with sonication in the range of 50 to 150 kHz. It is clear that the method according to the invention is ten times faster than a similar method without ultrasound.
Table 1: comparison of the synthesis times required according to the prior art and according to the method of the invention.
In addition to the protecting group bound to the N-or C-terminus, the amino acid may also have other protecting groups to block the reactive side chain. Attention must be paid here to the requirements of the chemical and physical environment during peptide synthesis, such as ultrasound and stability to bases or acids. Suitable protecting groups for the reactive side chains used in the process of the present invention are, for example, acid-labile protecting groups such as S-2,4, 6-trimethoxybenzyl (Tmob), trityl (Trt), tert-butyl (tBu), tert-butyloxycarbonyl (Boc) and 2,2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf).
With reference to the method according to the invention, for example, Fmoc amino acids selected from the group identified as one letter code for amino acids, 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, Fmoc-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), (Fmoc-Asn) -OH, (Tmob-Asn (Tmob) -OH (OB-2, 4, 6-trimethoxy-benzyl).
The Fmoc amino acid may exist in both L and D forms.
By using these, the synthesis time can be reduced by more than ten times without yield loss compared to the prior art.
Table 2 shows a typical synthesis procedure of the method of the invention based on an exemplary pipetting scheme using endomorphins in a preferred embodiment. This basically includes the steps (O-V) mentioned above, however, these steps are described in more detail in the following examples with sub-steps. The first step is the pre-swelling of the resin (O), followed by deprotection of the resin, e.g. with 20% piperidine in DMF, followed by washing with a solvent, e.g. DMF or DCM, coupling of the amino acid (I), again washing with a solvent, e.g. DMF or DCM, deprotection of the amino acid, and finally washing with a solvent, preferably DMF or DCM.
The cycle sequence here is always the same. After the last Amino Acid (AA) has been coupled, it is deprotected, washed and washed with a solvent (e.g. DMF or DCM). During each cycle, ultrasound with a frequency in the range of 25kHz to 2MHz is used to affect the reaction medium in the illustrated embodiment. In this example, the ultrasound (X) is also uninterrupted between the steps. Alternatively, the ultrasound may be interrupted between steps or during steps. However, continuous ultrasound has been shown to be particularly advantageous at test frequencies in the range of 40kHz to 2MHz, especially in the range of 50kHz to 200 kHz.
In the example shown, the steps of pre-swelling and final washing with a solvent, preferably Dichloromethane (DCM), are performed without sonication. However, this is only a preferred embodiment, and thus the ultrasonic waves can be provided in virtually all steps.
Table 2: in a preferred embodiment the synthetic sequence for solid phase peptide synthesis is performed using the method according to the invention.
The process 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 in table 3:
short-term synthesis
Long term synthesis
With ultrasonic deprotection
With ultrasonic deprotection
Deprotection duration: 30s
Deprotection duration: 1min
Number of deprotection steps: 1
Number of deprotection steps: 2
Ultrasonic-free washing
30-second ultrasonic wave washing
Each washing step: wash 5X
Each washing step: wash 3X
Mono-coupling of AA
Double coupling of AA
Table 3: description of short-and long-term Synthesis
The main difference between short-term and long-term synthesis is that the duration of deprotection is halved. Furthermore, the number of washing and deprotection steps is reduced. Although long-term synthesis requires longer synthesis times, it also provides a one-tenth reduction in synthesis time compared to the prior art.
Double coupling synthesis scheme (single frequency) in ultrasonic synthesis
TABLE 4
Total duration of double coupling cycles: 11min
This cycle is repeated until the entire sequence is synthesized (e.g., ACP: H-VQAAIDYING-NH2 → 10 amino acids → 10 cycles).
The preparation steps such as pre-swelling and washing and the final washing step are not listed here.
Single coupling synthesis scheme (Single frequency) in ultrasonic Synthesis
TABLE 5
Total duration of single coupling cycle: 8min
This cycle is repeated until the entire sequence is synthesized (e.g., ACP: H-VQAAIDYING-NH2 → 10 amino acids → 10 cycles).
The preparation steps such as pre-swelling and washing and the final washing step are not listed here.
Single coupling synthesis scheme in ultrasonic synthesis (several frequencies, e.g. 132kHz and 470kHz)
TABLE 6
Deprotection: 2 0.5 with 132+470 → 0.5min at 132kHz, then 0.5min at 470kHz
Addition of AA (+ HCTU, DIPEA):
1+2 has 132+470 → 1min at 132kHz, then 2min at 470kHz
Total duration of single coupling cycle: 8min
This cycle is repeated until the entire sequence is synthesized (e.g., H-PYLFWLAAI-NH2 → 9 amino acids → 9 cycles).
The preparation steps such as pre-swelling and washing and the final washing step are not listed here.
Synthetic protocol for LIPS Synthesis (3-fold coupling (3fach-Kopplung))
TABLE 7
Total duration of cycles with 3 times coupling: about 122min
This cycle is repeated until the entire sequence is synthesized (e.g., H-VQAAIDYING-NH2 → 10 amino acids → 10 cycles).
The time required to dispense a pen depends on several factors and is therefore only given an approximation here.
Synthesis scheme for ABI Synthesis without capping (acetylation) (1-fold coupling (1fach-Kopplung))
TABLE 8
Total duration of cycles with 1-fold coupling: about 80min
Time can only be given in approximations because the various modules may have different lengths, which in turn depends on the sequence to be synthesized. In addition, the internal sensor measures the proportion of deprotected Fmoc groups during deprotection.
This cycle is repeated until the entire sequence is synthesized (e.g., H-VQAAIDYING-NH2 → 10 amino acids → 10 cycles).
Within each module, additional washing steps are included, and therefore these are not shown separately.
Synthesis protocol for capped (acetylated) ABI Synthesis (2-fold coupling (2fach-Kopplung))
TABLE 9
Total duration of cycles with 2-fold coupling: about 130min
Time can only be given in approximations because the various modules may have different lengths, which in turn depends on the sequence to be synthesized. In addition, the internal sensor measures the proportion of deprotected Fmoc groups during deprotection.
This cycle is repeated until the entire sequence is synthesized (e.g., H-VQAAIDYING-NH2 → 10 amino acids → 10 cycles).
Within each module, additional washing steps are included, and therefore these are not shown separately.
ACP H-VQAAIDYING-NH2 M＝1063.2Da
The synthesis scale is as follows: 25 mu mol
Synthetic resin: knorr Amid resin LS 1% DVB
Activating agent: HCTU
Alkali: DIPEA
The amino acids used:
free amino-functional resin: amino acids: activating agent: ratio of alkali: 1:4:3.9:8
FIG. 11 compares ACP peptide H-VQAAIDYING-NH2While taking into account the synthesis strategy: 660-SL3 LIPS: ACP 3-fold coupling (standard protocol) duration in microtiter plate (MTP) LIPS robot: 22.5h
USPS 132kHz 50% power: ACP in ultrasound, 132kHz 1-fold coupling (average of 2 batches) duration: 2.5h
USPS 470kHz 50% power: ACP in ultrasound, 470kHz 1-fold coupling (average of 2 batches) duration: 2.5h
USPS 1000kHz 60% power: ACP in ultrasound, 1000kHz 1-fold coupling (average of 2 batches) duration: 2.5h
It can be seen that the level of frequency improves the quality of the product.
FIG. 12 graphically illustrates H-VQAAIDYING-NH synthesized according to FIG. 112Mass of 25. mu. mol.
Thus, it can be said that continuous ultrasound improves product quality, and an increase in frequency also improves product quality.
Figure 13 shows the testing of stock solutions of amino acids for different solution durations.
For different stock solutions, ACP peptide synthesis was performed using 1000kHz ultrasound, with different dissolution times for the amino acids used.
It can be seen that the shorter dissolution time of the amino acids used improves the product quality.
FIG. 14 is a graphical representation of the comparison of yield and quality in terms of coupling number (Kopplungszahl) at different frequencies H-VQAID.
It can be seen that at low frequencies (132kHz), the LCMS mass increases with increasing coupling number.
At high frequency (470kHz), LCMS quality hardly differs at all.
However, regardless of frequency, yield increases with increasing number of couplings.
For the experiment (fig. 15), a different peptide was selected than in the previous tests. The sequence is PYLFWLAAI-NH2, which is also a difficult peptide to synthesize.
ACP H-PYLFWLAAI-NH2 M＝1092.6Da
The synthesis scale is as follows: 25 mu mol
Synthetic resin: knorr Amid resin LS 1% DVB
Activating agent: HCTU
Alkali: DIPEA
The amino acids used:
free amino-functional resin: amino acids: activating agent: ratio of alkali: 1:4:3.9:8
Figure 15 shows a comparison of single and double couplings and amino acid excess of the peptide.
At the same frequency, there was no significant difference in the quality of the synthesis.
However, as the number of couplings was increased, the relative yield increased significantly.
Figure 16 shows a comparison of the average synthetic mass of peptides using different ultrasound frequencies.
It shows the synthesis of peptide PYLFWLAAI-NH2, which was synthesized using a single coupling at different ultrasound frequencies.
No ultrasonic wave:
conventional ABI synthesis with a 40-fold excess of amino acids.
Conventional ABI synthesis with 4-fold excess of amino acids.
The less the amino acid excess, the worse the LCMS quality in conventional ABI synthesis.
Simple 4-fold excess of amino acids at ultrasound frequency
Frequency: 40kHz, 132kHz, 470kHz
As the frequency increases, the LCMS quality improves.
Coupled ultrasound frequency (deprotection, coupling), washing only at lower frequencies
Frequency of coupling: 40kHz +470kHz and 132kHz +470kHz
Switching between frequencies results in a significant degradation of the quality of the synthesis.
Ultrasound VS classical ABI Synthesis
To achieve good to very good LCMS quality in conventional ABI synthesis, a very high excess of amino acids (40 fold) is required.
With the aid of ultrasound, a 4-fold excess of amino acids is sufficient. Here, the higher the frequency used, the higher the quality of the LCMS.
The equivalent results show the following parameters:
ABI (40X excess) and 470kHz (4X excess).
By ultrasonic synthesis, the use of solvents and amino acids can be reduced in a shorter time, with at least equivalent and generally better results.
Fig. 8 to 10 show the compositions of peptides synthesized by the solid phase peptide synthesis method, respectively. The peptides shown in figures 8 to 9 were prepared by a method according to the invention, whereas figure 10 is based on a peptide synthesized by tetra according to the prior art. All processes are carried out using the apparatus according to the invention.
The products obtained from the various methods were separated by HPLC and individual peaks were assigned by mass spectrometry and uv-vis spectrometry. Use of a device with the following parameters:
HPLC MS system
Dionex Binary HPLC pump
The running medium A: water + 0.1% formic acid
The running medium B: acetonitrile + 0.1% formic acid
Flow rate: 0.5ml/min
Gilson autosampler for up to 4 microtiter plates
Dionex column incubator
The temperature is 30 DEG C
Dionex ultraviolet detector
Measurement at 220nm
Dionex/Thermo Finnigan Surveyor MSQ single quadrupole mass spectrometer
Ionization mode: ESI
Sample temperature: 350 deg.C
Cone voltage: 50V
HPLC separation column: merk, Chromolith WP300, RP18, 100-4.6mm
FIG. 8 shows that the above-described protecting groups for blocking reactive side chains are stable in the process according to the invention. For this reason, the peptide synthesis results according to the method of the invention are shown for the three most common protecting groups.
Figure 8 shows the results of an analysis of endomorphin synthesis performed on the basis of a long-term synthesis procedure according to the method of the invention. Initial theoretical considerations indicate that oxidation-sensitive tryptophan can be ultrasonically oxidized during synthesis. However, this was not confirmed. In contrast, the synthesis was successful with a purity of 83%. Only a few by-products were identified.
The methionine, trityl and Tmob protecting groups also proved to be stable in individual tests during the process according to the invention.
FIG. 9 shows the synthesis of Acyl Carrier Protein (ACP) having the sequence VQAAIDYING-OH, prepared according to the methods of the invention with long-term synthesis.
Protecting groups Fmoc-Q (Tmob) -OH and Fmoc-N (Tmob) -OH were used. The synthetic peptides are substantially difficult to prepare due to their strong hydrophobicity. However, the synthetic peptide can be produced with a purity of 82% by the method according to the present invention. Compared to the synthesis of the same peptide (which can only reach 79% purity) using the prior art tetra method shown in fig. 10, it can be seen that the method according to the invention can achieve, among other advantages, an increase in yield. Furthermore, the synthesis time of the peptides produced according to the method of the invention was completed within 2.5 hours, whereas the comparative method according to the prior art required 25 hours. The method according to the invention is therefore ten times shorter.
The use of the method according to the invention and the device according to the invention advantageously allows the synthesis time to be reduced to at most one tenth of the synthesis time for a method according to the prior art without microwave assistance. It can be shown that this is in no way consistent with a reduction in yield, but that the process according to the invention produces the target peptide in a higher purity, in particular when using the apparatus according to the invention, as can be shown by a direct comparison with standard processes.
List of reference numerals
1 Synthesis device
2 clamping arm
3 working area
4 support
5 composite board
6 valve block
7 suction pump
8 washing comb
9 reaction chamber
10 irrigant supply line
11 synthetic pen
12 main body, hollow cylinder
13. 13a cover, screw cover and movable cover with bayonet cover
13b locking flap
14 suction nozzle
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 Material/frit
26 solid phase
27 sample plate
28 film
29 seal
30 clamping arm fixing part
31 quantitative cylinder
32 outlet valve
33 return spring
33a reset spring fastener
33b return spring, screw fastener
34 gap for dosing cartridge filling
35 union nut
36 dosing tube guide
37 quantitative tube
50 ultrasonic bath
Pre-expansion of O
I binding the amino acid protected by the protecting group at the N terminal to the solid carrier material through the C terminal of the amino acid,
II cleavage protecting group
III propagating at least one peptide
IV termination of the reaction by cleavage of the peptide from the support material
V washing
Effect of X-ray ultrasound
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