EP3365352A1 - Améliorations dans la synthèse peptidique en phase solide - Google Patents

Améliorations dans la synthèse peptidique en phase solide

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Publication number
EP3365352A1
EP3365352A1 EP16858322.7A EP16858322A EP3365352A1 EP 3365352 A1 EP3365352 A1 EP 3365352A1 EP 16858322 A EP16858322 A EP 16858322A EP 3365352 A1 EP3365352 A1 EP 3365352A1
Authority
EP
European Patent Office
Prior art keywords
deprotection
coupling
cycle
deprotecting
vessel
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP16858322.7A
Other languages
German (de)
English (en)
Other versions
EP3365352A4 (fr
Inventor
Jonathan M. Collins
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
CEM Corp
Original Assignee
CEM Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by CEM Corp filed Critical CEM Corp
Priority claimed from PCT/US2016/058181 external-priority patent/WO2017070512A1/fr
Publication of EP3365352A1 publication Critical patent/EP3365352A1/fr
Publication of EP3365352A4 publication Critical patent/EP3365352A4/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/045—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers using devices to improve synthesis, e.g. reactors, special vessels

Definitions

  • the present invention relates to improvements in the solid phase synthesis of peptides ("SPPS").
  • Peptides are linked chains of amino acids which in turn are the basic building blocks for most living organisms. Peptides are also the precursors of proteins! i.e., long complex chains of amino acids. Peptides and proteins are fundamental to human and animal life, and they drive, affect, or control a wide variety of natural processes.
  • peptides have been recently identified that can "keyhole" tumor specific mutations in certain cancers and thus act as tumor specific vaccines (e.g., SAMPSON, JH ET AL.
  • An epidermal growth factor receptor variant Ill-targeted vaccine is safe and immunogenic in patients with glioblastoma multiforme. Mol. Cancer Ther. 2009! 8: 2773- 2779; Li G, SlDDHARTHA M, WONG AJ.
  • solid phase synthesis is relatively simple and straightforward.
  • An amino acid is attached to a solid phase particle by a linking group on the acid side, and to a protecting group on the amine side.
  • the protecting group is removed so that the second acid (and in particular it's acid group) can be coupled to the amine group on the original acid.
  • the second (and succeeding) acids are also initially protected, and thus the general sequence is to deprotect, couple, and repeat until the desired peptide is completed, following which the completed peptide is cleaved from the solid phase resin.
  • the SPPS deprotection step is typically carried out by adding an organic base to the protected acid, then draining the reaction vessel—
  • one of the advantages of SPPS is that the organic compounds can be handled as if they were solids— then washing the deprotected chain. In most circumstances, a wash repeated five times is both typical and satisfactory to remove anything that might create different sequences or undesired byproducts.
  • the coupling step is then carried out followed by another draining step, and another repetitive wash, with five washes again being typical.
  • microwave assisted techniques have become widely accepted in the art, following their introduction about a decade ago (e.g., commonly assigned US Patent No. 7393920, the contents of which are likewise incorporated entirely herein by reference). Microwave techniques have reduced cycle times from hours to minutes, thus providing multiple advantages in SPPS and in research or commerce that depends upon SPPS.
  • the step of adding the deprotecting base is usually carried out by adding a sufficient volume of relatively low concentration that will cover the drained resin in the reaction vessel and the attached peptide after the coupling step to ensure that both the scavenging and deprotection reactions take place.
  • the invention is a method of deprotection in solid phase peptide synthesis in which the improvement comprises adding the deprotecting composition in high concentration and small volume to the mixture of the coupling solution, the growing peptide chain, and any excess activated acid from the preceding coupling cycle, and without any draining step between the coupling step of the previous cycle and the addition of the deprotection composition for the successive cycle.
  • the invention is a method of deprotection in solid phase peptide synthesis in which the improvement comprises deprotecting a protected amino acid by combining the protected amino acid and a liquid organic base in a reaction vessel and during or after the deprotection step reducing the ambient pressure in the vessel with a vacuum pull to remove the liquid organic base without any intermediate draining step.
  • the invention is a method of deprotection in solid phase peptide synthesis (SPPS) in which the improvement comprises deprotecting a protected amino acid at a temperature of at least about 60°C while providing a path for evaporating base to leave the reaction vessel
  • SPPS solid phase peptide synthesis
  • the invention is a system for microwave assisted solid phase peptide synthesis.
  • the system includes a microwave source positioned to direct microwave radiation into a microwave cavity, a microwave transparent reaction vessel in the cavity, and a vacuum source connected to the reaction vessel.
  • the invention is a method of deprotection in solid phase peptide synthesis in which the improvement comprises adding the deprotecting composition in high concentration and small volume to the mixture of the coupling solution, the growing peptide chain, and any excess activated acid from the preceding coupling cycle, and without any draining step between the coupling step of the previous cycle and the addition of the deprotection composition for the successive cycle, and thereafter reducing the ambient pressure in the vessel with a vacuum pull to remove the deprotecting composition without any draining step.
  • the invention is a method of deprotection in solid phase peptide synthesis which includes the steps of adding the deprotection composition in high
  • Figure 1 is a schematic diagram of the conventional steps of SPPS synthesis.
  • Figure 2 is a schematic diagram of an improved version of conventional SPPS peptide synthesis.
  • Figure 3 is a schematic diagram of a first embodiment of the present invention.
  • Figure 4 is a diagram illustrating the thermal advantages of the current invention.
  • Figure 5 is a schematic diagram of a second embodiment of the invention.
  • Figure 6 is a schematic diagram of an instrument used to carry out the method of the present invention.
  • FIG. 7 is a second schematic diagram of portions of the instrument used to carry out the present invention. Detailed Description
  • FIG. 1 is a schematic diagram of a conventional cycle repeated during solid phase peptide synthesis and broadly designated at 20.
  • the next acid to be added 21 is added in protected fashion to a reaction vessel schematically diagramed at 22.
  • the deprotection step 23 is carried out in the vessel 22 by adding an organic base in a concentration of about 20% by volume in dimethyl formamide (DMF).
  • organic bases include, but are not limited to piperidine (C5H11N; CAS No 110-89-4), pyrrolidine (C4H9N; CAS No 123-75- 1), and 4-methyl piperidine (CeFmN; CAS No. 626-58-4).
  • the deprotection solution 28 is added in advance of the next acid.
  • the deprotection solution is then drained (step 24) following which a washing liquid (e.g., methanol or isopropanol) is added to the vessel for a washing step 25 carried out repetitively with five repetitions being typical.
  • a washing liquid e.g., methanol or isopropanol
  • the washing solution is then removed in a second draining step 26 which allows the coupling step 27 to take place.
  • the coupling composition is then removed in a third draining step 30 followed by a second washing step 31, again repeated five times.
  • Figure 1 is schematic, and that there are many details about one SPPS cycle that could be added, but that Figure 1 illustrates the concept sufficiently for the skilled person to understand both it and the present invention.
  • Figure 1 represents a cycle that is neither the step of linking the resin to the first acid, nor does it illustrate cleaving a finished peptide from the resin.
  • FIG. 2 illustrates the improved conventional method referred to in the Background and broadly designated at 32.
  • the last washing step 31 can be omitted because any excess acid left after the coupling step 27 will be quenched by the deprotection solution (base) added at the start of the next cycle. Obviously, this requires that deprotection solution be added before the next acid 21 is added to the vessel 22.
  • Figure 3 illustrates a first embodiment of the invention in which the improvement comprises adding the deprotection composition in a high concentration and small volume to the mixture of the coupling solution, the growing peptide chain, and any excess activated acid from the preceding coupling cycle, and doing so without any draining step between the coupling step of the previous cycle and the addition of the deprotection composition for the successive cycle.
  • the use of a small volume in high concentration saves physical space (only a small bottle is needed), avoids the need to prepare a solution, and saves solvent.
  • the method additionally offers a thermal advantage (Figure 3).
  • an organic base is used as the deprotecting composition with piperidine or pyrrolidone or 4-methylpiperidine being typical (although not necessarily exclusive) for this purpose. It will be understood, of course, that additional organic bases that provide the deprotection function without otherwise interfering with the other steps in the method, the growing peptide chain, or the instrument, will be appropriate as well.
  • the piperidine or pyrrolidine or 4- methylpiperidine can be added neat! i.e. as an organic liquid and not in solution.
  • the piperidine or pyrrolidine or 4-methylpiperidine can be added as a highly concentrated solution of at least about 50% organic base by volume, typically in DMF.
  • the high concentration allows the organic base to be added in a proportionally small volume with a ratio of between about 1:20 and 1:3 being appropriate based upon the volume of the coupling solution.
  • Piperidine or pyrrolidone or 4- methylpiperidine can be added in the volume ratio of about 1:5 based upon the volume of the coupling solution when added neat.
  • the small volume of the deprotecting solution is typically less than 2 ml, and often less than one milliliter.
  • between about 0.4 and 1.0 ml of piperidine are added to between about 3.8 and 4.2 ml of the mixture of the coupling solution, the growing peptide chain and any excess activated acid.
  • the small volume of the deprotecting solution is 20% or less of the volume of the mixture of the coupling solution, the growing peptide chain, and any excess activated acid.
  • Figure 4 illustrates the thermal advantage offered by the invention which provides an additional time advantage in each SPPS cycle. As Figure 4 demonstrates, if the coupling step is carried out at temperatures of about 90°C, the conventional use of a room
  • Figure 5 illustrates another aspect of the invention in which the improvement comprises deprotecting a protected amino acid by combining the protected amino acid and liquid organic base in a reaction vessel, and then during or after the deprotection step reducing the ambient pressure in the vessel to below atmospheric pressure with a vacuum pull to remove the liquid organic base without any intervening draining step.
  • the boiling point of piperidine is approximately 106°C and that of DMF is about 153°C.
  • the vapor pressure of piperidine will be higher than the vapor pressure of DMF at any given temperature. Accordingly it has now been discovered that pulling a moderate vacuum from the vessel can selectively remove the piperidine and completely avoid the draining step.
  • Figure 5 illustrates this schematically by showing the deprotection step 23 followed by an evaporation step 36 followed by the draining step (of liquids other than the organic base) and then the coupling step 27.
  • the boiling point of 4-methylpiperidine is 123°C, offering similar advantages.
  • piperidine's vapor pressure is about 4 mm Hg at 25°C, about 39 mm Hg at 50°C, and about 55 mm Hg at 60°C.
  • the vapor pressure is about 8.4 mm Hg at 25°C and about 102 mm Hg at 60°C.
  • the method can further comprise accelerating the deprotection step by heating the combined protected amino acid and the liquid organic base in the vessel 22, and then accelerating the removal step further by pulling the vacuum 36 while heating the vessel contents.
  • the microwave radiation can be used to both accelerate the deprotection step and to accelerate the vacuum removal step.
  • the pressure can be reduced to below atmospheric pressure, or, expressed in terms of temperatures, the deprotection step can be carried out by heating the compositions to at least about 60°C, and in some cases to between about 81°C and 99°C, after which the vessel contents can be heated to between about 90° and 110° to accelerate the vacuum removal step.
  • the vacuum and the applied microwave power should provide the intended enhanced evaporation without otherwise adversely affecting the remaining materials in the vessel or causing problems in subsequent steps in the SPPS cycle.
  • the improvement includes the steps of adding the deprotecting composition in high concentration and small volume to the mixture of the coupling solution, the growing peptide chain, and any excess activated acid from the preceding coupling step, and doing so without any intervening draining step between the coupling step of the previous cycle and the addition of deprotection composition for the successive cycle. Thereafter, the ambient pressure in the vessel is reduced with a vacuum pull to remove the deprotecting composition without any draining step.
  • FIGs 6 and 7 are schematic illustrations of selected portions of a system for carrying out the improvements described herein.
  • the system includes a microwave source illustrated as the diode 40 positioned to direct microwave radiation into a microwave cavity 41, and with a vacuum source shown as the pump 42 connected to the reaction vessel 22 in the cavity 41.
  • the microwave source is illustrated as a diode (an IMPATT diode is exemplary)
  • a magnetron is a similarly acceptable source as is a klystron, each of these items being well understood in the art by the skilled person and can be selected as desired for purposes of convenience, design, or cost, and without undue experimentation.
  • FIG. 6 also shows that microwave radiation from the source 40 is typically directed through a waveguide 43 which provides support to the cavity 41.
  • the vacuum pump 42 pulls from the vessel 22 along line 44 and usually includes a trap 45 which is otherwise
  • the cavity 41 can support a single mode of microwave radiation at the microwave frequencies produced by the microwave source 40.
  • a temperature probe 46 (for which a fiber optic device is exemplary) is positioned to read the temperature of the reaction vessel 22 in the cavity 41.
  • a processor 47 (which can be either internal or external to the overall system), the measured temperature can be used to drive the source and to thus increase, decrease, or otherwise moderate the microwave radiation into the cavity in the most advantageous manner.
  • the microwave source 40 is driven by a power supply broadly designated at 50 which in preferred embodiments can be the switching power supply (and associated methods) set forth in US Patent No. 6288379, the contents of which are incorporated entirely herein by reference.
  • the basic circuits between the power supply and the diode 40 are likewise illustrated schematically at 51. Basic circuitry of the type required is well understood by those in the relevant arts, need not be described in detail herein, and can be built and operated by the skilled person without undue experimentation.
  • FIG. 7 schematically illustrates a few additional details of the system for carrying out the method of the invention.
  • the vessel is again designated at 22, and Figure 7 further illustrates that the vessel 22 includes a frit 52 (typically made of glass) and a spray head 53.
  • the frit 52 permits liquids to be drained from the reaction vessel 22 and the spray head 53 delivers compositions to the reaction vessel 22.
  • Other equivalent fixtures can be selected by the skilled person without undue experimentation.
  • Figure 7 illustrates a nitrogen supply 54 which is connected to a plurality of supply bottles 55 which for schematic purposes are illustrated as Erlenmeyer flasks.
  • a plurality of metered loops are schematically illustrated by lines 56, 57, and 58 and connect the nitrogen supply to the supply bottles 55; and corresponding lines 60, 61, and 62 then connect to a common line 63 that reaches the spray head 53 for delivery to the vessel 22.
  • a separate line 63 provides nitrogen from the source 54 to the liquids and resin in the vessel 22 to agitate (bubble) the contents of the vessel 22 to carry out appropriate mixing and circulation during deprotection, coupling, and cleavage reactions.
  • Nitrogen is helpful under these circumstances because it is relatively inexpensive, widely available, and inert to the reactions being carried out and to the equipment in the instrument or system. It will thus be understood that other inert gases, including the noble gases, can be used for this purpose, but in most cases will simply be more expensive and less widely available. In a functional sense, any gas that will avoid interfering chemically with the ongoing reactions or with the instrument will be appropriate.
  • the nitrogen supply and the metered loop can connect to the processor 47 so that the processor 47 can control the manner in which the compositions are dispensed from the vessels 55 to the reaction vessel 52.
  • DIEA Diisopropylethylamine
  • NMM N-methylmorpholine
  • TMP collidine
  • TMP piperidine
  • Trazine trifluoroacetic acid
  • TMP trifluoroacetic acid
  • thioanisole 1,2-ethanedithiol
  • phenol phenol
  • DCM Dichloromethane
  • DMF ⁇ , ⁇ -Dimethylformamide
  • NMP Nmethylpyrrolidone
  • anhydrous ethyl ether acetic acid
  • SPHERITIDETMresin Trityl linker was prepared using SPHERITIDETM resin (CEM Corporation; Matthews, NC; USA).
  • the SPHERITIDETM resin consists of polye -lysine cross- linked with multifunctional carboxylic acids.
  • the LIBERTYTM system (CEM Corporation, Matthews, NC) is a sequential peptide synthesizer capable of complete automated synthesis including cleavage of up to 12 different peptides.
  • the LIBERTYTM system uses the single-mode microwave reactor, DISCOVERTM, which has been widely used in the organic synthesis industry.
  • the LIBERTYTM synthesizer uses a standard 30 milliliter (ml) Teflon® glass fritted reaction vessel for 0.025-1.0 millimole (mmol) syntheses.
  • the reaction vessel features a spray head for delivery of all reagents and a fiber-optic temperature probe for controlling the microwave power delivery.
  • the system utilizes up to 25 stock solutions for amino acids and seven reagent ports that can perform the following functions: main wash, secondary wash, deprotection, capping, activator, activator base, and cleavage.
  • the system uses nitrogen pressure for transfer of all reagents and to provide an inert environment during synthesis. Nitrogen bubbling is used for mixing during deprotection, coupling, and cleavage reactions.
  • the system uses metered sample loops for precise delivery of all amino acid, activator, activator base, and cleavage solutions.
  • the LIBERTYTM synthesizer is controlled by an external computer, which allows for complete control of each step in every cycle.
  • a model peptide containing all 20 amino acids was synthesized under a variety of conditions using the CEM LIBERTYTM automated microwave peptide synthesizer on 0.152g SpheritideTM resin (0.66 meq/g substitution). Deprotection was performed in two stages using a fresh reagent each time with (i) 80% piperidine in DMF; or (ii) piperidine neat. In each case, 0.8 ml of the piperidine was added to 4.0 ml of the coupling solution remaining from the addition of the previous acid. An initial deprotection of 30 s at 50 W (5 min at 0 W for conventional synthesis) was followed by a 3-min deprotection at 50 W (15 min at 0 W for conventional synthesis) with a maximum temperature of 80 °C.
  • the piperidine was removed by applying a vacuum that reduced the ambient pressure in the reaction vessel to below atmospheric pressure. Removal was enhanced by applying microwave power at 50 W for 3 minutes.
  • HBTU:DIEA :AA (0.9 : 2 : 1); (ii) HBTU:HOBt: DIEA :AA (0.9 : 1 : 2 : 1); (iii) PyBOP : DIEA :AA (0.9 : 2 : 1); (iv) HBTU:NMM:AA (0.9 : 2 : 1); and (v) HBTU: TMP:AA (0.9 : 2 : l), double coupling on valine. Coupling reactions were for 5 min at 40 W (30 min at 0 W for
  • the invention presents a novel process whereby the coupling and deprotection steps occur within the same solvent.
  • concentrated base is added directly to the resin coupling solution after a desired period of time for the coupling to occur.
  • the deprotection step is then immediately started when the base is added. Therefore, the onset of the deprotection step is immediately after the coupling step without any time delay.
  • only a small volume of base is required since it can use the solvent present from the coupling reaction. This requires a sophisticated reagent delivery system for the base that is accurate at very small volumes (0.5mL) with rapid delivery.
  • a 20% solution of base (piperidine) in solvent is used for the deprotection step.
  • Excess base concentration can increase base-catalyzed side reactions and therefore significant solvent is required. This means that significant solvent can be saved from this process by adding concentrated base to the coupling solvent.
  • the peptides were synthesized at 0.05mmol scale with 10 equivalents of amino acid using CarboMAXTM coupling with AA/DIC/Oxyma (L2: i) based activation for 100 sec at 90°C.
  • ProTide resins CEM Corporation; Matthews, NC; USA
  • TentaGel® technology were used for synthesis with either a Rink Amide linker or a Cl-TCP(Cl) linker with unactivated loading of the first amino acid with DIEA at 90°C for 5 min.
  • the deprotection step was performed for 50 sec at 95°C and initiated by adding 0.5mL of 50% pyrrolidine directly to the coupling solution. A single lx4mL wash was used in between the deprotection and coupling steps. Peptides were cleaved with
  • TFA/TIS/H20/DODt 92.5:2.5:2.5:2.5
  • RAZOR cleavage system CEM Corporation; Matthews, NC; USA.
  • HIV-TAT (47-57) HIV/AIDS RA ProTide 93 0:31 Fmoc-YGRKKRRQRRR-NH 2 Research
  • HIV-TAT 48-60 HIV/AIDS RA ProTide 88 0:39

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un procédé amélioré de déprotection dans la synthèse peptidique en phase solide. En particulier la composition de déprotection est ajoutée à concentration élevée et sous un faible volume au mélange de la solution de couplage, de la chaîne peptidique en croissance, et de n'importe quel acide activé en excès provenant du précédent cycle de couplage, et sans aucune étape de drainage entre l'étape de couplage du cycle précédent et l'ajout de la composition de déprotection pour les cycles consécutifs. Ensuite, la pression ambiante dans le récipient est réduite par une aspiration sous vide afin d'éliminer la composition de déprotection sans aucune étape de drainage et sans nuire autrement aux matériaux restants dans le récipient ou provoquer de problèmes aux étapes ultérieures dans le cycle de SPPS.
EP16858322.7A 2015-10-23 2016-10-21 Améliorations dans la synthèse peptidique en phase solide Pending EP3365352A4 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201562245484P 2015-10-23 2015-10-23
PCT/US2016/058181 WO2017070512A1 (fr) 2015-10-23 2016-10-21 Améliorations dans la synthèse peptidique en phase solide

Publications (2)

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EP3365352A1 true EP3365352A1 (fr) 2018-08-29
EP3365352A4 EP3365352A4 (fr) 2019-06-05

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CN (1) CN108368152A (fr)

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EP3507299B1 (fr) * 2016-09-03 2022-07-27 CEM Corporation Procédé de recyclage de solvant in situ pour la synthèse de peptides en phase solide à des températures élevées
CA3232401A1 (fr) * 2021-09-17 2023-03-23 Cem Corporation Procedes de synthese de peptide en phase solide (spps) et systemes associes

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US8314208B2 (en) * 2006-02-10 2012-11-20 Cem Corporation Microwave enhanced N-fmoc deprotection in peptide synthesis
CN102002095A (zh) * 2010-05-20 2011-04-06 中国药科大学 微波促进固相合成苦瓜降血糖mc-jj0108多肽类似物及其应用
US9169287B2 (en) * 2013-03-15 2015-10-27 Massachusetts Institute Of Technology Solid phase peptide synthesis processes and associated systems
CN104788546A (zh) * 2015-03-12 2015-07-22 吉尔生化（上海）有限公司 一种含有24个氨基酸残基的线性直链肽的制备方法

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EP3365352A4 (fr) 2019-06-05

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