US20190194246A1 - Solid phase peptide synthesis - Google Patents
Solid phase peptide synthesis Download PDFInfo
- Publication number
- US20190194246A1 US20190194246A1 US16/143,917 US201816143917A US2019194246A1 US 20190194246 A1 US20190194246 A1 US 20190194246A1 US 201816143917 A US201816143917 A US 201816143917A US 2019194246 A1 US2019194246 A1 US 2019194246A1
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- United States
- Prior art keywords
- reaction vessel
- microwave
- coupling
- deprotection
- cycle
- 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.)
- Abandoned
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- 239000007790 solid phase Substances 0.000 title claims abstract description 17
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- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4723—Cationic antimicrobial peptides, e.g. defensins
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/57572—Gastrin releasing peptide
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
-
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/635—Parathyroid hormone, i.e. parathormone; Parathyroid hormone-related peptides
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/645—Secretins
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/81—Protease inhibitors
- C07K14/815—Protease inhibitors from leeches, e.g. hirudin, eglin
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- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/16—Oxytocins; Vasopressins; Related peptides
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- B01J2204/00—Aspects relating to feed or outlet devices; Regulating devices for feed or outlet devices
- B01J2204/005—Aspects relating to feed or outlet devices; Regulating devices for feed or outlet devices the outlet side being of particular interest
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- B01J2219/00049—Controlling or regulating processes
- B01J2219/00051—Controlling the temperature
- B01J2219/00054—Controlling or regulating the heat exchange system
- B01J2219/00056—Controlling or regulating the heat exchange system involving measured parameters
- B01J2219/00058—Temperature measurement
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- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
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- B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
- B01J2219/08—Processes employing the direct application of electric or wave energy, or particle radiation; Apparatus therefor
- B01J2219/12—Processes employing electromagnetic waves
- B01J2219/1203—Incoherent waves
- B01J2219/1206—Microwaves
- B01J2219/1248—Features relating to the microwave cavity
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- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16311—Human Immunodeficiency Virus, HIV concerning HIV regulatory proteins
- C12N2740/16322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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., S AMPSON , J H ET AL .
- An epidermal growth factor receptor variant III - targeted vaccine is safe and immunogenic in patients with glioblastoma multiforme. Mol. Cancer Ther. 2009; 8: 2773-2779; L I G, S IDDHARTHA M, W ONG A J.
- 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.
- Solid phase peptide synthesis had its genesis in 1963 when R. B. Merrifield published the synthesis of a four-acid chain using a solid phase method (R. B. M ERRIFIELD ; Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide; J. Am. Chem. Soc., 1963, 85 (14), pp 2149-2154).
- 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 U.S. Pat. No. 7,393,920, 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 concentration and small volume to the mixture of the coupling solution, the growing peptide chain, and any excess activated amino 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 which removes at least 50% of the volume of the previous cycle coupling solution; and with the coupling solution at least 30° C.
- FIG. 1 is a schematic diagram of the conventional steps of SPPS synthesis.
- FIG. 2 is a schematic diagram of an improved version of conventional SPPS peptide synthesis.
- FIG. 3 is a schematic diagram of a first embodiment of the present invention.
- FIG. 4 is a diagram illustrating the thermal advantages of the current invention.
- FIG. 5 is a schematic diagram of a second embodiment of the invention.
- FIG. 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.
- 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 (C 5 H 11 N; CAS No 110-89-4), pyrrolidine (C 4 H 9 N; CAS No 123-75-1), and 4-methyl piperidine (C 6 H 13 N; 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.
- FIG. 1 is schematic, and that there are many details about one SPPS cycle that could be added, but that FIG. 1 illustrates the concept sufficiently for the skilled person to understand both it and the present invention.
- FIG. 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 .
- FIG. 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 ( FIG. 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.
- FIG. 4 illustrates the thermal advantage offered by the invention which provides an additional time advantage in each SPPS cycle.
- the coupling step is carried out at temperatures of about 90° C.
- the conventional use of a room temperature (e.g. 25° C.) wash will have the expected thermal effect of lowering the temperature of peptide and the resin in the vessel in accordance with well understood and relatively simple relationships (e.g., the drop in temperature will be directly proportional to the mass of the added cooler liquid).
- a washing or draining step is carried out after coupling, there will be some time interval required to bring the reacting compositions back up to the 90° coupling temperature.
- FIG. 4 the conventional thermal profile is indicated by the solid line 34 and the thermal profile provided by the invention is indicated by the dotted line 35 . It will be understood, of course, that FIG. 4 is schematic, not drawn to scale, and illustrative rather than a precise track of any particular mixture.
- FIG. 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.
- FIG. 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.
- raising the temperature to 60° C. greatly encourages the desired evaporation.
- 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 conventional (e.g., a cold trap using liquid nitrogen) and positioned between the vessel and the vacuum pump 42 . In the absence of the trap 45 , the vacuum pump needs to be capable of handling the evaporated base and solvents while still operating as intended.
- 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 .
- 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 U.S. Pat. No. 6,288,379, 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
- FIG. 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.
- FIG. 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 .
- the simple schematic line connections ( 64 and 65 ) are in practice combination of tubes (pipes), valves, and controls for those lines; e.g., in practice line 64 represents a connection between a valve or manifold in line 58 , a controller for that line, and the processor 47 .
- Fmoc amino acids were obtained from Novabiochem (San Diego, CA) and contained the following side chain protecting groups: Asn(Trt), Asp(OtBu), Arg(Pbf), Cys(Trt), Gln(Trt), Glu(OtBu), His(Trt), Lys(Boc), Ser(tBu), Thr(tBu), Trp(Boc), and Tyr(tBu).
- Diisopropylethylamine (DIEM, N-methylmorpholine (NMM), collidine (TMP), piperidine, piperazine, trifluoroacetic acid (TFA), thioanisole, 1,2-ethanedithiol (EDT), and phenol were obtained from Sigma Aldrich (St. Louis, Mo.).
- DIEM Diisopropylethylamine
- NMM N-methylmorpholine
- TMP trifluoroacetic acid
- TMP trifluoroacetic acid
- thioanisole 1,2-ethanedithiol
- EDT 1,2-ethanedithiol
- phenol 1,2-ethanedithiol
- phenol 1,2-ethanedithiol
- phenol 1,2-ethanedithiol
- phenol 1,2-ethanedithiol
- phenol 1,2-ethanedithiol
- phenol 1,2-ethanedithiol
- phenol 1,2-ethanedithiol
- Trityl linker was prepared using SPHERITIDETM resin (CEM Corporation; Matthews, N.C.; USA).
- the SPHERITIDETM resin consists of poly-e-lysine cross-linked with multifunctional carboxylic acids.
- the LIBERTYTM system (CEM Corporation, Matthews, N.C.) 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 0.152 g 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.
- Coupling reactions were performed in the presence of a 5-fold molar excess of 0.2 M Fmoc-protected amino acids dissolved in DMF with various types of activation: (i) HBTU:DIEA:AA (0.9:2:1); 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 (0.9:2:1), double coupling on valine. Coupling reactions were for 5 min at 40 W (30 min at 0 W for conventional synthesis) with a maximum temperature of 80° C.
- 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 (0.5 mL) 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.
- peptides were synthesized using a Liberty Blue PRIME system (CEM Corporation; Matthews, N.C.; USA) allowing for automated in-situ solvent recycling and evaporation based washing.
- the peptides were synthesized at 0.05mmol scale with 10 equivalents of amino acid using CarboMAXTM coupling with AA/DIC/Oxyma (1:2:1) based activation for 100 sec at 90° C.
- ProTide resins (CEM Corporation; Matthews, N.C.; USA) based on 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.
- This new process provided a significant reduction in standard cycle time (2 minutes 57 seconds) from (a)—elimination of the coupling drain time, (b)—elimination of the deprotection delivery time between steps, and (c)—elimination of the temperature ramp time for the deprotection step thereby allowing a shorter deprotection time to be used. Additionally, significant solvent savings were possible with the complete elimination of the deprotection solvent during each cycle.
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Abstract
An improved method of deprotection in solid phase peptide synthesis is disclosed. In particular the deprotecting composition is added 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. Thereafter, the ambient pressure in the vessel is reduced with a vacuum pull to remove the deprotecting composition without any draining step and without otherwise adversely affecting the remaining materials in the vessel or causing problems in subsequent steps in the SPPS cycle.
Description
- This application is a divisional of Ser. No. 15/299,931 filed Oct. 21, 2016 and claims benefit of Serial No. PCT/US16/58181 filed Oct. 21, 2016 and Ser. No. 62/245,484 filed Oct. 23, 2015. This application is related to co-pending Ser. No. 15/490,090 filed Apr. 18, 2017 as a continuation-in-part of Ser. No. 15/299,931 and PCT/US17/28254 filed Apr. 19, 2017 which claim benefit of Ser. No. 62/383,397 filed Sep. 3, 2016.
- 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.
- As just one example, peptides have been recently identified that can “keyhole” tumor specific mutations in certain cancers and thus act as tumor specific vaccines (e.g., S
AMPSON , J HET AL . An epidermal growth factor receptor variant III-targeted vaccine is safe and immunogenic in patients with glioblastoma multiforme. Mol. Cancer Ther. 2009; 8: 2773-2779; LI G, SIDDHARTHA M, WONG A J. The epidermal growth factor variant III peptide vaccine for treatment of malignant gliomas. Neurosurg. Clin. N. Am. 2010; 21: 87-93; LI G, WONG A J. EGF receptor variant III as a target antigen for tumor immunotherapy. Expert Rev. Vaccines 2008; 7: 977-985). - As a result, the study of peptides and proteins and the capability to synthesize peptides and proteins are of significant interest in the biological sciences and medicine.
- In concept, 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.
- Solid phase peptide synthesis had its genesis in 1963 when R. B. Merrifield published the synthesis of a four-acid chain using a solid phase method (R. B. M
ERRIFIELD ; Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide; J. Am. Chem. Soc., 1963, 85 (14), pp 2149-2154). - At the time, it was generally recognized that organic reactions could be carried out in this manner, but it was assumed that the Merrifield method would be difficult to adapt to longer peptide sequences in any realistic purity. Specifically, Merrifield's suggestion that the isolation steps between and among coupling and deprotection steps could be carried out merely by washing and without identification of intermediates, was considered unlikely to offer long-term success. In peptide synthesis, two problems are characteristic: (1) the synthesis of unwanted byproducts; and (2) the synthesis of some fraction of an undesired sequence based on the presence of unremoved acid from a previous step or cycle. In particular, a residue of the recently added (“activated”) acid tends to remain after the coupling step and must accordingly be removed in some fashion.
- Nevertheless, as summarized by C
HAN AND WHITE , Fmoc Solid Phase Peptide Synthesis (Oxford University Press 2000), the washing steps provide acceptable purity and the general simplicity of those washing steps and of avoiding detailed characterization of intermediates gives the SPPS method its speed and efficiency advantages (e.g., page 1). - Accordingly, as generally well understood in the art, 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.
- More recently (e.g., US 20120041173; the contents of which are incorporated entirely herein by reference), it has become recognized that adding the deprotecting base for the next cycle will scavenge the activated acid remaining from the previous cycle, thus reducing or eliminating the number of washing cycles necessary to ensure purity and avoid unwanted sequences.
- To interject with a point well understood in this art, improving, accelerating or eliminating any of the SPPS steps becomes geometrically advantageous as longer peptide sequences are synthesized. In this regard, microwave assisted techniques have become widely accepted in the art, following their introduction about a decade ago (e.g., commonly assigned U.S. Pat. No. 7,393,920, 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.
- To the extent that a newer technique such as microwave assisted solid phase peptide synthesis can be called typical or conventional, 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.
- Doing so, however, creates a thermal slow down (so to speak) in that the volume of dilute organic base solution is added at room temperature (e.g., 25°) while the coupling step has just been carried out at an elevated temperature, of which temperatures of about 90° C. are exemplary (although not limiting). As expected in a normal heat transfer situation, this reduces the overall temperature of the components in the vessel, which then must be reheated to reach the reaction temperature required for the next deprotection and coupling cycle.
- Although these characteristics are disadvantageous only in the strictest sense, an overall advantage always exists when steps in the SPPS cycle are enhanced, accelerated, or simply rendered unnecessary. Such improvements become more and more advantageous (and conventional methods become more disadvantageous) as the peptide chain length increases. Thus, speed advantages that might remain proportionally meaningless in conventional organic solid phase reactions (i.e., those that require only a few, and perhaps only a single solid phase step) become increasingly important when peptides containing 10, 20, or more acids are synthesized using SPPS.
- In one aspect 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.
- In another aspect 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.
- In another aspect 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
- In another aspect the invention is a system for microwave assisted solid phase peptide synthesis. In this aspect, 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.
- In another aspect 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.
- In another aspect the invention is a method of deprotection in solid phase peptide synthesis which includes the steps of adding the deprotection composition in high concentration and small volume to the mixture of the coupling solution, the growing peptide chain, and any excess activated amino 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 which removes at least 50% of the volume of the previous cycle coupling solution; and with the coupling solution at least 30° C.
- The foregoing and other objects and advantages of the invention and the manner in which the same are accomplished will become clearer based on the followed detailed description taken in conjunction with the accompanying drawings.
-
FIG. 1 is a schematic diagram of the conventional steps of SPPS synthesis. -
FIG. 2 is a schematic diagram of an improved version of conventional SPPS peptide synthesis. -
FIG. 3 is a schematic diagram of a first embodiment of the present invention. -
FIG. 4 is a diagram illustrating the thermal advantages of the current invention. -
FIG. 5 is a schematic diagram of a second embodiment of the invention. -
FIG. 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. -
FIG. 1 is a schematic diagram of a conventional cycle repeated during solid phase peptide synthesis and broadly designated at 20. As set forth therein, the next acid to be added 21 is added in protected fashion to a reaction vessel schematically diagramed at 22. Thedeprotection step 23 is carried out in thevessel 22 by adding an organic base in a concentration of about 20% by volume in dimethyl formamide (DMF). Useful 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 (C6H13N; CAS No. 626-58-4). As indicated by the position of the relative arrows, thedeprotection 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. The washing solution is then removed in asecond draining step 26 which allows thecoupling step 27 to take place. The coupling composition is then removed in athird draining step 30 followed by asecond washing step 31, again repeated five times. - It will be understood that
FIG. 1 is schematic, and that there are many details about one SPPS cycle that could be added, but thatFIG. 1 illustrates the concept sufficiently for the skilled person to understand both it and the present invention. In particular, the skilled person already recognizes thatFIG. 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. In particular, thelast washing step 31 can be omitted because any excess acid left after thecoupling 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 thenext acid 21 is added to thevessel 22. -
FIG. 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 (
FIG. 3 ). - In exemplary versions of the claimed invention, 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.
- In the most exemplary embodiment, the piperidine or pyrrolidine or 4-methylpiperidine can be added neat; i.e. as an organic liquid and not in solution. In other circumstances, 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.
- As a further advantage, 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. In such circumstances, the small volume of the deprotecting solution is typically less than 2 ml, and often less than one milliliter. In exemplary circumstances, 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.
- Expressing the proportion as a percentage, 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.
-
FIG. 4 illustrates the thermal advantage offered by the invention which provides an additional time advantage in each SPPS cycle. AsFIG. 4 demonstrates, if the coupling step is carried out at temperatures of about 90° C., the conventional use of a room temperature (e.g. 25° C.) wash will have the expected thermal effect of lowering the temperature of peptide and the resin in the vessel in accordance with well understood and relatively simple relationships (e.g., the drop in temperature will be directly proportional to the mass of the added cooler liquid). Thus, when a washing or draining step is carried out after coupling, there will be some time interval required to bring the reacting compositions back up to the 90° coupling temperature. - In the invention, however, the addition of a small volume (mass) of concentrated base will greatly moderate the degree to which the temperature drops, thus making it easier and faster to return the compositions to the required coupling temperatures. In
FIG. 4 , the conventional thermal profile is indicated by thesolid line 34 and the thermal profile provided by the invention is indicated by the dottedline 35. It will be understood, of course, thatFIG. 4 is schematic, not drawn to scale, and illustrative rather than a precise track of any particular mixture. -
FIG. 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. - In general, and as can be confirmed by appropriate resources, the boiling point of piperidine is approximately 106° C. and that of DMF is about 153° C. As a result 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.
FIG. 5 illustrates this schematically by showing thedeprotection step 23 followed by an evaporation step 36 followed by the draining step (of liquids other than the organic base) and then thecoupling step 27. The boiling point of 4-methylpiperidine is 123° C., offering similar advantages. - Expressed alternatively, 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. For pyrrolidine, the vapor pressure is about 8.4 mm Hg at 25° C. and about 102 mm Hg at 60° C. Thus, raising the temperature to 60° C. greatly encourages the desired evaporation.
- Consistent with well understood principles of liquid and vapor pressure, 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. When using a microwave assisted process as described herein (and elsewhere), the microwave radiation can be used to both accelerate the deprotection step and to accelerate the vacuum removal step. - In exemplary methods, 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. Functionally, 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.
- These two improvements in overall SPPS cycles can, be combined, so that in another aspect, 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.
- Combining both improvements in this manner is illustrated by the differences between
FIG. 1 andFIG. 5 and can allow the cycle to avoid both the washing steps and two of the draining steps. As set forth in the Background, any such advantage in an individual cycle will be geometrically multiplied as a longer peptide chain is synthesized. -
FIGS. 6 and 7 are schematic illustrations of selected portions of a system for carrying out the improvements described herein. Most basically, the system includes a microwave source illustrated as thediode 40 positioned to direct microwave radiation into amicrowave cavity 41, and with a vacuum source shown as thepump 42 connected to thereaction vessel 22 in thecavity 41. Although 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 thesource 40 is typically directed through awaveguide 43 which provides support to thecavity 41. Thevacuum pump 42 pulls from thevessel 22 alongline 44 and usually includes atrap 45 which is otherwise conventional (e.g., a cold trap using liquid nitrogen) and positioned between the vessel and thevacuum pump 42. In the absence of thetrap 45, the vacuum pump needs to be capable of handling the evaporated base and solvents while still operating as intended. - As schematically illustrated in
FIG. 6 , in exemplary embodiments, thecavity 41 can support a single mode of microwave radiation at the microwave frequencies produced by themicrowave source 40. A temperature probe 46 (for which a fiber optic device is exemplary) is positioned to read the temperature of thereaction vessel 22 in thecavity 41. In conjunction with 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. - As further schematic details, 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 U.S. Pat. No. 6,288,379, the contents of which are incorporated entirely herein by reference. The basic circuits between the power supply and thediode 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. InFIG. 7 the vessel is again designated at 22, andFIG. 7 further illustrates that thevessel 22 includes a frit 52 (typically made of glass) and a spray head 53. The frit 52 permits liquids to be drained from thereaction vessel 22 and the spray head 53 delivers compositions to thereaction vessel 22. Other equivalent fixtures can be selected by the skilled person without undue experimentation. - In particular,
FIG. 7 illustrates anitrogen supply 54 which is connected to a plurality ofsupply bottles 55 which for schematic purposes are illustrated as Erlenmeyer flasks. A plurality of metered loops are schematically illustrated bylines supply bottles 55; andcorresponding lines common line 63 that reaches the spray head 53 for delivery to thevessel 22. Aseparate line 63 provides nitrogen from thesource 54 to the liquids and resin in thevessel 22 to agitate (bubble) the contents of thevessel 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.
- In a manner consistent with the diagram of
FIG. 6 , the nitrogen supply and the metered loop can connect to theprocessor 47 so that theprocessor 47 can control the manner in which the compositions are dispensed from thevessels 55 to thereaction vessel 52. Although not illustrated, the skilled person will recognize that the simple schematic line connections (64 and 65) are in practice combination of tubes (pipes), valves, and controls for those lines; e.g., inpractice line 64 represents a connection between a valve or manifold inline 58, a controller for that line, and theprocessor 47. The same relationships hold true for theline 65 between thenitrogen supply 54 and theprocessor 47. - Experimental (Predictive)
- Materials and Methods
- Reagents
- All Fmoc amino acids were obtained from Novabiochem (San Diego, CA) and contained the following side chain protecting groups: Asn(Trt), Asp(OtBu), Arg(Pbf), Cys(Trt), Gln(Trt), Glu(OtBu), His(Trt), Lys(Boc), Ser(tBu), Thr(tBu), Trp(Boc), and Tyr(tBu). N-[(1H-Benzotriazol-1-yl)(dimethylamino)methylenel-N-methylmethanaminium hexafluorophosphate Noxide (HBTU), N-hydroxybenzotriazole (HOBO, and benzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), were also obtained from Novabiochem. Diisopropylethylamine (DIEM, N-methylmorpholine (NMM), collidine (TMP), piperidine, piperazine, trifluoroacetic acid (TFA), thioanisole, 1,2-ethanedithiol (EDT), and phenol were obtained from Sigma Aldrich (St. Louis, Mo.). Dichloromethane (DCM), N,N-Dimethylformamide (DMF), Nmethylpyrrolidone (NMP), anhydrous ethyl ether, acetic acid, HPLC grade water, and HPLC grade acetonitrile were obtained from VWR (West Chester, Pa.).
- SPHERITIDE™resin: Trityl linker was prepared using SPHERITIDE™ resin (CEM Corporation; Matthews, N.C.; USA). The SPHERITIDE™ resin consists of poly-e-lysine cross-linked with multifunctional carboxylic acids.
- CEM LIBERTYTM Automated Microwave Peptide Synthesizer
- The LIBERTY™ system (CEM Corporation, Matthews, N.C.) is a sequential peptide synthesizer capable of complete automated synthesis including cleavage of up to 12 different peptides. The LIBERTY™ system uses the single-mode microwave reactor, DISCOVER™, which has been widely used in the organic synthesis industry. The LIBERTY™ 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 LIBERTY™ synthesizer is controlled by an external computer, which allows for complete control of each step in every cycle.
- Peptide Synthesis: VYWTSPFMKLIHEQCNRADG-NH2
- A model peptide containing all 20 amino acids was synthesized under a variety of conditions using the CEM LIBERTY™ automated microwave peptide synthesizer on 0.152 g Spheritide™ 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.
- No draining step was carried out between the coupling step of a previous cycle and the addition of the piperidine for the successive cycle.
- After deprotection, 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.
- Coupling reactions were performed in the presence of a 5-fold molar excess of 0.2 M Fmoc-protected amino acids dissolved in DMF with various types of activation: (i) HBTU:DIEA:AA (0.9:2:1); 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 (0.9:2:1), double coupling on valine. Coupling reactions were for 5 min at 40 W (30 min at 0 W for conventional synthesis) with a maximum temperature of 80° C. In later experiments, coupling conditions of cysteine and histidine were altered to 2 min at 0 W followed by 4 min at 40 W with a maximum temperature of 50° C. Cleavage was performed using 10 ml of Reagent K (TFA/phenol/water/thioanisole/EDT; 82.5/5/5/5/2.5) for 180 min. Following cleavage, peptides were precipitated out and washed using ice-cold anhydrous ethyl ether.
- Peptide Analysis
- Prior to LC-MS analysis, all peptides were dissolved in 10% acetic acid solution and lyophilized to dryness. Analytical HPLC of peptide products was performed using a Waters Atlantis dC18 column (3 μm, 2.1×100 mm) at 214 nm. Separation was achieved by gradient elution of 5-60% solvent B (solvent A=0.05% TFA in water; solvent B=0.025% TFA in acetonitrile) over 60 min at a flow rate of 0.5 ml/min. Mass analysis was performed using an LCQ Advantage ion trap mass spectrometer with electrospray ionization (Thermo Electron, San Jose, Calif.). Racemization analysis of amino acids was performed by C.A.T. GmbH & Co. (Tuebingen, Germany) using a published GC-MS method that involves hydrolysis of the peptide in 6 N DCl/D2O (The Peptides: Analysis, Synthesis, Biology, E
RHARD GROSS editor). - In another embodiment, the invention presents a novel process whereby the coupling and deprotection steps occur within the same solvent. In this process 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. Additionally, 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 (0.5 mL) with rapid delivery. Typically, 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.
- To demonstrate the effectiveness of this new process a batch of 24 peptides were assembled using an automated peptide synthesizer modified to perform the in-situ solvent recycling step during each cycle.
- Materials and Methods
- All peptides were synthesized using a Liberty Blue PRIME system (CEM Corporation; Matthews, N.C.; USA) allowing for automated in-situ solvent recycling and evaporation based washing. The peptides were synthesized at 0.05mmol scale with 10 equivalents of amino acid using CarboMAX™ coupling with AA/DIC/Oxyma (1:2:1) based activation for 100 sec at 90° C. ProTide resins (CEM Corporation; Matthews, N.C.; USA) based on 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 0.5 mL of 50% pyrrolidine directly to the coupling solution. A single 1×4 mL wash was used in between the deprotection and coupling steps. Peptides were cleaved with TFA/TIS/H2O/DODt (92.5:2.5:2.5:2.5) for 30 min at 38° C. using a RAZOR cleavage system (CEM Corporation; Matthews, N.C.; USA).
- Results and Discussion:
- All peptides synthesized in Table 1 gave the desired target as the major peak with a standard cycle time of 2 minutes and 58 seconds. The in-situ solvent recycling process allowed for 0.5 mL of a concentrated pyrrolidine (BP 87° C.) solution to be added to the end of the coupling step (without draining) An advantage of this setup was that the deprotection immediately proceeded very close to the desired temperature (95° C.) since the coupling solution was already at 90° C. During the deprotection process a vacuum was applied and the pyrrolidine was evaporated and subsequently condensed in the waste container. This allowed only a single wash step (1×4 mL) to be required at the end of the deprotection step.
-
TABLE 1 Automated Sequential Batch Synthesis of 24 Peptides UPLC Synthesis # Peptide Disease Area Resin Used Purity (%) Time 1 GRP Regulates Gastrin RA ProTide 81 1:22 VPLPAGGGTVLTKMYPRGNHWAVGHLM-NH 2 Release 2 Glucagon Hypoglycemia RA ProTide 75 1:28 H- HSQGTFTSDYSKYLDSRRAQDFVQWLMNT- NH2 3 Bivalirudin Blood thinner C1-2-C1-Trt 71 1:05 H-fPRPGGGGNGDFEEIPEEYL-OH 4 Angiotensin Vasoconstrictor C1-2-C1-Trt 82 0:30 H-NRVYVHPF-OH 5 PTH 1-34 Osteoporosis RA ProTide 70 1:43 H- SVSEIQLMHNLGKHLNSMERVEWLRKKLQD VHNF-NH2 6 Gonadorelin Fertility RA ProTide 91 0:35 pEHWSYGLRPG-NH2 7 Triptorelin Breast Cancer, RA ProTide 73 0:35 pEHWSYwLRPG-NH2 Prostrate Cancer, Fertility 8 Liraglutide Diabetes RA ProTide 80 1:31 H-HAEGTFTSDVSSYLEGQAAK(γ-E- palmitoyl)EFIAWLVRGRG-NH2 9 Exenatide Diabetes RA ProTide 74 1:58 H- HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGP SSGAPPPS-NH2 10 MOG (35-55) Multiple Sclerosis RA ProTide 71 1:05 H-MEVGWYRSPFSRVVHLYRNGK-NH2 11 Secretin Osmoregulation RA ProTide 70 1:19 H-HDGTFTSELSRLRDSARLQRLLQGLV-NH2 12 Teriparatide Osteoporosis RA ProTide 60 1:43 H- SVSEIQLMHNLGKHLNSMERVEWLRKKLQD VHNF-NH2 13 GLP-1 (7-37) Diabetes RA ProTide 74 1:34 H- HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR G-NH2 14 Magainin 1 Antibiotic RA ProTide 79 1:11 H-GIGKFLHSAGKFGKAFVGEIMKS-NH2 15 Tetracosactide Adrenal Cortex RA ProTide 77 1:13 H-SYSMEHFRWGKPVGKKRRPVKVYP-NH2 stimulant 16 [Arg8]-Vasopressin Hormone (blood RA ProTide 94 0:32 H-CYFQNCPRG-NH2 vessel contraction) 17 Ubiquitin Protein signaling RA ProTide ≥60 3:44 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQD agent KEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKE STLHLVLRLRGG-NH2 18 Parasin I Antibiotic RA ProTide 87 0:59 H-KGRGKQGGKVRAKAKTRSS-NH2 19 Dynorphin A Opioid Research RA ProTide 71 0:53 H-YGGFLRRIRPKLKWDNQ-NH2 20 ACP Fatty Acid RA ProTide 92 0:32 H-VQAAIDYING-NH2 Synthesis 21 BAM 3200 Opioid Research RA ProTide 70 1:16 H-YGGFMRRVGRPEWWMDYQKRYGGFL- NH2 22 HIV-TAT (47-57) HIV/AIDS RA ProTide 93 0:31 Fmoc-YGRKKRRQRRR-NH2 Research 23 HIV-TAT (48-60) HIV/AIDS RA ProTide 88 0:39 Fmoc-GRKKRRQRRRPPQ-NH2 Research 24 Pramlintide Diabetes RA ProTide 72 1:52 KCNTATCATQRLANFLVHSSNNFGPILPPTN VGSNTY--NH2 - Total Synthesis Time for Entire Batch: 32.6 Hours
- This new process provided a significant reduction in standard cycle time (2
minutes 57 seconds) from (a)—elimination of the coupling drain time, (b)—elimination of the deprotection delivery time between steps, and (c)—elimination of the temperature ramp time for the deprotection step thereby allowing a shorter deprotection time to be used. Additionally, significant solvent savings were possible with the complete elimination of the deprotection solvent during each cycle. - In the drawings and specification there has been set forth a preferred embodiment of the invention, and although specific terms have been employed, they are used in a generic and descriptive sense only and not for purposes of limitation, the scope of the invention being defined in the claims.
Claims (8)
1. A system for microwave assisted solid phase peptide synthesis comprising:
a microwave source positioned to direct microwave radiation into a microwave cavity;
a microwave transparent reaction vessel in said cavity; and
a vacuum source connected to said reaction vessel.
2. A method according to claim 1 further comprising a trap between said reaction vessel and said vacuum Source.
3. A method according to claim 1 wherein said cavity can support a single mode of microwave radiation at the microwave frequencies produced by said microwave source.
4. A method according to claim 3 where wherein said reaction vessel comprises:
a (glass) frit for draining liquids from said reaction vessel; and
a spray head for delivery of reagents to said reaction vessel.
5. A system according to claim 1 further comprising a fiber optic temperature probe positioned to read the temperature of said reaction vessel in said cavity (for controlling the microwave power delivered to said reaction vessel).
6. A system according to claim 4 that incorporates nitrogen pressure to transfer all reagents and to provide an inert environment during peptide synthesis.
7. A system according to claim 6 further comprising a nitrogen source in communication with said reaction vessel to bubble the contents of said reaction vessel for mixing during deprotection, coupling, and cleavage reactions.
8. A system according to claim 1 further comprising a processor for controlling every step in every SPPS cycle carried out in said system.
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US15/299,931 US10125163B2 (en) | 2015-10-23 | 2016-10-21 | Solid phase peptide synthesis |
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WO2023044004A1 (en) * | 2021-09-17 | 2023-03-23 | Cem Corporation | Solid phase peptide synthesis (spps) processes and associated systems |
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US10125163B2 (en) | 2015-10-23 | 2018-11-13 | Cem Corporation | Solid phase peptide synthesis |
US10239914B2 (en) | 2015-10-23 | 2019-03-26 | Cem Corporation | In-situ solvent recycling process for solid phase peptide synthesis at elevated temperatures |
USRE49961E1 (en) | 2016-10-21 | 2024-05-07 | Cem Corporation | Solid phase peptide syntheses |
WO2019241903A1 (en) * | 2018-06-19 | 2019-12-26 | Shanghai Space Peptides Pharmaceutical Co., Ltd. | Synthetic method of bivalirundin |
CN108912208A (en) * | 2018-06-20 | 2018-11-30 | 南京肽业生物科技有限公司 | A kind of adjustable Solid-phase synthesis peptides device of temperature |
CN110642936B (en) * | 2018-06-26 | 2023-01-17 | 深圳翰宇药业股份有限公司 | Method for preparing teriparatide |
KR20230019120A (en) | 2020-06-03 | 2023-02-07 | 추가이 세이야쿠 가부시키가이샤 | Efficient peptide condensation of difficult sequences |
JP2023541403A (en) | 2020-09-09 | 2023-10-02 | ソーシャル・プロフィット・ネットワーク | Methods and compositions for delivering biotin to mitochondria |
WO2024058924A1 (en) * | 2022-09-16 | 2024-03-21 | Cem Corporation | Peptide synthesis processes |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4065781A (en) * | 1974-06-21 | 1977-12-27 | Westinghouse Electric Corporation | Insulated-gate thin film transistor with low leakage current |
US5614608A (en) * | 1995-01-20 | 1997-03-25 | Selectide Corporation | Apparatus and method for multiple synthesis of organic compounds on polymer support |
US6329139B1 (en) * | 1995-04-25 | 2001-12-11 | Discovery Partners International | Automated sorting system for matrices with memory |
US6828389B2 (en) * | 2000-05-12 | 2004-12-07 | Lonza Ag | Method for producing polymer-bonded 2-chlorotrityl-chloride |
US6890491B1 (en) * | 1997-06-10 | 2005-05-10 | Pharmacopeia Drug Discovery, Inc. | Method and apparatus for universal fluid exchange |
US20140033297A1 (en) * | 2007-05-11 | 2014-01-30 | Nagrastar Llc | Apparatus for controlling processor execution in a secure environment |
US10125163B2 (en) * | 2015-10-23 | 2018-11-13 | Cem Corporation | Solid phase peptide synthesis |
US10239914B2 (en) * | 2015-10-23 | 2019-03-26 | Cem Corporation | In-situ solvent recycling process for solid phase peptide synthesis at elevated temperatures |
Family Cites Families (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6084226A (en) | 1998-04-21 | 2000-07-04 | Cem Corporation | Use of continuously variable power in microwave assisted chemistry |
US20040238794A1 (en) * | 2003-05-30 | 2004-12-02 | Karandikar Prashant G. | Microwave processing of composite bodies made by an infiltration route |
US7902488B2 (en) * | 2003-06-23 | 2011-03-08 | Cem Corporation | Microwave-assisted peptide synthesis |
US7393920B2 (en) * | 2003-06-23 | 2008-07-01 | Cem Corporation | Microwave-assisted peptide synthesis |
US7803351B2 (en) * | 2004-08-20 | 2010-09-28 | Washington University | Blood brain barrier permeation peptides |
US8314208B2 (en) * | 2006-02-10 | 2012-11-20 | Cem Corporation | Microwave enhanced N-fmoc deprotection in peptide synthesis |
CA2808270A1 (en) * | 2010-08-16 | 2012-02-23 | Cem Corporation | Water soluble solid phase peptide synthesis |
US8906681B2 (en) * | 2011-08-02 | 2014-12-09 | The Scripps Research Institute | Reliable stabilization of N-linked polypeptide native states with enhanced aromatic sequons located in polypeptide tight turns |
EP2703073A1 (en) * | 2012-08-31 | 2014-03-05 | Biotage AB | Apparatus and method for solid phase synthesis |
US9169287B2 (en) * | 2013-03-15 | 2015-10-27 | Massachusetts Institute Of Technology | Solid phase peptide synthesis processes and associated systems |
WO2015028599A1 (en) | 2013-08-30 | 2015-03-05 | Biotage Ab | Cleavage of synthetic peptides |
-
2016
- 2016-10-21 US US15/299,931 patent/US10125163B2/en not_active Ceased
- 2016-10-21 JP JP2018521074A patent/JP6763948B2/en active Active
- 2016-10-21 CA CA3002871A patent/CA3002871C/en active Active
- 2016-10-21 WO PCT/US2016/058181 patent/WO2017070512A1/en active Application Filing
-
2018
- 2018-09-27 US US16/143,917 patent/US20190194246A1/en not_active Abandoned
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4065781A (en) * | 1974-06-21 | 1977-12-27 | Westinghouse Electric Corporation | Insulated-gate thin film transistor with low leakage current |
US5614608A (en) * | 1995-01-20 | 1997-03-25 | Selectide Corporation | Apparatus and method for multiple synthesis of organic compounds on polymer support |
US6329139B1 (en) * | 1995-04-25 | 2001-12-11 | Discovery Partners International | Automated sorting system for matrices with memory |
US6890491B1 (en) * | 1997-06-10 | 2005-05-10 | Pharmacopeia Drug Discovery, Inc. | Method and apparatus for universal fluid exchange |
US6828389B2 (en) * | 2000-05-12 | 2004-12-07 | Lonza Ag | Method for producing polymer-bonded 2-chlorotrityl-chloride |
US20140033297A1 (en) * | 2007-05-11 | 2014-01-30 | Nagrastar Llc | Apparatus for controlling processor execution in a secure environment |
US10125163B2 (en) * | 2015-10-23 | 2018-11-13 | Cem Corporation | Solid phase peptide synthesis |
US10239914B2 (en) * | 2015-10-23 | 2019-03-26 | Cem Corporation | In-situ solvent recycling process for solid phase peptide synthesis at elevated temperatures |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023044004A1 (en) * | 2021-09-17 | 2023-03-23 | Cem Corporation | Solid phase peptide synthesis (spps) processes and associated systems |
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