Patent Application
20170226152
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., SAMPSON, 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; 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. MERRIFIELD; 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 CHAN 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., U.S. 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.
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 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.
It will be understood that
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 (
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.
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
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.
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
As schematically illustrated in
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 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.
In particular,
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
Experimental (Predictive)
Materials and Methods
Reagents
All Fmoc amino acids were obtained from Novabiochem (San Diego, Calif.) 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)methylene]-N-methylmethanaminium hexafluorophosphate Noxide (HBTU), N-hydroxybenzotriazole (HOBt), and benzotriazol-1-yl-N-oxy-tris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP), were also obtained from Novabiochem. Diisopropylethylamine (DIEA), 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 LIBERTY™ 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 (0.9:21), 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, ERHARD 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 0.05 mmol 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.
VPLPAGGGTVLTKMYPRGNHWAVGHLM-NH2
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.
| Number | Date | Country | |
|---|---|---|---|
| 62245484 | Oct 2015 | US |
