Patent Publication Number: US-2019194246-A1

Title: Solid phase peptide synthesis

Description:
RELATED APPLICATIONS 
     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. 
    
    
     BACKGROUND 
     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 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.  The epidermal growth factor variant III peptide vaccine for treatment of malignant gliomas.  Neurosurg. Clin. N. Am. 2010; 21: 87-93; L I  G, W ONG  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&#39;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&#39;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  W HITE   , 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. 
     SUMMARY 
     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. 
    
    
     
       BRIEF DESCRIPTION OF THE 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. 
     
    
    
     DETAILED DESCRIPTION 
       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 . 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). Useful 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). As indicated by the position of the relative arrows, 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. 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  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. In particular, the skilled person already recognizes that  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 . In particular, 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 ). 
     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. As  FIG. 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 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. 
     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 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. 
     Expressed alternatively, piperidine&#39;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  and  FIG. 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 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 . 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 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. 
     As schematically illustrated in  FIG. 6 , in exemplary embodiments, 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 . 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 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. In  FIG. 7  the vessel is again designated at  22 , and  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. 
     In particular,  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. 
     In a manner consistent with the diagram of  FIG. 6 , 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 . 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., in practice line  64  represents a connection between a valve or manifold in line  58 , a controller for that line, and the processor  47 . The same relationships hold true for the line  65  between the nitrogen supply  54  and the processor  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 &amp; 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  G ROSS  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-NH 2   
                   
                   
                   
                   
               
               
                   
               
               
                 6 
                 Gonadorelin 
                 Fertility 
                 RA ProTide 
                 91 
                 0:35 
               
               
                   
                 pEHWSYGLRPG-NH 2   
                   
                   
                   
                   
               
               
                   
               
               
                 7 
                 Triptorelin 
                 Breast Cancer, 
                 RA ProTide 
                 73 
                 0:35 
               
               
                   
                 pEHWSYwLRPG-NH 2   
                 Prostrate Cancer, 
                   
                   
                   
               
               
                   
                   
                 Fertility 
                   
                   
                   
               
               
                   
               
               
                 8 
                 Liraglutide 
                 Diabetes 
                 RA ProTide 
                 80 
                 1:31 
               
               
                   
                 H-HAEGTFTSDVSSYLEGQAAK(γ-E- 
                   
                   
                   
                   
               
               
                   
                 palmitoyl)EFIAWLVRGRG-NH 2   
                   
                   
                   
                   
               
               
                   
               
               
                 9 
                 Exenatide 
                 Diabetes 
                 RA ProTide 
                 74 
                 1:58 
               
               
                   
                 H- 
                   
                   
                   
                   
               
               
                   
                 HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGP 
                   
                   
                   
                   
               
               
                   
                 SSGAPPPS-NH 2   
                   
                   
                   
                   
               
               
                   
               
               
                 10 
                 MOG (35-55) 
                 Multiple Sclerosis 
                 RA ProTide 
                 71 
                 1:05 
               
               
                   
                 H-MEVGWYRSPFSRVVHLYRNGK-NH 2   
                   
                   
                   
                   
               
               
                   
               
               
                 11 
                 Secretin 
                 Osmoregulation 
                 RA ProTide 
                 70 
                 1:19 
               
               
                   
                 H-HDGTFTSELSRLRDSARLQRLLQGLV-NH 2   
                   
                   
                   
                   
               
               
                   
               
               
                 12 
                 Teriparatide 
                 Osteoporosis 
                 RA ProTide 
                 60 
                 1:43 
               
               
                   
                 H- 
                   
                   
                   
                   
               
               
                   
                 SVSEIQLMHNLGKHLNSMERVEWLRKKLQD 
                   
                   
                   
                   
               
               
                   
                 VHNF-NH 2   
                   
                   
                   
                   
               
               
                   
               
               
                 13 
                 GLP-1 (7-37) 
                 Diabetes 
                 RA ProTide 
                 74 
                 1:34 
               
               
                   
                 H- 
                   
                   
                   
                   
               
               
                   
                 HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR 
                   
                   
                   
                   
               
               
                   
                 G-NH 2   
                   
                   
                   
                   
               
               
                   
               
               
                 14 
                 Magainin 1 
                 Antibiotic 
                 RA ProTide 
                 79 
                 1:11 
               
               
                   
                 H-GIGKFLHSAGKFGKAFVGEIMKS-NH 2   
                   
                   
                   
                   
               
               
                   
               
               
                 15 
                 Tetracosactide 
                 Adrenal Cortex 
                 RA ProTide 
                 77 
                 1:13 
               
               
                   
                 H-SYSMEHFRWGKPVGKKRRPVKVYP-NH 2   
                 stimulant 
                   
                   
                   
               
               
                   
               
               
                 16 
                 [Arg8]-Vasopressin 
                 Hormone (blood 
                 RA ProTide 
                 94 
                 0:32 
               
               
                   
                 H-CYFQNCPRG-NH 2   
                 vessel 
                   
                   
                   
               
               
                   
                   
                 contraction) 
                   
                   
                   
               
               
                   
               
               
                 17 
                 Ubiquitin 
                 Protein signaling 
                 RA ProTide 
                 ≥60 
                 3:44 
               
               
                   
                 MQIFVKTLTGKTITLEVEPSDTIENVKAKIQD 
                 agent 
                   
                   
                   
               
               
                   
                 KEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKE 
                   
                   
                   
                   
               
               
                   
                 STLHLVLRLRGG-NH 2   
                   
                   
                   
                   
               
               
                   
               
               
                 18 
                 Parasin I 
                 Antibiotic 
                 RA ProTide 
                 87 
                 0:59 
               
               
                   
                 H-KGRGKQGGKVRAKAKTRSS-NH 2   
                   
                   
                   
                   
               
               
                   
               
               
                 19 
                 Dynorphin A 
                 Opioid Research 
                 RA ProTide 
                 71 
                 0:53 
               
               
                   
                 H-YGGFLRRIRPKLKWDNQ-NH 2   
                   
                   
                   
                   
               
               
                   
               
               
                 20 
                 ACP 
                 Fatty Acid 
                 RA ProTide 
                 92 
                 0:32 
               
               
                   
                 H-VQAAIDYING-NH 2   
                 Synthesis 
                   
                   
                   
               
               
                   
               
               
                 21 
                 BAM 3200 
                 Opioid Research 
                 RA ProTide 
                 70 
                 1:16 
               
               
                   
                 H-YGGFMRRVGRPEWWMDYQKRYGGFL- 
                   
                   
                   
                   
               
               
                   
                 NH 2   
                   
                   
                   
                   
               
               
                   
               
               
                 22 
                 HIV-TAT (47-57) 
                 HIV/AIDS 
                 RA ProTide 
                 93 
                 0:31 
               
               
                   
                 Fmoc-YGRKKRRQRRR-NH 2   
                 Research 
                   
                   
                   
               
               
                   
               
               
                 23 
                 HIV-TAT (48-60) 
                 HIV/AIDS 
                 RA ProTide 
                 88 
                 0:39 
               
               
                   
                 Fmoc-GRKKRRQRRRPPQ-NH 2   
                 Research 
                   
                   
                   
               
               
                   
               
               
                 24 
                 Pramlintide 
                 Diabetes 
                 RA ProTide 
                 72 
                 1:52 
               
               
                   
                 KCNTATCATQRLANFLVHSSNNFGPILPPTN 
                   
                   
                   
                   
               
               
                   
                 VGSNTY--NH 2   
               
               
                   
               
            
           
         
       
     
     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.