Patent Publication Number: US-7902488-B2

Title: Microwave-assisted peptide synthesis

Description:
This application incorporates by reference the sequence listing submitted concurrently herewith on paper. This paper copy of the sequence listing is entitled “Sequence Listing.” 
     RELATED APPLICATIONS 
     This application is a continuation in part of Ser. No. 10/604,022 filed Jun. 23, 2003 now U.S. Pat. No. 7,393,920. This application is also related to applications Ser. Nos. 11/235,027; 11/235,328; and 11/235,329; all filed on Sep. 26, 2005 and all of which are divisional applications of Ser. No. 10/604,022. 
    
    
     BACKGROUND OF THE INVENTION 
     The present invention relates to solid-phase peptide synthesis (SPPS), and in particular relates to microwave-assisted techniques for SPPS. 
     The early part of the twentieth century saw the birth of a novel concept in scientific research in that synthetically produced peptides could greatly facilitate the study of the relationship between chemical structure and biological activity. Until that time, the study of structure-activity relationships between peptides and their biological function had been carried out using purified, naturally occurring peptides. Such early, solution-based techniques for peptide purification were plagued with problems, however, such as low product yield, contamination with impurities, their labor-intensive nature and the unpredictable solubility characteristics of some peptides. During the first half of the twentieth century some solution-based synthesis techniques were able to produce certain “difficult” peptides, but only by pushing known techniques to their limits. The increasing demand for higher peptide yield and purity resulted in a breakthrough technique first presented in 1963 for synthesizing peptides directly from amino acids, now referred to as solid-phase peptide synthesis (SPPS). 
     The drawbacks inherent in solution-based peptide synthesis have resulted in the near-exclusive use of SPPS for peptide synthesis. Solid phase coupling offers a greater ease of reagent separation, eliminates the loss of product due to conventional chemistries (evaporation, recrystalization, etc.), and allows for the forced completion of the reactions by adding excess reagents. 
     Peptides are defined as small proteins of two or more amino acids linked by the carboxyl group of one to the amino group of another. Accordingly, at its basic level, peptide synthesis of whatever type comprises the repeated steps of adding amino acid molecules to one another or to an existing peptide chain of acids. 
     The synthetic production of peptides is an immeasurably valuable tool in the field of scientific research for many reasons. For example, some antiviral vaccines that exist for influenza and the human immunodeficiency virus (HIV) are peptide-based. Likewise, some work has been done with antibacterial peptide-based vaccines (diphtheria and cholera toxins). Synthetically altered peptides can be labeled with tracers, such as radioactive isotopes, and used to elucidate the quantity, location, and mechanism of action of the native peptide&#39;s biological acceptor (known as a receptor). This information can then be used to design better drugs that act through that receptor. Peptides can also be used for antigenic purposes, such as peptide-based antibodies to identify the protein of a newly discovered gene. Finally, some peptides may be causative agents of disease. For example, an error in the biological processing of the beta-amyloid protein leads to the “tangling” of neuron fibers in the brain, forming neuritic plaques. The presence of these plaques is a pathologic hallmark of Alzheimer&#39;s Disease. Synthetic production of the precursor, or parent molecule, of beta-amyloid facilitates the study of Alzheimer&#39;s Disease. 
     These are, of course, only a few of the wide variety of topics and investigative bases that make peptide synthesis a fundamental scientific tool. 
     The basic principle for SPPS is the stepwise addition of amino acids to a growing polypeptide chain that is anchored via a linker molecule to a solid phase particle which allows for cleavage and purification once the coupling phase is complete. Briefly, a solid phase resin support and a starting amino acid are attached to one another via a linker molecule. Such resin-linker-acid matrices are commercially available (e.g., Calbiochem, a brand of EMD Biosciences, an affiliate of Merck KGaA of Darmstadt, Germany; or ORPEGEN Pharma of Heidelberg, Germany, for example). The starting amino acid is protected by a chemical group at its amino terminus, and may also have a chemical side-chain protecting group. The protecting groups prevent undesired or deleterious reactions from taking place at the alpha-amino group during the formation of a new peptide bond between the unprotected carboxyl group of the free amino acid and the deprotected alpha-amino of the growing peptide chain. A series of chemical steps subsequently deprotect the amino acid and prepare the next amino acid in the chain for coupling to the last. Stated differently, “protecting” an acid prevents undesired side or competing reactions, and “deprotecting” an acid makes its functional group(s) available for the desired reaction. 
     When the desired sequence of amino acids is achieved, the peptide is cleaved from the solid phase support at the linker molecule. This technique consists of many repetitive steps making automation attractive whenever possible. 
     Many choices exist for the various steps of SPPS, beginning with the type of reaction. SPPS may be carried out using a continuous flow method or a batch flow method. Continuous flow is useful because it permits real-time monitoring of reaction progress via a spectrophotometer. However, continuous flow has two distinct disadvantages in that the reagents in contact with the peptide on the resin are diluted, and scale is more limited due to physical size constraints of the solid phase resin. Batch flow occurs in a filter reaction vessel and is useful because reactants are accessible and can be added manually or automatically. 
     Other choices exist for chemically protecting the alpha-amino terminus. A first is known as “Boc” (N{acute over (α)}-t-butoxycarbonyl). Although reagents for the Boc method are relatively inexpensive, they are highly corrosive and require expensive equipment. The preferred alternative is the “Fmoc” (9-fluorenylmethyloxycarbonyl) protection scheme, which uses less corrosive, although more expensive, reagents. 
     For SPPS, solid support phases are usually polystyrene suspensions; more recently polymer supports such as polyamide have also been used. Preparation of the solid phase support includes “solvating” it in an appropriate solvent (dimethyl formamide, or DMF, for example). The solid phase support tends to swell considerably in volume during salvation, which increases the surface area available to carry out peptide synthesis. As mentioned previously, a linker molecule connects the amino acid chain to the solid phase resin. Linker molecules are designed such that eventual cleavage provides either a free acid or amide at the carboxyl terminus. Linkers are not resin-specific, and include peptide acids such as 4-hydroxymethylphenoxyacetyl-4′-methylbenzyhydrylamine (HMP), or peptide amides such as benzhydrylamine derivatives. 
     Following the preparation of the solid phase support with an appropriate solvent, the next step is to deprotect the amino acid to be attached to the peptide chain. Deprotection is carried out with a mild base treatment (picrodine or piperidine, for example) for temporary protective groups, while permanent side-chain protecting groups are removed by moderate acidolysis (trifluoroacetic acid, or TFA, as an example). 
     Following deprotection, the amino acid chain extension, or coupling, is characterized by the formation of peptide bonds. This process requires activation of the C-alpha-carboxyl group, which may be accomplished using one of five different techniques. These are, in no particular order, in situ reagents, preformed symmetrical anhydrides, active esters, acid halides, and urethane-protected N-carboxyanhydrides. The in situ method allows concurrent activation and coupling; the most popular type of coupling reagent is a carbodiimide derivative, such as N,N′-dicyclohexylcarbodiimide or N,N-diisopropylcarbodiimide. 
     After the desired sequence has been synthesized, the peptide is cleaved from the resin. This process depends on the sensitivity of the amino acid composition of the peptide and the side-chain protector groups. Generally, however, cleavage is carried out in an environment containing a plurality of scavenging agents to quench the reactive carbonium ions that originate from the protective groups and linkers. One common cleaving agent is TFA. 
     In short summary SPPS requires the repetitive steps of deprotecting, activating, and coupling to add each acid, followed by the final step of cleavage to separate the completed peptide from the original solid support. 
     Two distinct disadvantages exist with respect to current SPPS technology. The first is the length of time necessary to synthesize a given peptide. Deprotection steps can take 30 minutes or more. Coupling each amino acid to the chain as described above requires about 45 minutes, the activation steps for each acid requires 15-20 minutes, and cleavage steps require two to four hours. Thus, synthesis of a mere twelve amino acid peptide may take up to 14 hours. To address this, alternative methods of peptide synthesis and coupling have been attempted using microwave technology. Microwave heating can be advantageous in a large variety of chemical reactions, including organic synthesis because microwaves tend to interact immediately and directly with compositions or solvents. Early workers reported simple coupling steps (but not full peptide synthesis) in a kitchen-type microwave oven. Such results are not easily reproducible, however, because of the limitations of a domestic microwave oven as a radiation source, a lack of power control, and reproducibility problems from oven to oven. Others have reported enhanced coupling rates using microwaves, but have concurrently generated high temperatures that tend to cause the solid phase support and the reaction mixtures to degenerate. Sample transfer between steps has also presented a disadvantage. 
     Another problem with the current technology is aggregation of the peptide sequence. Aggregation refers to the tendency of a growing peptide to fold back onto itself and form a loop, attaching via hydrogen bonding. This creates obvious problems with further chain extension. Theoretically, higher temperatures can reduce hydrogen bonding and thus reduce the fold-back problem, but such high temperatures can create their own disadvantages because they can negatively affect heat-sensitive peptide coupling reagents. For this reason, SPPS reactions are generally carried out at room temperature, leading to their characteristic extended reaction times. 
     SUMMARY OF THE INVENTION 
     In one aspect the invention is an instrument for the accelerated synthesis of peptides by the solid phase method. In this aspect the invention comprises a microwave cavity; a microwave source in communication with the cavity; a column in the cavity formed of a material that is transparent to microwave radiation; a solid phase peptide support resin in the column; respective filters for maintaining the solid phase support resin in the column; a first passageway for adding starting compositions to the column; a second passageway for removing compositions from the column; and a third passageway for circulating compositions from the column into the third passageway and back to the column. 
     In another aspect the invention is a method of solid phase support peptide synthesis. In this aspect, the invention comprises deprotecting a first amino acid linked to a solid phase resin in a column by removing protective chemical groups from the first amino acid while applying microwave energy during the deprotecting step; activating chemical groups on a second amino acid to prepare the second amino acid for coupling with the first amino acid linked to the solid phase resin in the column; coupling the activated second amino acid to the deprotected first amino acid to form a peptide on the solid phase resin from the first and second amino acids while applying microwave energy during the coupling step; and successively deprotecting, activating and coupling a third and succeeding amino acids into a peptide on the solid phase resin in the column. 
     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 illustrating certain aspects of solid phase peptide synthesis. 
         FIG. 2  is a perspective view of a synthesis instrument according to the present invention. 
         FIGS. 3 ,  4  and  5  are perspective views of a reaction vessel and adapter according to the present invention. 
         FIG. 6  is a flow circuit diagram illustrating aspects of the present invention. 
         FIG. 7  is a cut-away perspective view of the cavity and waveguide of the present invention. 
         FIG. 8  is the mass spectrum of one peptide synthesized according to the method of the invention. 
         FIG. 9  is the mass spectrum of a second peptide synthesized according to the method of the invention. 
         FIG. 10  is a schematic diagram of another embodiment of the invention. 
     
    
    
     DETAILED DESCRIPTION 
     The invention is an apparatus and method for the solid phase synthesis of one or more peptides, specifically utilizing microwave energy to accelerate the method. 
       FIG. 1  is a schematic diagram illustrating some aspects of the solid phase peptide synthesis process. It will be understood that  FIG. 1  is general in nature and is not limiting of the invention.  FIG. 1  illustrates a first amino acid  10  that includes an N-alpha protective group  11  and a side chain protective group  12  attached to it. A linking molecule  13  is attached to a resin support  14 . In a first step designated by the arrow  15 , the first acid and its protective groups  11  and  12  are attached to the linker  13  and the resin support  14 . In a second step indicated by the arrow  17 , the N-alpha protective group is removed (“deprotected”) to produce the structure in which the first acid  10  and its side chain-protecting group  12  are linked to the support  14  through the linker molecule  13 . In the next step, indicated by the arrow  21 , the first amino acid  10  is coupled to a second amino acid designated at  20 , which similarly has an N-alpha protective group  11  and an activation group  22  attached to it to encourage the coupling. Following the coupling step  21 , the resulting structure includes the first acid  10  and the second acid  20  connected to one another and still including the N-alpha protective group  11  attached to the second acid  20  and the side chain protective group  12  attached to the first acid  10  with the connected acids being in turn linked to the support  14  through the linking molecule  13 . Additional acids, represented by the broken rectangle  25  are added in the same manner (arrow  21 ′) to lengthen the peptide chain as desired. 
     In the final step, the connected acids  10 ,  20  and  25  are cleaved, represented by the arrow  23 , from the protective groups and the support to result in the desired peptide separate from the resin support  14 . The coupling steps can, as indicated a number of times elsewhere herein, be repeated as many times as desired to produce a resulting peptide. 
       FIG. 2  illustrates one commercial embodiment of the present invention broadly designated at  30 .  FIG. 2  illustrates some of the broad structural aspects of the invention, the details of which will be explained with respect to  FIGS. 3 and 6 . 
     First,  FIG. 2  illustrates the microwave portion of the device  31 . The portion of the instrument that applies microwave irradiation to the vessel is preferably a single-mode cavity instrument that can be controlled to apply suitable amounts of power to the sample sizes and materials used in the method of the invention. In the preferred embodiment of the invention, the microwave portion of the instrument has the design and operation that is set forth in a number of commonly assigned U.S. Patents and applications. These include U.S. Pat. Nos. 6,713,719; 6,630,652; 6,744,024; and 6,867,400; and U.S. published application No. 20030199099. The disclosures of all of these references are incorporated entirely herein by reference. Commercial versions of such single-mode microwave instruments are available from the assignee of the present invention, CEM Corporation, of Matthews, N.C., under the DISCOVERY™, VOYAGER™, and EXPLORER™ trade names. 
     With those considerations in mind,  FIG. 2  illustrates the location of the cavity  32 , the housing  33 , and an appropriate display  34 , for providing instructions or information during operation. A plurality of amino acid source containers or bottles are each respectively indicated at  35 . The respective resin containers are illustrated at  36 , and the product peptide containers are designated at  37 . A series of fluid passageways are illustrated by the portions of tubing broadly designated at  40  and will be discussed in more detail with respect to  FIG. 6 . Similarly, the instrument  30  includes an upper housing portion  41 , which includes an appropriate manifold, for physically transporting the fluids and resins in the manner described herein. Although the manifold is not illustrated, it can comprise any series of passageways and valves that serve to direct the fluids in the manner described herein and particularly described with respect to the circuit diagram of  FIG. 6 . 
     Thus, in the embodiment illustrated in  FIG. 2 , up to  20  different amino acids can be incorporated in the respective containers  35 , and up to  12  different peptides can be produced and placed in the respective containers  37  in automated fashion. It will be understood that these are commercial embodiment numbers, however, and that the invention is neither limited to this number nor does it need to have as many sources or product containers as are illustrated. 
       FIG. 2  also illustrates a complimentary series of passageways shown as the tubing broadly designated at  42  that are immediately connected to the reaction vessel adapter  43 , which is partially illustrated in  FIG. 2 , but is described in more detail with respect to  FIGS. 3 ,  4 , and  5 . 
       FIG. 3  is a partial perspective view of the reaction vessel  45  and the vessel adapter  43 , portions of which were also illustrated in  FIG. 2 . The reaction vessel  45  is preferably pear-shaped and formed of a material that is transparent to microwave radiation. Preferred materials include, but are not limited to, glass, Teflon®, and polypropylene. A first passageway, shown as the tubing  46 , is in fluid communication with the reaction vessel (or “cell,” the terms are used interchangeable herein)  45  for transferring solid phase resin between a resin source external to the cell  45  and the cell  45 . A second passageway  47  is in fluid communication between at least one amino acid source ( FIG. 6 ) and the cell  45  for adding amino acids to the cell  45 . A third passageway  50  is in gas communication with an inert gas source ( FIG. 6 ) and with a vent ( FIG. 6 ) for applying and releasing gas pressure to and from the cell  45 , so that the controlled flow of gas in the manifold and to and from the cell  45  can be used to add and remove fluids and flowing solids to and from the cell  45 . 
       FIG. 3  also illustrates that the second passageway  47  also includes a filter, shown as the frit  51 , for preventing solid-phase resin from entering the second passageway  47  from the cell  45 . 
     In preferred embodiments, the invention further comprises a fourth passageway  52 , in fluid communication between an external solvent source ( FIG. 6 ) and the cell  45  for flushing the cell  45  with solvent. As illustrated in  FIG. 3 , the fourth passageway  52  includes a spray head  53  or equivalent structure for adding the solvent to the cell  45 . 
     The adapter  43  is formed of a microwave transparent and chemically inert material, which is preferably formed of a polymer, such as a fluorinated polymer (e.g., PTFE) or an appropriate grade of polypropylene. The adapter  43  is preferably a solid cylinder with the passageways  46 ,  47 ,  50 , and  52  drilled or bored there through. The passageways  46 ,  47 ,  50 ,  52  can simply comprise the bore holes through the adapter  43 , but preferably may also include tubing, which again is formed of a microwave transparent, chemically inert material such as PTFE, PTFE variations, or polypropylene. The tubing is preferably ⅛ inch outside diameter and 1/16 inch inside diameter. 
     Although not illustrated in  FIG. 3  (to reduce the complexity of the drawing), the vessel neck  54  preferably is externally threaded and engages an internally threaded bore hole  55  in lower portions of the adapter  43 . The threaded engagement between the vessel  45  and the adapter  43  permits secure engagement between these two items, and also permits the vessel  45  to be easily engaged and disengaged to and from the adapter  43 . In particular, differently sized vessels or vessels formed of different materials can be substituted and still fit the adapter  43 , provided the necks are of the same size and threading. 
     As some final details,  FIG. 3  also includes threaded fittings  56 ,  57 ,  60 , and  62  to the respective first, second, third and fourth passageways  46 ,  47 ,  50  and  52 . These permit the entire adapter  43  and vessel  45  to be easily connected to and removed from the remainder of the instrument  30 . 
       FIGS. 4 and 5  are respective assembled and exploded perspective drawings of the adapter of  FIG. 3 , and thus illustrate the same elements. Both figures include the adapter  43  and the cell  45 . The threaded fittings  57 ,  60 ,  56 , and  62  are visible in  FIG. 5 , with  57 ,  60  and  56  also visible in  54 . The exploded view of  FIG. 5  also illustrates portions of the first and second passageways,  46 ,  47 , as well as the threaded vessel neck  54  and the board opening  55  in the lower portions of the adapter  43 . 
       FIG. 6  is a flow circuit diagram for the present invention. Wherever possible, the elements illustrated in  FIG. 6  will carry the same reference numerals as in the other drawings. Because most of the elements symbolized in  FIG. 6  are commonly available and well understood, they will not be described in particular detail, as those of skill in this art can practice the invention based on  FIG. 6  without undue experimentation. 
     Accordingly,  FIG. 6  illustrates a vessel system for the accelerated synthesis of peptides by the solid-phase method. The vessel system comprises the reaction cell (or vessel)  45 , which is indicated in  FIG. 6  schematically as a square. Otherwise, the reaction cell  45  has all of the characteristics already described and which will not be repeated with respect to  FIG. 6 . The first passageway  46  is in fluid communication with the cell  45  for transferring solid phase resin between an external resin source  36  and the cell  45 . Three resin sources,  36  (A, B and C) are illustrated in  FIG. 6  and correspond to the resin sources  36  illustrated in  FIG. 2 . As set forth with respect to  FIG. 2 , the number of resin sources is elective rather than mandatory with  12  being shown in the embodiment of  FIG. 2 , and three illustrated in  FIG. 6  for purposes of simplicity and schematic understanding. Each of the resin sources  36  is in communication with a respective three-way valve  64 , A, B and C, and in turn, to an appropriate resin line  65 , A, B and C and then another three-way valve  66  adjacent to cell  45  for delivering resin through the first passageway  46  into the cell  45 . The three-way valve  66  is immediately in communication with another three-way valve  67 , the purpose of which will be described shortly. 
       FIG. 6  also shows the second passageway  47 , which is in communication with at least one of the amino acid sources  35 , which are illustrated again as rectangles in the upper portions of  FIG. 6 . The schematically illustrated amino acid sources or containers  35  correspond to the containers  35  illustrated in  FIG. 2 . 
     The third passageway  50  is in gas communication with an inert gas source  70  and with a vent  71  for applying gas pressure to and releasing gas pressure from the cell  45 , so that the controlled flow of gasses to and from the cell  45  can be used to add and remove fluids and flowing solids to and from the cell. The third passageway  50  accomplishes this in conjunction with at least one valve  72  which, depending upon its orientation, permits the third passageway  50  to communicate with either the gas source  70  or the vent  71 . The gas source can be any gas that can appropriately be pressurized and that does not otherwise interfere with the chemistry of the peptide synthesis or the elements of the instrument itself. Thus, a number of inert gases are suitable, with pressurized nitrogen being typically favored for reasons of wide availability, lower cost, ease of use, and lack of toxicity.  FIG. 6  illustrates that the nitrogen supply  70  is controlled through a two-way valve  72  and an appropriate regulator  73 , which also may include a filter. In the orientation of  FIG. 6 , the gas line from the two-way valve  72  to the vent  71  is labeled at  74 , and the passageway from the valve  72  to the regulator  73  is designated at  75 . 
       FIG. 6  also illustrates the filter  51  in the second passageway  47  for preventing the solid phase resin from entering the second passageway from the cell  45 . 
       FIG. 6  also illustrates the fourth passageway  52  along with the spray head  53 . As described with respect to  FIG. 3 , the fourth passageway  52  is in fluid communication with one or more external solvent sources three of which are illustrated at  76 , A, B and C. Two other external solvent sources  77  and  80  are separately labeled because of their optionally different fluid paths. 
       FIG. 6  also illustrates the manner in which the pressurized gas from the source  70  can be used to both deliver compositions to, and then remove them from, the reaction cell  45  as desired whether they be peptides, solvent, wastes, or resin. Thus, in one aspect of such delivery,  FIG. 6  illustrates a gas passage  81  that communicates with several items. First, the gas passageway  81  communicates with a series of two-way valves designated at  82 A, B, C and D that each provide a gas passage when the respective valve is open to its corresponding amino acid container  35 . Pressurized gas entering a container  35  pushes the acid through the respective delivery lines  83 A, B, C or D, which in turn communicates with a respective acid valves  84 A, B, C and D and then with the second passageway  47  and its respective two-way valve  85  and three-way valve  86 . To illustrate, when valves  82 A and  84 A are open, and the remaining valves  82 B, C and D are closed, gas from the source  70  can be directed through the gas passage  81 , through valve  82 A, into amino acid bottle  35 A, from the bottle  35 A through the valves  84 A,  85  and  86 , and then into the cell  45 . 
     The respective valves are automated in order to provide the cell with the desired composition (e.g. resin, solvent, acid) at the appropriate point in the synthesis, as well as to remove compositions from the cell (peptides, waste) at other appropriate points. The required programming and processor capacity is well within the capability of a personal computer-type processor (e.g. PENTIUM III®), and the use of automated controls and sequences is generally well understood in this and related arts, e.g. Dorf, The Electrical Engineering Handbook, 2d Ed. (CRC Press 1997). 
     It should be understood that while many amino acids exist, the twenty source containers of this apparatus are intended, but not limited to, contain the twenty “common” amino acids for synthesizing proteins that are well known to those skilled in this art. These commercially available common amino acids can be purchased in chemically “protected” form (also from Sigma-Aldrich) to prevent unwanted and/or deleterious reactions from occurring. 
     Solvent can be delivered to the cell in an analogous manner. The solvents communicate with the gas passage  81  through the valves  87 A, B and C and  90  and  91 . This places the gas in direct communication with the external solvent tanks  76 A, B and C and  77  and  80 . External solvent tanks  76 A, B and C are further in communication with respective two-way valves  92 A, B and C and respective three-way valves  93  and  94 . These all lead, when the valves are appropriately oriented, to the second passageway  47  for delivering solvent to the reaction vessel  45  using gas pressure in the same manner that the acids are delivered. A TFA solvent is used in external reservoirs  76 C and thus can be directed through alternative lines for optional isolation. 
       FIG. 6  also indicates that the gas source  70  can be used to drive items from the cell  45  directly by closing all of the valves to the amino acids and the external solvent reservoirs, and then directing the gas through the regulator and filter  92  and its associated passageway  93  directly to valves  67  and  66  and then into the first passageway  46  and the cell  45 . 
     Alternatively, the first passageway  46  can be used to empty the cell  45 . In this aspect, valve  72  is set to direct gas from the source  70  and through the passage  75  to the valve  72  and through the third passageway  50  and into the cell. The gas pressure then directs fluids in the cell  45  through either second passageway  47  or first passageway  46  depending upon the orientation of the valves  86 ,  66  and  67 .  FIG. 6  also illustrates an additional three-way valve  95  that can direct product to the product containers  37 A, B and C, which correspond to the product containers  37  illustrated in  FIG. 2 . An appropriate set of product valves  96 A, B and C can be opened or closed as desired to direct the desired peptide product to the desired product container  37 A, B or C. 
     Alternatively, depending upon the orientation of valves  86 ,  66 ,  67  and  95 , and together with additional two-way valve  100  and three-way valve  101  adjacent to waste containers  102 A and  102 B, materials can be directed from the cell  45  to either of the waste containers  102 A and B. 
     The pressurized gas from the source  70  can also be used to deliver resin. In this aspect, the pressurized gas is directed through the gas passage  81  and through the three-way valves  103  and  104 . With respect to delivery of resin, however, when both of the valves  103  and  104  are open to the resin containers, they direct the pressurized gas to three respective valves  105 A, B and C which in turn are in communication with the resin containers  36  and the exit valves  64 A,  64 B and  64 C which then use the gas pressure delivered to force the resin through the resin line  65  and eventually to the first passage  46  for delivery into the reaction vessel  45 . 
     The resin sources may contain variable amounts and kinds of resins, including, but not limited to, Wang resins, Trityl resins, and Rink acid labile resins; the resins are commercially available from vendors such as Sigma-Aldrich Corp., Saint Louis, Mo. 63101. 
     Solvent can be directed to the resin containers  36 A, B, C, from the external reservoirs  77 ,  80  using the valves  103 ,  104  between the solvent reservoirs and the resin containers. 
       FIG. 6A  is a more detailed illustration of the valving system adjacent the reaction vessel  45 . In particular,  FIG. 6A  shows a series of liquid sensors  106 ,  107  and  110  in conjunction with a series of three-way valves  111 ,  112 ,  113 ,  114  and  115 . The operation of the valves in accordance with the sensors permits a metered amount of liquid to be added to the reaction vessel  45  as may be desired or necessary. For example, with the valves  111 ,  113  and  114  shown in the orientation of  FIG. 6A , fluid can flow directly from valve  86  all the way to those portions of second passageway  47  that extend immediately into the reaction vessel  45 . Alternatively, if valve  111  is open towards valve  112 , liquid will flow through valves  111  and  112  until it reaches the liquid sensors  107  and  110 . The liquid sensors inform the system when a proper or desired amount of liquid is included, which can then be delivered by changing the operation of valve  112  to deliver to the valve  113 , and then to the valve  114 , and then to the second passageway  47  and finally into the cell  45  as desired. 
     Thus, in overall fashion,  FIG. 6  illustrates the delivery of precursor compositions (amino acids, solvents, resin, deprotectants, activators) from their respective sources to the single reaction cell and the further delivery of products and by-products (peptides, waste, cleaved resin) from the cell to their respective destinations. It will be understood that the particular flow paths and valve locations illustrate, rather than limit, the present invention. 
     As noted earlier, the microwave instrument portions of the synthesis instrument can essentially be the same as those set forth in a number of commonly assigned and co-pending U.S. patent applications. Accordingly,  FIG. 7  is included to highlight certain aspects of the microwave portion of the instrument without overly burdening the specification herein. In particular,  FIG. 7  is essentially the same as  FIG. 1  in previously incorporated U.S. Pat. No. 6,744,024.  FIG. 7  illustrates a microwave cavity  117  shown in cutaway fashion for clarity. The cavity is attached to a wave guide  120 , which is in microwave communication with an appropriate source (not shown). Microwave sources are widely available and well understood by those of ordinary skill in this art, and include magnetrons, klystrons, and solid state diodes.  FIG. 7  illustrates a test tube-shaped cell  121  in the cavity  117  and such can be used if desired for the reactions of the present invention, although the pear-shaped vessel  45  is generally preferred. 
     In order to carry out the simultaneous cooling, the instrument includes a cooling gas source (not shown) which delivers the cooling gas to the inlet fitting  122  on the flow valve  123  (typically a solenoid). During active cooling, the solenoid  123 , which is typically software controlled, directs cooling gas through the tubing  124  and to the cooling nozzle  125 , which directs the cooling gas on to the reaction vessel  121 . 
     It should be pointed out, however, that other cooling mechanisms may be adapted to this method, such as a stream of refrigerated air or a liquid cooling mechanism that circulates refrigerated liquid around the reaction cell in a manner that would not interfere with the transfer of microwave energy. 
       FIG. 7  also illustrates a cylindrical opening  126 , which is typically used to permit temperature observation of the reaction vial  121 . Such temperature observation can be carried out with any appropriate device, which can normally include a fiber optic device of the type that can measure the temperature of an object by reading the infrared radiation produced by the object. Such devices are well understood in the art, and will not be discussed in further detail herein, some aspects having already been discussed in the incorporated references. 
     In preferred embodiments, the microwave source is capable of, but not limited to, “spiking” microwave energy. In other words, the microwave source is capable of generating high power for a short length of time as opposed, but not limited to, low power for a longer period of time. This feature aids in preventing the undesirable effect of overheating the contents of the reaction vessel and appears to increase the rate of reaction as well. 
     The apparatus optionally includes an infrared photosensor for measuring temperature. The infrared sensor does not contact the reaction cell contents, yet still accurately measures the average temperature of the reaction cell contents and not merely the air temperature surrounding the contents. Infrared temperature analysis is more accurate, non-intrusive, and allows for a more simplified apparatus design compared to a probe or the like, which measures only a localized area and would require physical contact of the contents. 
     The second passageway is further characterized by a filter which prevents the passage of resin. Additionally, the first and second passageways are in fluid communication with each other with respect to the movement of liquid solvents and flowing solids; herein the term “flowing solids” refers to resin, with or without amino acids or peptides attached, and suspended in an appropriate solvent. 
     In another aspect, the invention is a method for the solid phase synthesis of one or more peptides that incorporates the use of microwave energy. Microwave energy applied to the contents of the reaction cell during the deprotecting, activating, coupling, and cleaving steps greatly decreases the length of time necessary to complete these reactions. The method for applying microwave energy may be moderated by the microwave source in such a way as to provide the fastest reaction time while accumulating the least amount of heat, thus more microwave energy may be applied and heat-associated degradation of the reaction cell contents does not occur. This method includes, but is not limited to, spiking the microwave energy in large amounts for short lengths of time. 
     The method optionally includes the synthesis of a complete peptide of two or more amino acids in a single reaction vessel, and may include the coupling of one or more amino acids to one or more amino acids that are attached to the solid phase resin. 
     The method includes cooling the reaction cell, and thus its contents, during and between applications of microwave energy up to and including the final cleaving step. The cooling mechanism of the method operates during amino acid extension cycles, the term “cycle” used herein to refer to the deprotection, activation, and coupling necessary to link one amino acid to another. The cooling system can also operate during and between applications of microwave energy in a given cycle to keep the bulk temperature of the reaction cell contents down. The cooling system can also operate when the complete peptide is cleaved from the resin. 
     Alternatively, it has also been discovered that controlling the power, rather than strictly controlling the temperature, can also provide a desired control over the progress of a reaction. As noted elsewhere herein, the use of a variable or switching power supply can help serve this purpose, an example of which is given in commonly assigned U.S. Pat. No. 6,288,379; the contents of which are incorporated entirely herein by reference. 
     The method includes agitating the contents of the reaction cell with nitrogen gas in order to promote maximal exposure of the resin and any attached amino acids or peptides to solvents and free amino acids. 
     In a preferred embodiment, the method comprises transferring a first common amino acid linked to a resin of choice, both suspended in an appropriate solvent, to the reaction cell via pressurized nitrogen gas. A deprotection solution is then pumped into the reaction cell. This process is accelerated by the application of microwave energy, and the heat generated by the microwave energy is minimized by a cooling mechanism. Multiple deprotection steps may be executed. The deprotection solution is then withdrawn from the reaction cell, leaving the deprotected, common amino acid linked to the resin. After several (three to five) resin washes of approximately one resin volume each using an appropriate solvent and removing the wash solvent, the next “free” common amino acid or acids (dissolved in solution) is added to the reaction cell along with an activating solution. The activation of the free amino acid is accelerated by the application of microwave energy, and the reaction cell temperature is controlled by a cooling mechanism as described above. The method further comprises coupling the free amino acid or acids to the deprotected, linked amino acid, forming a peptide, using microwave energy to accelerate the method. As above, heat generated by the microwave energy is minimized by a cooling mechanism. The coupling step is further preferred to include nitrogen agitation of the reaction cell contents. Completion of this step represents one cycle of one or more amino acid addition. Following the coupling step, the activation solution is withdrawn and the resin is washed as above. The cycle is repeated until the desired peptide sequence is synthesized. Upon completion of peptide synthesis, a further deprotection step may be carried out to remove protective chemical groups attached to the side chains of the amino acids. This deprotection step is carried out as described above. The resin containing the attached, completed peptide is then washed as above with a secondary solvent to prepare the peptide for cleavage from the resin. Following the removal of the secondary solvent, cleaving solution is added to the reaction cell and cleaving is accelerated by the application of microwave energy, and the heat generated by the microwave energy is minimized by a cooling mechanism. Upon completion of cleaving, the peptide product is transferred to a product tube. Optionally, the peptide may be “capped” at any point during the synthesis process. Capping is useful to terminate incompletely coupled peptides, assist in proper folding of the peptide sequence, and to provide a chemical identification tag specific to a given peptide. However, these modifications decrease the solubility of synthetic peptides and thus must be carefully considered. Capping is carried out for example, but not limited to, using acetic anhydride or fluorous capping in solid phase synthesis, or by attaching any of a large variety of chemical groups such as biotin to either the N-terminal, C-terminal or side chain of a peptide. 
     In another embodiment, the invention comprises de-protecting first amino acid linked to a solid phase resin by removing protective first chemical groups, activating chemical groups on a second amino acid to prepare the second amino acid for coupling with the first amino acid, coupling the activated the second amino acid to the de-protected first amino acid to form a peptide from the first and second amino acids, cleaving the peptide from the solid phase resin, applying microwave energy to accelerate the de-protected, activating and coupling cycle, and applying microwave energy to accelerate the cleaving step. 
     It is, of course, the usual procedure to add a number of amino acids to one another to form a peptide sequence. Accordingly, the method can, and usually, comprises repeating the de-protecting, activating and coupling cycle to add third and successive acids to form a peptide of a desired sequence. 
     In that regard, it will be understood that as used herein, terms such as “first,” “second,” or “third” are used in a relative rather than absolute sense. 
     In a particularly preferred embodiment, the method comprises successively de-protecting, activating and coupling a plurality of amino acids into a peptide in a single vessel without removing the peptide from the solid phase resin between the cycles. This, and additional aspects, of the invention will be understood with regard to the discussion of the figures. 
     In another embodiment, the method comprises proactively cooling the vessel and its contents during the application of microwave energy to thereby prevent undesired degradation of the peptide or acids by limiting heat accumulation that would otherwise result from the application of the microwave energy. 
     As is typical in peptide synthesis, the de-protecting step comprises de-protecting the alpha-amino group of the amino acid, but can also comprise de-protecting side chains on the amino acids of the peptide, both under the microwave and radiation. Similarly, the activating step typically comprises activating the alpha-carboxyl group of the second amino acids. 
     Because the amino acids and peptides are sensitive to excessive heat, and in addition to the proactive cooling step just described, the step of applying the microwave energy can comprise “spiking” the application of microwave energy to relatively short-time intervals to thereby prevent undesired degradation of the peptidal acids by limiting heat accumulation that could be encouraged by the continuous application of the microwave energy. As used herein, the term “spiking” refers to the limitation of the application of microwave energy to the relative short time intervals. Alternatively, the microwave power can be supplied from a switching power supply as set forth in commonly assigned U.S. Pat. 6,288,379, the contents of which are incorporated entirely herein by reference. 
     In other embodiments, the peptide synthesis process can comprise activating and coupling in situ using a carbodiimide type coupling free agent. 
     In another aspect, the invention is a process for accelerating the solid phase synthesis of peptides. In this aspect, the method comprises deprotecting a protected first amino acid linked to a solid phase resin by admixing the protective linked acid with a deprotecting solution in a microwave, transparent vessel while irradiating the admixed acid and solution with microwaves, and while cooling the admixture (or alternatively controlling the applied power, or both) to prevent heat accumulation from the microwave energy from degrading the solid phase support or any of the admixed compositions. In particular, the method comprises deprotecting the alpha-amino group of the amino acid, and most typically with a composition suitable for removing protective chemicals selected from the group consisting of fmoc and boc. As is known to those familiar with this chemistry, the deprotecting step can also comprise deprotecting the side chain of the amino acid. In those circumstances, the deprotecting step comprises using a composition suitable for removing t-butyl-based side chain protecting groups. 
     Following the deprotecting step, the method comprises activating a second amino acid by adding this second amino acid and an activating solution to the same vessel while irradiating the vessel with microwaves and while simultaneously cooling the vessel to prevent heat accumulation from the microwave energy from degrading the solid face support or any of the admixed compositions. 
     The method next comprises coupling the second amino acid to the first acid while irradiating the composition in the same vessel with microwaves, and while cooling the admixture to prevent heat accumulation from the microwave energy from degrading the solid phase support or any of the admixed compositions. 
     Finally, the method comprises the step of cleaving the linked peptide from the solid phase resin by admixing the linked peptide with a cleaving composition in the same vessel while irradiating the composition with microwaves, and while cooling the vessel to prevent heat accumulation from the microwave energy from degrading the solid phase support or the peptide. 
     The activating step can also comprise activating and coupling the second amino acid using an in situ activation method and composition such as phosphorium or uranium activators, HATU, HBTU, PyBOP, PyAOP, and HOBT. 
     Once again, because the synthesis of peptides almost always includes the addition of three or more acids into the chain, the method can comprise cyclically repeating the steps of deprotecting, activating and coupling for three or more amino acids in succession to thereby synthesize a desired peptide. 
     In a particular embodiment of the invention, the successive steps of deprotecting, activating, coupling and pleading are carried out in the single reaction vessel without removing the peptide from the solid phase resin or from the vessel between cycles. 
     The method can further comprise agitating the admixture, preferably with nitrogen gas during one or more of the deprotecting, activating, coupling and pleading steps. Any gas can be used for the agitation, provided it does not otherwise interfere with the synthesis chemistry, the peptides or the amino acids, but nitrogen is typically preferred for this purpose because of its wide availability, low cost and chemical inertness with respect to the particular reactions. 
     Experimental: 
     Peptide: Asn-Gly-Val 
     MW=288 
     Scale=0.10 mmol 
     Resin used=Fmoc-Val-Wang Resin 
     Resin substitution=0.27×10−3 moles/gram resin 
     Microwave Protocol: 
     For all reactions in this peptide the microwave power was initially set at 50 W then regulated to maintain the temperature below 60° C. 
     Deprotection: A 20% Piperidine in DMF solution was used for deprotection. The reaction was performed for 30 seconds in microwave, and then repeated with new deprotection solution for 1:00 minute in microwave. 
     Coupling: Activation was performed with 0.40 mmol HCTU, 0.80 mmol DIPEA, and 0.40 mmol of each Fmoc-amino acid for each coupling in the synthesis. Approximately 10 mL of DMF was used to dissolve the mixture. The reaction was performed for 5:00 min. in the microwave. 
     Washing: The vessel was filled with approximately 10 mL of DMF and rinsed 5 times after each deprotection and coupling step. 
     Cleavage: Cleavage was performed with 95% TFA and 5% H2O for 90:00 min. 
     Peptide was precipitated in 50 mL of cold ethyl ether overnight. Product was collected and dried. Mass Spectrum was obtained of crude product from electrospray ionization mass spectrometry using a ThermoFinnigan Advantage LC/MS. 
     Results: The Electrospray Ionization Mass Spectrum ( FIG. 8 ) showed a single peak at 289.1 corresponding to the MW of Asn-Gly-Val. No other peaks corresponding to incomplete couplings were observed. 
     Peptide: Gly-Asn-Ile-Tyr-Asp-Ile-Ala-Ala-Gln-Val 
     MW=1062 
     Scale=0.25 mmol 
     Resin used: Fmoc-Val-Wang Resin 
     Resin substitution=0.27×10−3 moles/gram resin 
     Microwave Protocol: 
     This peptide was synthesized with a power time control method. 
     Deprotection: A 20% Piperidine in DMF solution was used for deprotection. The deprotection was performed with 25 W of microwave power for 30 seconds, and then repeated with new deprotection solution for 1:00 min. in the microwave. 
     Coupling: Activation was performed with 0.9/1.0 mmol of HBTU/HOBt respectively, 2 mmol of DIPEA, and 1.0 mmol of Fmoc-amino acid for each coupling in the synthesis. Approximately 15 mL of DMF was used to dissolve the mixture. The coupling reaction was done in 5:00 min. in the microwave with power alternating between on for 15 seconds and off for 45 seconds. The first cycle of power was 25 W, and the remaining four were each 20 W. 
     Washing: The vessel was filled with approximately 15 mL of DMF and rinsed 5 times after each deprotection and coupling step. 
     Cleavage: Cleavage was performed with 95% TFA, 2.5% H2O, and 2.5% TIS. 
     Peptide was precipitated in 100 mL of cold ethyl ether overnight. Product was collected and dried. Mass Spectrum was obtained of crude product from electrospray ionization mass spectrometry using a ThermoFinnigan Advantage LC/MS. 
     Results: The Electrospray ionization mass spectrum ( FIG. 9 ) shows a peak at 1063.3 that corresponds to the desired peptide mass. No peaks were detected for incomplete couplings. A second peak was obtained at 1176.5 that corresponds to the desired peptide with an extra Ile amino acid. This corresponds to incomplete removal of the deprotection solution before one of the Ile coupling reactions and allowing two Ile amino acids to be added to the peptide. 
       FIG. 10  is a schematic diagram of a flow through embodiment of the invention. In this embodiment, the invention includes the microwave cavity designated by the dotted rectangle  130 . A column broadly designated at  131  is positioned within the cavity  130 . It will be understood that the cavity can be of the type described previously with appropriate arrangements for the microwave source, the waveguide (if desired or necessary), the establishment of single or multiple modes, and the appropriate feedback and control elements referred to in the other embodiments. For the sake of clarity, some of these have not been illustrated in  FIG. 10 , but it will be understood that this is for purposes of illustration rather than limitation. 
     In this embodiment, a microwave-transparent column  131  typically (but not necessarily) formed of glass is filled with the solid phase resin  132 . The resin is the same as or equivalent to the resins described with respect to the earlier embodiments. Respective filters  133  and  134  define each end of the column  131 . Typical columns are between about 15 and 25 millimeters in diameter, between about 100 and 150 millimeters in length, and hold between about 2.5 and 5.0 grams of the resin. 
     A source reservoir  135  is in fluid communication with the column  131  through the tubing portions  136  and  137  and the valve  140 . In a similar manner, an output reservoir  141  is in communication with the column  131  through the tubing  142 ,  143 , and the valve  144 . The respective valves  140  and  144  can be controlled to add source compositions from the reservoir  135  and collect output compositions in the reservoir  141 . The valves  140  and  144  can also be adjusted, however, to circulate compositions through the loop tubing  145  potentially in connection with a pump  146  (typically a positive displacement pump). 
     It will accordingly be understood that this embodiment will generally include a plurality of source reservoirs or their equivalents. In a manner analogous to the previous embodiments, these can include positions or stations for amino acids or protected amino acid derivatives, activator solutions, washing compositions, capping compositions, and solvent exchange compositions. 
     In the same manner, in actual practice the instrument will include a plurality of output reservoirs, and a plurality of valves and associated tubing. Each of these will generally operate, however, in the manner illustrated in  FIG. 10 . 
     Similarly, it will be understood that the potential exists to use multiple columns in a single cavity or multiple cavities with individual columns, or some combination of both. In each case, however, the basic system and its operation are illustrated in  FIG. 10  and the more complex systems would represent additional arrangements that are either identical or similar to  FIG. 10 . 
     If desired or necessary, each respective reaction can be carried out with the relevant compositions isolated in the column  131  using the valves  140 ,  144 , or the reaction can proceed with the relevant compositions circulating through the column  131  and the loop tubing  145  again depending upon the desired orientation of the valves  140 ,  144 . In some cases, the reaction can proceed using both arrangements; i.e. for some period of time isolated in the column  131  and for some other period of time circulating through the columns  141  and the tubing  145 . At the end of the desired number of cycles (or the end of any particular cycle), the valves  140 ,  144  can be adjusted to deliver the peptide product. 
     The flow through system of this embodiment provides a convenient opportunity for real time spectroscopic analysis of the compositions.  FIG. 10  illustrates this schematically as the source  147  and detector  148  that in actual practice are generally spectroscopes that operate in the ultraviolet or infrared frequencies, or potentially both. The real-time monitoring can identify the extent to which a given reaction (typically deprotection) has progressed and, when used in conjunction with a control circuit (or its equivalent) can extend any given step using the loop tubing  145  until it is sufficiently complete. 
     In the drawings and specification there have been disclosed typical embodiments of the invention. The use of specific terms is employed in a descriptive sense only, and these terms are not meant to limit the scope of the invention being set forth in the following claims.