Patent Abstract:
A device capable of synthesizing a plurality of selected peptides by automatically mixing various amino acids, solvents, and activators and adding these to resins contained in a plurality of individual reaction vessels. A plurality of amino acids are contained in vessels within a carousel which is rotated into position where a syringe is inserted into a selected vessel to transport the amino acid within to a pre-reaction vessel for mixing with other selected amino acids which were previously drawn from the carousel. The mixture of amino acids is then transported to a reaction vessel containing the resin balls for growth of the selected peptide. The device includes a computer, controllable valves, at least one pump, pressurized gas such as nitrogen for transporting fluids, various vessels containing amino acids, solvents, activators, resins, and tubing connecting these elements. The computer is programmable to sample, mix selected components, and apply the mixture to resins for growing peptides.

Full Description:
CROSS REFERENCES TO RELATED APPLICATIONS 
     This application claims priority from U.S. Provisional Application Ser. No. 61/688,931 filed on May 24, 2013 which is incorporated by reference herein. 
    
    
     TECHNICAL FIELD 
     The present invention relates to the field of machines capable of synthesizing selected peptides. 
     BACKGROUND OF THE INVENTION 
     Peptide synthesis is the process by which amino acids are linked by amide bonds to produce peptides. The biological process of making long peptides, that is proteins, is known as protein photosynthesis. 
     Liquid-phase peptide synthesis is a classical approach to peptide synthesis and has been replaced in most labs by solid-phase synthesis. However, liquid-phase peptide synthesis retains usefulness in large-scale production of peptides for industrial purposes. 
     Solid-phase peptide synthesis (SPPS), is now the accepted method for creating peptides and proteins in the lab in a synthetic manner. SPPS allows the synthesis of natural peptides which are difficult to express in bacteria, the incorporation of unnatural amino acids, peptide/protein backbone modification, and the synthesis of D-proteins, which consist of D-amino acids. The process typically utilizes small solid insoluble porous beads which are treated with functional units on which peptide chains can be built. The resulting peptide chain will remains covalently attached to the bead until cleaved from that bead by a reagent such as anhydrous hydrogen fluoride or trifluoroacetic acid. The peptide is thus ‘immobilized’ on the solid-phase media or bead and can be retained during a filtration process, whereas liquid-phase reagents and by-products of synthesis are flushed away. 
     Repeated cycles of coupling-wash-deprotection-wash creates the desired peptide chain. The free N-terminal amine of a solid-phase attached peptide is coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which a further amino acid may be attached. The ability to perform wash cycles after each reaction provides a means to remove excess reagent with all peptide product remaining covalently attached to the insoluble resin bead. The objective is to generate high yield in each step. Thus each amino acid is added in major excess (2˜10×) and coupling amino acids together is optimized by the selection of agents. There are two major forms of SPPS utilized in labs and industry, Fmoc and Boc. Unlike ribosome protein synthesis, solid-phase peptide synthesis proceeds in a C-terminal to N-terminal fashion. The N-termini of amino acid monomers is protected by either of these two groups and added onto a deprotected amino acid chain. 
     SPPS is limited by yields, and typically peptides and proteins in the range of 70 amino acids are pushing the limits of synthetic accessibility. Synthetic difficulty also is sequence dependent and amyloid peptides and proteins are difficult to make. 
     SUMMARY OF THE INVENTION 
     In accordance with the present invention, there is presented herein an automated peptide synthesizing machine comprising a cabinet or housing containing a plurality of reagent containers, a plurality of pre-reaction vessels, a plurality of reaction vessels, at least one waste container, a power supply, a plurality of motor controllers, a computer, a motorized amino acid syringe/needle probe assembly, a motorized rotatable amino acid carousel, a fluid metering assembly, and a plurality of fluid and gas control valves and lines connecting the fluid handling elements included above. The computer is capable of controlling valves, motors, and a pump for the purpose of delivering fluids and gases to particular vessels. The computer receives inputs from fluid sensing photo cells and flag sensing photo cells and is programmed to carry out given processes necessary for the synthesizing of peptides and for the delivering of particular selected fluids and gases to particular selected pre-reaction vessels and selected reaction vessels to resulting in synthesizing of distinct peptides within separate distinct reaction vessels so that a different and distinct peptide is synthesized in each of the reaction vessels. 
     The automated peptide synthesizer is capable of synthesizing differing and distinct peptides in the plurality of reaction vessels simultaneously, each distinct peptide being synthesized in a separate and distinct the reaction vessel. The motorized amino acid needle probe assembly is capable of moving a needle probe down into or up out of an amino acid bottle or a needle probe cleaning agent bottle whereupon fluid is drawn up into the needle probe and on through a connected line to a selected pre-reaction vessel. Further, the needle probe assembly is capable of rotating a needle probe arm to a horizontal position centered over the amino acid bottle or the needle probe cleaning agent bottle. 
     The needle probe is mounted on a first vertically movable carriage moved by a first motor and belt driven threaded rod. The first vertically moveable carriage is moved to a given vertical position by the motor, belt, and threaded rod wherein the rotation of the rod and therefore the vertical position of the first carriage is sensed by a photocell monitoring a slotted disc rotating on the end of the threaded rod. The motorized amino acid needle probe assembly is controlled by the computer. 
     The motorized rotatable amino acid carousel contains a plurality of bottles with various amino acids and wherein the rotary position of the carousel is controlled by the computer. The fluid metering assembly includes a clear metering tube with a fluid level sensing photocell fixed within a second vertically moveable carriage wherein the fluid sensing photocell is capable of sensing a fluid level visible through the clear metering tube. The vertical movement of the second vertically moveable carriage is controlled by a second motor, a second belt and a second threaded rod wherein the rotation of the second rod is sensed by a photocell monitoring a slotted disc on the end of the second threaded rod, and movement of the second motor is controlled by the computer. 
     A plurality of fluid and gas control valves and lines connect the pre-reaction vessels, the reaction vessels, the reagent bottles, the amino acid needle probe assembly, the at least one waste container and the metering vessel, for the purpose of delivering required fluids to vessels for the synthesizing of peptides. The pre-reaction vessels provide a location for the pre-reaction of amino acids and reagents prior to transfer of the amino acids and reagents to the reaction vessel. The reaction vessel provides a location for the reaction of the amino acids and the reagents with resins contained within the reaction vessel to produce desired peptides. The plurality of fluid and gas control valves are controlled by the computer. 
     It is an object of this invention to provide an automated peptide production machine which is programmed to produce a multiplicity of different peptides, each in an individual reaction vessel, simultaneously. 
     It is an object of this invention to provide an automated peptide production machine wherein selected amino acids and activators are transferred into a pre-reaction vessel for a selected period of time (for example approximately five minutes), then the mixture is transferred to a reaction vessel containing resin balls comprising small solid insoluble porous beads onto which peptides are grown. 
     It is an object of this invention to provide an automated peptide production machine including a carousel containing selected amino acids held within vessels and an amino acid transfer arm containing a needle probe which is inserted into a selected amino acid vessel, the amino acid is withdrawn from the vessel and transferred to a pre-reaction vessel to be mixed with other selected amino acids and activators for a selected amount of time which is around five minutes and the needle probe can be rinsed if required between selections. 
     It is an object of this invention to provide an automated peptide production machine which transfers a premixed combination of amino acids and activators to a reaction vessel containing resin beads which may or may not have amino acid chains grown thereon previously. 
     It is an object of this invention to provide an automated peptide production machine which contains a plurality of pre-reaction and reaction vessels wherein separate and possibly different peptides are being synthesized simultaneously according to a program contained within the computer wherein that program may be changed as desired. The number of different pre-reaction and reaction vessels is only limited by the practicality and capability of the hardware to mix, process, and transfer the elements within the machine in an effective amount of time. A preferable range is 4 to 12 pre-reaction and reaction vessels. 
     Other objects, features, and advantages of the invention will be apparent with the following detailed description taken in conjunction with the accompanying drawings showing a preferred embodiment of the invention. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       A better understanding of the present invention will be had upon reference to the following description in conjunction with the accompanying drawings in which like numerals refer to like parts throughout the views wherein: 
         FIG. 1  is a piping schematic of that portion of the automated peptide synthesizer which includes the pre-reaction vessels and the hardware used in the pre-reaction portion of the peptide synthesis and a top view of the amino acid carousel and transfer conduits in fluid connection with a selected number “twelve” pre-reaction vessels; 
         FIG. 2  is a piping schematic of the portion of the automated peptide synthesizer which includes the reaction vessels including the hardware which delivers the amino acids, activators, and reagents from the pre-reaction vessels to resins in reaction vessels for synthesizing peptides; 
         FIG. 3  is a rear view of the automated peptide synthesizer cabinet/housing showing the location of the stepper controller, interface boards, power supply cabinet vent, three way valves, amino acid needle delivery assembly, delivery pump location and waste block; 
         FIG. 4  is a view of the right side of the automated peptide synthesizer cabinet showing the reagent bottles, reagent bottles tube connections, connectors for DMF and PIP and nitrogen connector, access door to valves, waste blocks, and access door to the electronics; 
         FIG. 5  is a rear view of the automated peptide synthesizer showing the solvent measuring assembly; 
         FIG. 6  is a top view of the automated peptide synthesizer showing the amino acid containers, amino acid carousel for holding the bottles, and weight plates, reagent bottle block tube connections, and reagent bottles; 
         FIG. 7  is front view of the amino acid delivery assembly including the z-axis stop, home position sensor and holder block, rotary motor movement, z-movement assembly section, rotary probe holder, probe, guide rods and lead screw for z-axis, belt and pulleys; 
         FIG. 8  is front perspective view of the amino acid delivery assembly including the lead screw encoder and sensor (photo cell), stepper motor for rotary movement, belt for rotary needle movement, rotary home position sensor (photo cell), rotary movement/needle holder plate, and probe;. 
         FIG. 9  is a left side view of the amino acid delivery assembly showing the z-axis motor, lead screw encoder, motor for rotary action, liquid detection sensor, and motor mount, tube connection tho the pump, amino acid container vent, probe rotary holder, and probe; 
         FIG. 10  is a front view of the measuring vessel assembly shows the assembly guide rods, and lead screws, photocell/optical coupler with photo transistor housing, stepper motor and measuring vessel; 
         FIG. 11  is a right side perspective view from underneath the measuring vessel assembly showing the guide rods and lead screw, measuring vessel, and a photocell carriage member driven by a toothed pulley for the purpose of lifting and lowering the photocell carriage; 
         FIG. 12  is a left side perspective view from above the measuring vessel assembly showing the measuring vessel, photocell holder block, lead screw encoder, motor, lead screw nut holder block, bearing for guide rods, home photo cell, and home photo cell tang; 
         FIG. 13  is a right side perspective view of a reaction vessel and holder showing the top grip to release the reaction vessel, top seal spring loaded holder, reaction top seal, bottom reaction vessel seal, filter holder inside of the reaction vessel, glass reaction vessel, pivot rod and cabinet attachment block; 
         FIG. 14  is a front view of a reaction vessel and holder; 
         FIG. 15  is a front perspective view of a reaction vessel and holder showing the bottom seal and tube connection of the reaction vessel; 
         FIG. 16  is a perspective view of a reaction vessel and holder; 
         FIG. 17  is a perspective view of a bottle cap insert composed of TEFLON, the snap ring to hold the cap in place, the o-ring seal of the bottom and tube insert; 
         FIG. 18  is a piping schematic of the four reaction vessel embodiment of the present invention; 
         FIG. 19  is a perspective view of the peptide synthesizer; 
         FIG. 20  is a left side view of the peptide synthesizer of  FIG. 18  showing the waste blocks, electronics access panel, pip bottle, DMF gas and liquid connections, and nitrogen connection; 
         FIG. 21  is a front view of the peptide synthesizer of  FIG. 18  showing the nitrogen and DMF tube connections, robotic needle assembly, reaction vessel assembly, and amino acid carousel; 
         FIG. 22  is a top view of the peptide synthesizer of  FIG. 18  showing the tube connections for the gas and DMF, reagent bottle PIP bottle, amino acids weight bottles, amino acid bottles, reaction vessel assembly, and robotic needle assembly for delivering amino acids and reagents; 
         FIG. 23  is a rear view of the peptide synthesizer of  FIG. 18  showing the measuring vessel assembly, valve connections, bottom valve panel, waste block, power supply location, electronics location, stepper driver boards location, tube connections for the gas and DMF, amino acid pump location, robotic needle assembly and top valve panel; 
         FIG. 24  is a top view of the peptide synthesizer of  FIG. 18  with to top cover removed showing the amino acid pump, robotic needle assembly, reagent bottle PIP bottle, amino acid weight guide, amino acid carousel, top valve panel, valve connections, waste block and photo cell connections; 
         FIG. 25  is a left side view of the peptide synthesizer showing the waste connection, USB connection, power entry module fan location, and reagent bottle tube connection; 
         FIG. 26  is a front view of the peptide synthesizer showing the cabinet vent, valve and robotics assembly compartment, waster connections, and electronics compartment; 
         FIG. 27  is a perspective view of the peptide synthesizer showing the reaction vessels and prereaction vessels, amino acid weight guide and amino acid carousel, amino acid needle assembly, reagent bottles; 
         FIG. 28  is a right side view of the peptide synthesizer showing the reagent bottles and tube connections, waste connection, electronic compartment, solvent and reagent bottle connection, and nitrogen connection; 
         FIG. 29  is a top view of the peptide synthesizer showing the valves compartment, reagent bottles and connections, vent cabinet and weight plate indicator and weight plates; 
         FIG. 30  is a front view of the peptide synthesizer showing the delivery valves, solvent/piperidine delivery assembly, stepper driver location, power supply location, waste connection, amino acid regents delivery needle assembly and waste connections; 
         FIG. 31  is a perspective view of the amino acid delivery system; 
         FIG. 32  is an enlargement of the of the amino acid rotary delivery system of  FIG. 31  showing the z-axis stop, home position photo cell sensor, and liquid detection sensor; 
         FIG. 33  is a bottom perspective view of the amino acid rotary delivery system showing the drive belts; 
         FIG. 34  is an enlarged view showing the drive belt assembly of  FIG. 33 ; and 
         FIG. 35  is a perspective view showing the amino acid delivery needle probe assembly and encoder wheel assembly; 
         FIG. 36  is a top view of a carousel holding amino acid containers within a subtray. 
         FIG. 37  is a front view of the automated peptide synthesizer cabinet showing the reaction vessel assembly for  12  units, pre-reaction vessels, reagent bottles, amino acid needle delivery assembly and amino acid turn table and containers therein of  FIG. 3 ; 
         FIG. 38  is a view of the left side of the automated peptide synthesizer showing the cooling fan, power module switch, communication cable connection, and reagents bottle connections of  FIG. 4 ; 
         FIG. 39  is a perspective view showing an enlarged view of the weight plate having cylindrical bores or sleeves for holding removable bottles therein of  FIG. 6 ; 
         FIG. 40  is an enlarged view of the depth encoder and sensor (photo cell) of the amino acid delivery assembly of  FIG. 7 ; 
         FIG. 41  is a perspective top and front view of the amino acid delivery assembly of  FIG. 7 ; 
         FIG. 42  is a perspective bottom and front view of the amino acid delivery assembly of  FIG. 7 ; 
         FIG. 43  is a perspective view of the photo cell holder and lead screw movement encoder consisting a wheel and photo cell of  FIG. 10 ; 
         FIG. 44  is an enlarged view of the lower portion of the measuring vessel assembly of  FIG. 11  showing the motor, motor mount, belt to drive the lead screw and pulleys; 
         FIG. 45  is a top view of a carousel subtray; 
         FIG. 46  is a side view of the reaction vessel of  FIG. 15 ; 
         FIG. 47  is a perspective view of a bottle cap insert from just above; and 
         FIG. 48  is an enlarged view of the encoder wheel assembly of  FIG. 35 . 
     
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     The automated peptide synthesizer  10 , shown in the figures includes a cabinet  7 , reaction vessels  101 - 112 , pre-reaction vessels  201 - 212 , reagent bottles  90 - 93 , a carousel  80 , a carousel motor  88  (shown in  figure 36 ), an amino acid delivery needle probe assembly  85 , a metering assembly  120 , a fluid pump  5 , solenoid valves  1 -  4 ,  8 ,  9  and  11 -  75 , and a control system including a power supply  434 , and a computer  430  and stepper motor drivers  432  which control the motors in the carousel  80 , the amino acid delivery needle probe assembly  85 , the metering assembly  120 , and the fluid pump  5 . 
     In this specification, it is understood that the valves are all electrically controlled solenoid valves. Where shown in the schematics, the valves are drawn in the de-energized state. The valves have three ports: A, B and C. As drawn, fluid flows into port A and out through port Band port C is closed. If the valve becomes energized, fluid flows into port A and out through port C and port B is closed. 
     It is also understood that, as shown in  FIGS. 13, 14, 15, 46, 16, 17 and 47 , the reaction vessels  101 - 112  are removably held within a bracket assembly  136  and are manually removed and replaced as follows: while holding the reaction vessel  106 , for example, with one hand, use the other hand to urge top seal holder  130  toward the top grip  132  to release and free the top of reaction vessel  106 , thus allowing vessel  106  to be removed. At this point, a user either replaces vessel  106  with another selected vessel or prepares vessel  106  to be returned to the original vessel holder  136  by emptying, cleaning and replacing new resins into vessel  106  for a new peptide synthesizing procedure. Replacing vessel  106  into vessel holder  136  is the reverse of the removal process. Reaction vessels of varying volumes are provided, all of which are capable of being held in vessel holder  136 . The reaction vessels  106  are cylindrical and the volumes depend on the particular diameter of a given reaction vessel. Pre-reaction vessels  201 - 212  are not intended to be removable but are used and cleaned automatically by the automated peptide synthesizer  10  by way of the connected fluid lines and valves. 
     A two part schematic of the automated peptide synthesizer  10  is shown in figures +a  36  and  2 .  Figure 36  shows the pre-reaction portion of the synthesizer. Different amino acids are held in amino acid containers  82  within carousel  80 , shown in  figure 36 . Carousel  80  holds up to  24  amino acid containers, each containing a different amino acid. Shown in  FIGS. 6 and 39 , carousel  80  is a circular turn table tray holding four sub-trays  95 . Each sub-tray  95  includes a knob type handle for lifting the sub-tray  95  from or into the turn table tray. As shown in  figures 6 and 39 , each sub-tray  95  is capable of holding up to six amino acid containers  82  within the acid container receptacles  96 . The sub-trays  95  provide a quick and easy method for a user to supply and replenish amino acids to synthesizer  10 . As shown in  FIGS. 1 and 36 , carousel motor  88  causes carousel  80  to rotate on pin  81  whereby the tip of needle probe  84  is brought into horizontal alignment with the center of the top opening of a selected acid container  82 , thus selecting a particular amino acid to be drawn to a particular pre-reaction vessel, as shown in  figure 37 . Sub-trays  95  are located and supported in the turn table tray by a lip  98  at the top marginal edge of sub-tray  95 . 
     The amino acid delivery needle probe assembly  85 , shown in  FIGS. 7, 40, 41, 42, 8 , and  9 , includes a frame  184 , a threaded lead screw  194 , a threaded carriage block  191 , guide rods  187 , a lead screw toothed belt  186 , a toothed drive pulley  183 , a toothed driven pulley  189 , a z-axis motor  86 , and a sub-frame  193  which holds a rotary motor  182 , a rotary arm  188 , and an amino acid needle  84 . Amino acid needle  84  includes two nipples at the top, a suction line nipple  180  and a vent line nipple  177  as shown in  FIG. 9 . Z-axis motor  86  turns to drive lead screw  194  which in turn moves carriage block  191  up or down. Carriage block  191  carries sub-frame  193  along with rotary motor  182 , rotary arm  188  and amino acid needle  84 , all as one unit, up and down. Therefore, when needle probe  84  needs to plunge downward into an amino acid container  82 , stepper rotary motor  86  runs, turning lead screw  194 , which causes sub- frame  193  to move needle probe  84  downward. There is a home flag or tang  199  which is sensed by home photocell  197  when the needle assembly is at the top of the range of vertical movement. There is also an encoder wheel  190  with slits  192  which are counted by photocell  185  to provide precise vertical positioning of needle  84 . With respect to the schematic in  figure 1 , nipple  180  of needle probe  84  is connected by tubing  83  to valves  1 ,  2  and  3  and to pump  5 . Pump  5  is, in turn, connected to a top inlet of a selected one of pre-reaction vessels  201 - 212  by energizing a selected one of valves  14 - 25 . 
     With respect to  FIG. 39 , carousel  80  contains four sub-trays  95 , each with six amino acid containers  82   a  and  82   b  and a knob-type handle  87 . Looking at the overall carousel  80 , there are  16  outer amino acid container  82   a  forming an outer circle and there are eight inner amino acid containers  82   b , forming an inner circle. As shown in  FIG. 37 , rotary arm  188  is positioned, with amino acid needle  84  over amino acid container  82   a . In this position, carousel  80  can be rotated to locate any one of the  16  outer amino acid containers directly under needle  84 , at which time, Z-axis motor  86  can be driven to cause needle  84  to plunge down into the selected amino acid container  82   a . In order to access any one of the eight amino acid containers in the inner circle of the carousel  80 , rotary motor  182  is driven to rotate needle  84  out to a position where the carousel  80  can be rotated to a position where a selected one of the inner amino acid containers  82   b  is directly under needle  84 . 
     When the amino acid has been drawn from any one of containers  82   a  or  82   b , needle  84  needs to be removed from the container and cleaned. Z-axis motor  86  is driven in reverse to raise needle  84  from the container. A cleaning station  195  is located toward the rear side of synthesizer  10  just behind carousel  80 . Therefore, rotary motor  182  is driven to rotate rotary arm  188  toward the rear of the synthesizer  10  to a position directly over cleaning station  195 . At this time, z-axis motor  86  is driven to plunge needle  84  into a solvent within cleaning station  195 . Solvent is drawn in and out of needle  84 . Needle  84  is now raised out of cleaning station  195  and is ready to be used again. It can be seen that there are three stationary positions for rotary arm  188 : the first position being with needle  84  located over the cleaning station  195 , the second position being with the needle  84  over the outer circle of amino acid containers  82   a  and the third position being with needle  84  over the inner circle of amino acid containers  82   b .  FIG. 8  shows rotary arm  188  connected to a home position wheel  196  containing one slit. Home position wheel  196  therefore rotates with rotary arm  188 . Home position photocell  198  senses the slit in home position wheel  196  when rotary arm  188  causes needle  84  to be positioned over cleaning station  195 . 
     To deliver, for example, a selected amount of the amino acid in acid container  82   a  into the pre-reaction vessel  206 , motor  88  rotates carousel  80  so that the selected amino acid container  82   a is directly under needle probe  84 . Motor  86  lowers needle probe  84  down into amino acid container  82   a . With respect to  FIG. 36 , valve  19  is energized to open the top right inlet port of pre-reaction vessel  206  to the fluid line  83 . Valve  3  must also be energized to allow fluid to the pump  5 . Pump  5  is now started. Amino acid is drawn from amino acid container  82   a , through fluid line  83 , valves  1 ,  2 ,  3 ,  4 ,  11 , and  14 - 18 , whereupon energized valve  19  diverts the amino acid into the top right inlet of pre-reaction vessel  206 . Pump  5  runs until the desired amount of amino acid is delivered. The needle is then withdrawn from amino acid container  82   a , and is rotated and plunged into a solvent within cleaning station  195  to be cleaned. In this same manner, any of the amino acids contained within the  24  amino acid containers  82  held within carousel  80  may be added to any of the pre-reaction vessels  201 - 212  by energizing the proper one of the diverter valves  14 - 25 . 
     Further, to deliver a selected amount of Activator  1  or  2 , contained in vessels  91  and  90  respectively, to the pre-reaction vessel  206 , either valve  1  or valve  2  must be energized to allow the desired activator fluid to be pumped from either vessel  90  or  91 , after which, pump  5  is started to deliver the activator through valves  3 ,  4 ,  11 ,  14 - 18  and then the fluid is diverted by valve  19  into the top right inlet port of pre-reaction vessel  206 . As stated in the paragraph above, Activators  1  or  2  may be pumped to any of the pre-reaction vessels  201 - 212  by energizing the proper one of the diverter valves  14 - 25 . 
     After the amino acids and activators are added to the selected pre-reaction vessel, vessel  206  in this example, the mixture is allowed a selected amount of time, approximately 5 minutes, to react. 
     A selected amount of resin has previously been placed within reaction vessel  106  by hand. Referring to  FIGS. 13, 14, 15, 46, 16, 17 and 47 , this is accomplished by urging top seal holder  130  toward top grip  132  to release reaction vessel  106 . Reaction vessel  106  is then lifted and removed by hand and a selected amount of resin is added to the reaction vessel  106 . Reaction vessel top seal  134  is rigidly fixed to the bottom of the top seal holder  130 . As top seal holder  130  is urged upward and rotated about pivot pin  131 , top seal  134  is raise out of and above the top opening of reaction vessel  106 , for example. Now, reaction vessel  106  is grasped and raised up and out of the bottom of reaction vessel holder  136 . The bottom reaction vessel seal  135  includes a rubber stopper  137  with a central drain hole and a TEFLON filter  133  above the stopper  137 . When resin is added to reaction vessel  106 , the TEFLON filter  133  prevents resin from escaping through the drain hole in stopper  137 . Further, when amino acids and solvents are added and then drained from reaction vessel  106 , the TEFLON filter  133  prevents the resins and attached peptides from draining out of the reaction vessel  106 . Now, reaction vessel  106 , along with the resins which were added, is returned to reaction vessel holder  136 . 
     With reference to  FIGS. 13, 14, 15, 46, 16, 17 and 47 , the top seal  134  comprises a TEFLON stopper-like seal with two parallel axial apertures to receive incoming fluid lines. TEFLON is trademark of the DuPont Corporation of Wilmington, Delaware. Top seal  134  includes an integral exterior shoulder  334  and parallel slot  353  with a snap ring  352 . Top seal  134  is inserted into an aperture within the top seal holder  130  and snap ring  352  is applied so that top seal  134  is captured between shoulder  334  and snap ring  352  to hold top seal  134  snugly onto top seal holder  130 . Below shoulder  334  is another slot  355  wherein resides an elastomeric O-ring  350  to form a pressure tight seal between the fluid lines and the reaction vessel. 
     After the pre-reaction time of five minutes or so, the fluid mixture is delivered from the pre-reaction vessel  206  to the reaction vessel  106 . To accomplish this, valves  4  and  11  must be energized to put pressurized nitrogen to the top port of valve  31 . Valves  31  and  43  are then energized to allow the pressurized nitrogen to force the mixture out of the bottom outlet of pre-reaction vessel  206  to a fluid line. In  FIG. 2 , the fluid line is connected directly to the top right inlet port of reaction vessel  106 . Therefore, the mixture flows directly into the top right inlet port of reaction vessel  106 . It should be noted that valves  26  through  37  are dual valves with one half of the valve being connected above the adjoining pre-reaction vessel and the other half of the valve being connected below the adjoining pre-reaction valve. Therefore, it can be seen in  FIG. 1  that valves  14 - 25  are energized to add fluid to the respective pre-reaction vessels  201 - 212  and that valves  26 - 37  are energized to remove or empty fluid from the respective pre-reaction vessels  201 - 212 . 
     After the fluid mixture has been added to the resin in reaction vessel  106  as described above, a reaction takes place wherein peptides are grown onto the resin particles. This reaction typically takes around 45 minutes to one hour or more. After this reaction is complete, the fluid residue is removed by opening drain valve  67 . 
     If desired, more amino acid fluid mixtures may be applied to the same resin and peptides to grow longer peptide polymers, using the same steps as described. Further steps in the process include cleaning vessels, resins and peptides with solvents such as DMF.(dimethylformamide). 
     Solvents and reagents such as DMF, MeOH, and piperidine are used in the process and delivered to reaction vessels by valves  51 - 75 . It can be noted that MeOH container  220  and piperidine container  222  can be vented or pressurized with nitrogen by control valves as needed but that DMF container  226  is always pressurized. As needed, any of these is routed to metering vessel  120  to be measured precisely, and then delivered to the desired reaction vessel. For example, to deliver a precise amount of piperidine to reaction vessel  106 , valve  55  is energized to pressurize piperidine vessel  222 . Valve  53  and  56  are energized to send piperidine through valve  53 ,  54  and  56  into metering vessel  120  until a photocell  330  within the fluid measuring assembly  300  senses the liquid, indicating that enough liquid has been sent into metering vessel  120 . Photocell  330  was previously placed at the proper vertical position with respect to vessel  120  by stepper motor  301  as follows. Now valve  56  is de-energized, valve  58  is energized to apply pressurized nitrogen to the top of metering vessel  120  and valves  57 ,  59  and  67  are energized to route the fluid from metering vessel  120  to reaction vessel  106 . 
     As best shown in  FIGS. 10, 43, 11, 44, and 12   10   a  - 12 , metering assembly  300  includes a frame  300 , a metering vessel  120  which is a vertical clear tube, a photocell carriage frame  320  which surrounds metering vessel  120  and moves vertically while carrying an internal photocell  330  capable of sensing the fluid level within vessel  120 , a photocell carriage member  302  with female threads being threaded onto a threaded vertical rod  312  driven by a toothed pulley  311  for the purpose of lifting and lowering the photocell carriage  320 , and a stepper motor  301  with a toothed driving pulley  316 , a toothed belt rotatably connecting pulleys  311  and  313 . At the top of threaded rod  312  is a disc  306  with eight slots  310  and a photocell  308  for the purpose of counting revolutions of threaded rod  312  and therefore providing feedback as to the distance which the photocell carriage has moved. There is also a home tang or flag  320  which is sensed by a home photocell  318  when the carriage is at a bottom position. Upon power up, the stepper motor  301  drives the carriage to the home photocell  318 . From this point forward, the computer drives the motor  301  and counts pulses from photocell  308  to determine the precise vertical position of the photocell carriage. When a specific amount of fluid is required, the computer causes the stepper motor  301  to drive the metering photocell  330  to the proper height corresponding to the specific amount of fluid required, then, the proper valves are energized to fill the metering tube  120  until photocell  330  senses the fluid. Then the valves are de-energized because the proper amount of fluid has been delivered to the metering vessel. 
     As can be seen in  FIGS. 36 and 2-6 , there are 12 sets of pre-reaction vessels, reaction vessels, and fluid control valves which provide the user with the capability of programming 12 separate and different processes for synthesizing 12 different peptides. One such set of the mentioned twelve sets has been used as an example process in the preceding discussion and includes: 
     Set 6. pre-reaction vessel  206  with connected valves  19 ,  31 , and  43 , reaction vessel  106  with connected valve  67 . 
     The other eleven sets are as follows: 
     Set 1. pre-reaction vessel  201  with connected valves  14 ,  26 , and  38 , reaction vessel  101  with connected valve  62 ; 
     Set 2. pre-reaction vessel  202  with connected valves  15 ,  27 , and  39 , reaction vessel  102  with connected valve  63 ; 
     Set 3. pre-reaction vessel  203  with connected valves  16 ,  28 , and  40 , reaction vessel  103  with connected valve  64 ; 
     Set 4. pre-reaction vessel  204  with connected valves  17 ,  29 , and  41 , reaction vessel  104  with connected valve  65 ; 
     Set 5. pre-reaction vessel  205  with connected valves  18 ,  31 , and  42 , reaction vessel  105  with connected valve  66 ; 
     Set 7. pre-reaction vessel  207  with connected valves  20 ,  32 , and  44 , reaction vessel  107  with connected valve  68 ; 
     Set 8. pre-reaction vessel  208  with connected valves  21 ,  33 , and  45 , reaction vessel  108  with connected valve  69 ; 
     Set 9. pre-reaction vessel  209  with connected valves  22 ,  34 , and  46 , reaction vessel  109  with connected valve  70 ; 
     Set 10. pre-reaction vessel  210  with connected valves  23 ,  35 , and  47 , reaction vessel  110  with connected valve  71 ; 
     Set 11. pre-reaction vessel  211  with connected valves  24 ,  36 , and  48 , reaction vessel  111  with connected valve  72 ; 
     Set 12. pre-reaction vessel  212  with connected valves  25 ,  37 , and  49 , reaction vessel  112  with connected valve  73 . 
     These 12 sets of vessels and valves are intended to operate independent of one another according to the program which is stored within the onboard computer  434  to synthesize as many as twelve separate and different peptides simultaneously. 
     Other embodiments of this peptide synthesizer include the same elements but have fewer sets of pre-reaction vessels, reaction vessels and connected valves. For example, one embodiment has only four such sets and therefore can only be used to synthesize four independent peptides simultaneously. Another embodiment contains 16 sets of pre-reaction vessels, reaction vessels and connected valves and therefore can be used to synthesize up to sixteen independent peptides simultaneously. An even higher number of sets of pre-reaction vessels, reaction vessels and connected valves is possible but higher numbers of components become impractical when there are too many processes taking place for the moving mechanical components such as the carousel, needle probe and metering assembly to keep satisfied. In other words, in order to keep 12 processes running simultaneously, each individual process needs amino acids and reagents delivered to pre-reaction and reaction vessels at the proper times. This requires a minimum amount of time to perform each of these deliveries. If the amount of time to deliver these to each pre-reaction and reaction vessel is, on average, five minutes per process, and each synthesizing process takes, on average, one hour (60 minutes), then at most, 12 processes can be simultaneously satisfied by the automated synthesizer of the present invention (5×12=60). If, however, the average amount of time to deliver these amino acids and reagents is four minutes, then an automated synthesizer of the present invention with 15 sets of pre-reaction vessels, reaction vessels and connected valves is practical (4×15=60). Thus, it can be seen that there is a practical upper limit to number of simultaneous processes, and therefore, the number of sets of pre-reaction vessels, reaction vessels and connected valves which are practical to include in any embodiment of the present invention. 
     The schematic of still another embodiment of the automated peptide synthesizer  400  is shown in  FIG. 18 . This automated peptide synthesizer  400  contains a cabinet  407 , only 4 sets of reaction vessels RV 1 - 4  and connected valves  1 - 10 ,  18  and  19 , a pump  23 , a motorized amino acid carousel  80 , a needle probe assembly  85 , a fluid metering assembly  303 , reagent bottles  90  and  91 , and flow monitoring photo cells PC  2 - 9 , but does not include pre-reaction vessels as do the previously discussed embodiments. The omission of the pre-reaction vessels simplifies the processing and is the primary difference between peptide synthesizer  400  and peptide synthesizer  10  of  FIGS. 3   a.  The down side, however, is the loss of the advantageous pre-reacting of the amino acids and reagents. 
     With reference to  FIGS. 18-24 , in order to process peptides within reaction vessel  402 , for example, the user must first remove reaction vessel  402  from vessel holder  136 , and place a selected amount of resin in the vessel  402 . The user then returns the vessel  402  to holder  136 . Now the computer  434  causes the carousel to align a particular amino acid bottle  82   a  directly under the needle probe of the amino acid delivery needle probe assembly  85 . The needle probe is thrust downward into the amino acid bottle  82   a  by driving needle probe motor  86 . Now, valves  5 ,  10  and  20  are energized to open a fluid path from the needle probe assembly  85  to reaction vessel  402 , and pump  23  is started until the amino acid is delivered to vessel  402 . 
     Now, the needle probe is withdrawn and rotated and plunged into a cleaning solution whereupon fluid is pumped into and out of the probe. If another amino acid is needed, the carousel  80  is rotated to the proper position and the needle probe assembly  85  thrusts the needle probe into the next amino acid bottle  82   a  to draw the proper amount of the that amino acid into vessel  402 . Then the needle is cleaned as before. If a reagent is needed in vessel  402 , valves  5 ,  10  and either  21  (for bottle  91 ) or  22  (for bottle  90 ) are energized and pump  23  is started until the proper amount of reagent is pumped into vessel  402 . Now, the mixture in reaction vessel  402  is allowed to react for a specific amount of time (around 45 minutes to one hour) during which time peptides will grow on the resin beads. Now, the remainder of fluid in vessel  402  is drained by energizing valve  1  and  18 . Valve  18  supplies pressurized nitrogen and valve  1  provides a fluid path from vessel  402  to a waste bottle. 
     At this point, the resins along with the attached peptides may be removed from the vessel  402  or, if needed, additional peptides may be grown onto the peptides already on the resins. To do this, repeat the previous paragraph. 
     The foregoing detailed description is given primarily for clearness of understanding and no unnecessary limitations are to be understood therefrom, for modification will become obvious to those skilled in the art upon reading this disclosure and may be made upon departing from the spirit of the invention and scope of the appended claims. Accordingly, this invention is not intended to be limited by the specific exemplification presented herein above. Rather, what is intended to be covered is within the spirit and scope of the appended claims.

Technology Classification (CPC): 1