Abstract:
A instrument for performing synthesis of small molecules such as Oligos and Peptides when using solid phase synthesis techniques to synthesize small molecules. The system and methods include a mechanism for controlling the pressure differential across the reaction vessels that contain the solid support used in solid phase synthesis. Reaction vessels are held in a holder that provides a sealable chamber at the outlet ends of the reaction vessels. The rotor containing the reaction vessels is placed within a sealable chamber. The sealable chamber is fitted with a means for engaging the rotor and draining the reaction vessels to waste. The sealable chamber is also fitted with a means for engaging the rotor to drain the reaction vessels at a slower, variable rate.

Description:
TECHNICAL FIELD 
       [0001]    This invention relates to instruments for control over the reactions performed during the synthesis of small molecules when using solid phase synthesis chemistry. 
       BACKGROUND 
       [0002]    Solid phase synthesis of small molecules has been performed for many years. The instrumentation used to perform the solid phase synthesis chemistry varies in design based on the type of synthesis and the scale of synthesis to be performed. Very early instruments were completely enclosed and were capable of synthesis of only small numbers of different molecules in a single synthesis run. The consumption of reagents of these instruments was very inefficient. They consumed too much reagent and took too much time to make them viable for synthesizing the growing need for more different molecules to be produced in shorter time and at a lower cost. 
         [0003]    As the scientific community developed processes that called for larger and larger numbers of different molecules, the molecules had to be synthesized faster and at a lower cost. 
         [0004]    Instruments were developed to produce larger numbers of different molecules in a single synthesis run. A design that incorporated open ended reaction vessels (typically called columns or wells) allowed more economical synthesis of larger numbers of different molecules. This general design has been used in various instruments in more recent years. However this design has always been implemented with relatively inefficient control over the reaction process because reagents are moved through the reaction vessels using only one mechanism, namely gas pressure from above. In one common usage the reaction vessels have an open top and an open bottom. Between the open top and open bottom is a solid support material held in the solid support by a top filter and a bottom filter. Such a reaction vessel is held in a holder in which the open top end is held in a first area and the open bottom end is held at a second area. The only flow between the first area and the second area is through the reaction vessel. Once liquid is dispensed into a reaction vessel, the liquid can act as a plug in the reaction vessel as it moves through the reaction vessel. The only way to equalize the pressure between the first area and the second area is to migrate the liquid through the reaction container. By using one gas pressure from one gas pressure source and one drain in a chamber holding a plurality of reaction vessels, liquid is drained from the reaction vessels at only one flow rate. This is not ideal for optimization of reactions. 
         [0005]    One result is that the reagents move through the reaction vessels too quickly. This in turn may require the addition of greater volumes of reagents. Since flow rate through the reaction vessel can be controlled only in a limited manner, additional reagent is used to ensure that sufficient reagent will react on the solid substrate. A considerable amount of reagent may flow through a reaction vessel without adding to the product being synthesized. These systems waste reagents, time and process gas that drives the reaction reagents through a solid phase. 
         [0006]    The scientific and commercial community has continued to increase its need for larger and larger numbers of different synthesized molecules. There is pressure in the market to produce these molecules at lower cost. One means of achieving this lower cost is by making the synthesis process more efficient. 
         [0007]    For open ended reaction vessels, reagents are dispensed into the open top of reaction vessels. The reaction vessel have open bottoms so reagent can be forced out the bottom of the reaction vessels. In between the open top and open bottom is a solid phase material where reactions take place. Reactants adhere to this solid phase. At the end of the reaction, an eluting reagent is added to remove the reaction products from the solid phase. These reaction products are collected in a collection container, such as a multiwell plate. 
         [0008]    It is an objective to provide a more efficient system and method for production of solid phase synthesis reaction products. 
       SUMMARY 
       [0009]    In present solid phase reaction systems, reaction vessels are held on a rotor. The open top ends extend from the top of the rotor and the open bottom ends are contained with one or more sealed chamber within the rotor. Reagents are dispensed into the reaction vessels. Gas pressure is then introduced, but the enclosed area above the rotor must be equalized with the sealed camber within the rotor. This is achieved by allowing gas to flow through the reaction vessels until the pressure is equalized. However, this will rapidly drive the fluid through the reaction vessel wasting costly fluid. To drain the sealed chamber, a drain is opened. Such a drain will drain the chamber at a single speed. This may not produce the flow rate through the reaction vessels that is most efficient. Utilization of both the process gas and the reaction reagents is not ideal. 
         [0010]    The various embodiments of system and methods relate to a solid phase synthesizer reaction system. This type utilizes a number of flow through reaction vessels, each reaction vessel having an open top to allow introduction of reagents through said reaction vessel, an open bottom of each reaction vessel to allow liquid to flow from said reaction vessel and a solid phase reaction material within each of said reaction vessel between said open top end and open bottom end. Such systems use a rotor that holds a number of reaction vessels. These may be grouped into banks. A cover is positioned above the rotor, enclosing an upper sealable chamber. This is considered the “upper chamber” because it contains open tops of the reaction vessels. Within the rotor is a lower chamber that holds the open bottom ends of the reaction vessels. In some embodiments, multiple lower chambers are used. Each lower chamber includes a passageway through the rotor, allowing gas communication between the upper chamber and the lower chamber. When the area around the rotor is pressurized, in prior systems, the only pathway to the lower chamber would be through the reaction vessel. The present embodiment, this passageway through the rotor allows equalization of the pressure between the upper and lower chambers without flow through the reaction vessels. 
         [0011]    The “upper chamber” will include both the area above the open top of the reaction vessels, and the area between the gas source and reaction vessels. This may include some area around the rotor. The rotor may include a priming port that flows to a drain. 
         [0012]    In one embodiment, this passageway through the rotor into the lower chamber is part of a second drain system that connects to tubing and valve elements to allow variable speed draining of gas from the lower chamber. A first drain system at the bottom of the sealable lower chamber is a more rapid speed drain of both gas and liquids. In one embodiment, this first drain is a ball check valve. This in turn, allows greater control of the flow rate through reaction vessels, allowing more efficient reactions by regulating the flow rate of reagents through a solid support material. In one embodiment, this variable speed drain includes a manifold attached to multiple calibration tubes, each of which is controlled by a valve. When one valve is opened, gas can flow through a calibration tube, through the manifold, and through the tube connecting this drain to the lower chamber. This provides a simple, low cost device which still greatly optimizes the flow through the reaction vessels. In one embodiment, the first drain, as a variable speed drain, drains gas at a lower rate and the second drain drains both liquid and gas at a rate that is, for example, two to one hundred times greater. By activation of the different drain systems, a user is allowed control of the flow rate through the reaction vessels and a more rapid drain to clear both reaction vessels and the lower chamber of liquid. In one embodiment, the variable rate drain is not in line with the longitudinal axis of the reaction vessels. 
         [0013]    In one embodiment, the cover over the rotor will hold a number of reagent dispense nozzles. A motor linked to the rotor allows the rotor to be turned such that various reagent dispense nozzles align with the open top ends of the reaction vessels. The motor is mounted on a motor mount. In one embodiment, this motor mount includes a spill channel that collects spilled waste and a drain to drain this spill channel, preventing spills from compromising the motor or other sensitive system elements. 
         [0014]    To reduce the requirement of process gas, in one embodiment a spacer is used on top of the rotor. This reduces the amount of gas that fills the space between the rotor and the cover that encloses the rotor into an interior space. 
         [0015]    In one embodiment a method of fluid control is described. This method utilizes the above described system embodiments. In this method, pressure is equalized between the sealable interior chamber where the reaction vessels terminate, and the area surrounding the rotor where the open top ends of the reaction vessels terminate without requiring that the entire flow move through the reaction vessel. This reduces the flow through the reaction vessels to a minimal level (gravitational flow). A variable rate drain can then be opened, to allow a selectable flow rate of liquid reagent through the reaction vessel. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0016]      FIG. 1  is a top perspective view of the sealable reaction vessel inlet chamber with the reaction vessel holder installed. The top of the sealable chamber is shown in the open position. 
           [0017]      FIG. 2  is the view of  FIG. 1  with the top of the sealable chamber shown in the closed position. 
           [0018]      FIG. 3  is a side view of  FIG. 2  showing three of the four drain stations. 
           [0019]      FIG. 4  is a bottom view of  FIG. 3 , showing all four drain stations. Each drain station has 2 waste connection ports. 
           [0020]      FIG. 5  is a top perspective detail of the sealable reaction vessel inlet chamber with the rotor installed. The top of the chamber has the reagent dispensing nozzles mounted in it (dispensing nozzles not shown). 
           [0021]      FIG. 6  is a top perspective view of the rotor showing a sealable vent hole into each bank lower chamber. 
           [0022]      FIG. 7  is a side perspective view of  FIG. 6  showing the chamber formed at the outlets of the reaction vessels. 
           [0023]      FIG. 8  is a cross sectional view of the chamber shown in  FIG. 7 . The sealable vent hole is detailed. 
           [0024]      FIG. 9  is a cross section of  FIG. 3  showing the rotor mounted in the chamber and 2 of the 4 drain stations completely plumbed. 
           [0025]      FIG. 10  is a cross section of part of  FIG. 3 , showing one of the waste stations shown in  FIG. 9 . The bank vent hollow shaft is shown disengaged from the rotor and the fast drain shaft is also shown disengaged from the rotor. The external gas source, valves and tubing are shown in this figure however they are not cross sectioned. 
           [0026]      FIG. 11  is a cross section of part of  FIG. 3 , showing one of the drain stations shown in  FIG. 9 . The bank vent hollow shaft is shown engaged with the rotor thereby connecting the reaction vessel outlet chamber for one bank to the hollow shaft and subsequently through a solenoid valve to waste. The fast drain shaft is shown disengaged from the rotor. The external gas supply, valves and tubing are shown in this figure however they are not cross sectioned. 
           [0027]      FIG. 12  is a cross section of part of  FIG. 3 , showing one of the drain stations shown in  FIG. 9 . The bank vent hollow shaft is shown engaged with the rotor thereby connecting the reaction vessel outlet chamber for one bank to the hollow shaft and subsequently through a solenoid valve to waste. The fast drain shaft is shown engaged with the rotor thereby connecting the reaction vessel outlet chamber for one bank to the fast drain hollow shaft and subsequently through a pneumatic valve to waste. The external gas source, valves and tubing are shown in this figure however they are not cross sectioned. 
           [0028]      FIG. 13  is a cross section of part of  FIG. 3 , showing one of the drain stations shown in  FIG. 9 . The bank vent hollow shaft is shown disengaged from the rotor. The fast drain shaft is shown engaged with the rotor thereby connecting the reaction vessel outlet chamber for one bank to the fast drain hollow shaft and subsequently through a pneumatic valve to waste. The external gas source, valves and tubing are shown in this figure however they are not cross sectioned. 
           [0029]      FIG. 14  is a top perspective view of the gearbox mounting bracket showing the trough that captures any spilt waste reagent. 
           [0030]      FIG. 15  is a cross section of the system showing the gearbox mounting bracket mounted in the system. The drain path for spilt waste reagent is identified in this figure. 
       
    
    
     DETAILED DESCRIPTION 
       [0031]    With reference to  FIG. 1 , an exploded view shows a top plate  1  and a top window  2 . Top plate  1  is hinged or fixed to a frame (not shown) such that it is movable by the operator to allow access to reaction vessels  17 . During operation top plate  1  is bolted or clamped onto a lower plate  6  forming a leak proof seal between top plate  1  and lower plate  6  at O-Ring seal  26 . Top plate  1  and lower plate  6  are contained within a housing (not shown). Rotor  4  in lower plate  6  is shaped to define a sealable upper chamber  3 . The reaction vessels  17  are held in the sealable upper chamber  3 . The rotor  4  is housed on bowl  5 . 
         [0032]    As shown in  FIG. 2 , top plate  1  is bolted onto to lower plate  6 . Mounted on top window  2  are reagent dispense nozzles  7 , as seen in  FIG. 5 . Rotor  4  may be rotated to position reagent dispense nozzles  7  above reaction vessels  17 . Dispense nozzles are stationary, held on top cover  2 . 
         [0033]    With reference to  FIGS. 3 and 4 , bowl  5  holds drain stations  8 ,  9 ,  10 ,  11 ,  12 ,  13 ,  14  and  15 . As explained below, drain stations have components that make up the high flow waste system and the variable flow waste system. Also mounted centrally on plate  6  is motor system  16 . This system includes a motor, a gear box, an encoder, a home sensor, a motor drive controller, and a power supply. The rotor is coupled to motor system  16  through hub  23  and collet  24  (as shown in  FIG. 9 ), allowing rotation and positioning of the rotor. This allows selective placement of the reaction vessels  17  below a selected dispense nozzle  7 . This is controlled by an automated control system utilizing a programmed computer and software (not shown). 
         [0034]    With reference to  FIGS. 6 and 7 , the rotor is shown divided into banks  48  for holding groups of reaction vessels  17 . These are enclosed to form upper chamber  3  shown in  FIG. 1 . As shown,  12  reaction vessels  17  are grouped in each bank  48 . In alternative embodiments, more or fewer reaction vessels or banks could be used, depending on need and available space (rotor size). 
         [0035]    A vent port  18  selectively sealed to hollow shaft  27  by a seal  25  is used with each bank. Vent port  18  extends from a sealable lower chamber  19  to a passage connecting to sealable upper chamber  3 . A priming port  50  is also used to dispense reagents to waste while bypassing all reaction columns. A single priming port is used for the rotor. A partial cutaway view shows the high flow waste system check valve  20  which allows draining of the sealable lower chamber  19 . The open bottom ends of the reaction vessels  17  extend into this lower chamber  19 . Each of bank  48  has its own high flow waste system check valve  20  that drains one lower chamber  19 . Each of the lower chambers  19  are isolated from each other. When reaction vessels  17  are installed in the rotor  4 , the lower chambers  19  and the upper chamber  3  (shown in  FIG. 1 ) are separated by the reaction vessels  17 . The vent ports  18  are also associated with each bank  48  of reaction vessels  17 . These vents serve two purposes. First, they provide an alternative means by which the upper chamber  3  and the lower chamber  19  can be connected during gas flow. This allows regulation of pressure between the upper chamber and the lower chamber, which stops the flow in both directions through the reaction vessels which would result from pressure equalization between the upper chamber and the lower chambers. Second, vent port  18  may be connected to a variable flow waste system. This allows liquid to flow through the reaction vessels at selectable different flow rates. 
         [0036]    As described, pressure the described embodiments allow the pressure source that pressurizes the upper chamber to also pressurize the lower chamber. It should be realized that this can work in the opposite way: the passageway between the upper and lower chamber can be used to relieve pressure from the lower chamber. It is equally beneficial to have a mechanism to release the pressure in the lower chamber. It is not optimal for gas pressure in the lower chamber to flow up through the reaction vessels. In the disclosed embodiments, the same vent hole allows both vent the lower chamber and pressurizing it. 
         [0037]    If gas flows up through the reaction vessels, reagent expelled into the lower chamber from multiple reaction vessels, all of which may have different reagents in them, may also flow into the reaction vessels. These reagents can be drawn back up through the reaction vessels causing cross contamination between reaction vessels. In the disclosed embodiments, the system allows venting the lower chamber through the port on the side of the rotor (one for each bank). This substantially eliminates cross contamination due to reverse flow through the reaction vessels. 
         [0038]    With reference to  FIG. 8 , lower chamber  19  is shown in this cutaway having a port  18  covered by a seal  25 . An O-ring seal  51  between the rotor top half  21  and the rotor bottom half  22  form a pressure tight seal for each bank  48  in the rotor  4 . Also shown is high flow waste system drain check valve  20 . Each high flow waste system drain check valve  20  illustrated in these embodiments utilizes a ball valve in which a ball  53  is biased by a spring  54  and retained against an O-ring  52 . 
         [0039]    Tubing inside diameter (ID) and length of the high flow waste system and the variable flow waste system is selected to achieve the flow rate difference between the High Flow Waste Systems and the Low Flow Waste Systems. In one example, one quarter inch OD× 3/16 inch ID tubing was used on drain of the High Flow Waste System, where the ball is biased against the drain opening. 
         [0040]    The cross section of  FIG. 9  shows a number of the major systems including: 
       The Rotor 
       [0041]    The rotor  4  is driven by the motor  16 , allowing circumferential positioning of the reaction vessels  17 . The rotor consists of the top spacer  49 , a reaction vessel holder  21  and a rotor bottom  22 . Reaction vessel holder  21  and rotor bottom  22  are bolted together, and top spacer  49  is placed into position and is located using a locating pin (not shown) or other means. The top spacer minimizes the space required for the sealable upper chamber. It extends proximate to window  2  with the exception of the area above the reaction vessels  17 . The reaction vessel holder  21  includes a number of grouped holes that the reaction vessels  17  extend through. The area surrounding the rotor assembly is the sealable upper chamber  3  and the area between rotor part  21  and rotor part  22  is the sealable lower chamber  19 . Upper chamber  3  includes the area above and around the open top end of the reaction vessel  17 . It also include the rest of the space connecting this chamber to the gas source. As indicated, this includes the passageway on the side of the rotor, the space on the top of the rotor between the cover  2  and the spacer  49 , and other space around the rotor. The rotor  4  includes one or more banks of the sealable lower chambers  19  for each bank of reaction vessels  17 . The sides of rotor reaction vessel holder  21  include one port  18  and seal  25  for each bank of reaction vessels. 
       Rotor Rotation 
       [0042]    As noted, motor  16  turns rotor. Part of motor  16  is an encoder, allowing rotational positioned to be determined. This allows the rotor to position the reaction vessels below a nozzle for reagent dispensing. The rotor can also be selectively positioned such that the port  18  having seal  25  is brought into a position to allow sealed connection with the center passage of shaft  27 . Rotor hub  23  is an interconnection mechanism to connect rotor  4  to the transmission shaft. Collet  24  is also part of the connection mechanism between the transmission shaft and rotor  4 . In one embodiment, the rotor is bolted onto the connection mechanism. 
       Dispensing of Reaction Reagents 
       [0043]    Reactions occur on a solid support located within reaction vessels  17 . As explained with regard to  FIG. 1 , the dispense nozzles  7  are aligned with open top ends of reaction vessels  17  held on rotor  4 . Reagents may then be dispensed into the reaction vessels  17 . 
         [0000]    Control of Flow Rate through the Reaction Vessels 
         [0044]    A mechanism that includes pneumatic cylinders, hollow shafts, solenoid valves, calibrated tubing and manifolds is used to selectively engage the rotor to connect the desired calibrated tube from the reaction vessel outlet chambers to waste. This system is referred to as the “variable flow waste system”. 
         [0045]    Control of flow rate during the reaction is controlled by a variable flow waste system. In this system pneumatic cylinders  29  are engaged to move hollow shafts  27  horizontally to connect or disconnect the center passage of hollow shaft  27  with port  18  on the rotor  4 . This is indicated by arrow  28 . Tube  30  connects the center passage of the horizontal hollow shaft  27  to a manifold  31 . Each port on the manifold is connected to a calibration tube  32  which in turn connects to a two way solenoid valve  36 . Two way solenoid valves connect to tubes  37  which connect to waste  38 . Each variable drain station has one set each of these components. The system as illustrated thus would have four variable drain systems, one for each bank of reaction vessels. This mechanism would allow the passage of gas from the lower sealed chamber to the upper sealed chamber. Each of the valves and pneumatic cylinder are able to operate separately under automated control. 
         [0046]    In one embodiment the calibration tubes  32  include a first tube of 1/16inch OD×0.020 inch ID and a second tube of 1/16 inch OD×0.0155 inch ID. These can be used as alternative pathways for this drain, or could both be used together. This provides three different drain rates. 
         [0047]    In one embodiment the system uses a fixed pressure. The pressure may be in a range from 2 psi to 7 psi. In one embodiment, the pressure using currently available commercial solid supports was 3 to 4 psi (for example 3.5 psi). 
       High Flow Waste System to Rapidly Drain Reaction Vessels 
       [0048]    A mechanism that includes pneumatic cylinders, hollow shafts, tubing, solenoid valves and pneumatic valves is used to selectively engage the rotor and quickly remove all waste reagents from the reaction vessels and the reaction vessel outlet chamber and send waste reagent to a vented waste container. This system is referred to as the “High Flow Waste System”. 
         [0049]    When the reaction is complete and it is desired to drain the reaction vessels and the lower chamber, hollow shaft  34  is moved in the direction of arrow  33 . A tip on the end of shaft  34  displaces the ball of the rapid flow check valve  20 . The components of this valve where discussed in respect to  FIG. 8 . The liquid and gas in the lower chamber  19  flow into hollow shaft  34  and into tube  39  when valve  40  is opened. The liquid then flows into tube  41  and to waste. 
         [0050]    For a given pressure the High Flow Waste System is designed to flow at a rate that is at least several times to about 100 times that of the variable flow Waste Systems. In one embodiment, a pressure value is chosen that will allow the High Flow Waste System to empty all reaction vessels and reaction vessel outlet chambers within 2 seconds. 
         [0051]    The initial positioning of elements is shown in  FIG. 10 . The following description occurs for each of the lower chambers in the rotor. If each lower chamber is provided with a high flow rate waste system and a variable flow rate waste system, the process described occur simultaneously for each of the lower chambers in the rotor. Horizontal shaft  27  is moved to the disengaged position indicated by arrow  56 . Shaft  27  is thus positioned so that it does not engage rotor  4 . The vertical drain shaft  34  is moved to the down position indicated by arrow  57 . In this position drain shaft  34  does not engage with rotor  4 . Solenoid valves  36  and  41  are closed, so there is no flow in the connected pipes. Rotor  4  may then be rotated by motor  16  such that a selected dispense nozzle  7  is positioned above the open tops of reaction vessel  17 . A separate system for dispensing reagents using pressurized reagent containers, valves, and tubing connected to the dispense nozzles  7  is conventional and known in this art. 
         [0052]    After the reagent has been dispensed, rotor  4  is rotated to align the sealed lower chamber with the drains of the variable flow waste system. As noted, each of the banks of reaction vessels is separately sealable and have a high flow rate drain and a variable flow rate drain. Upper chamber  3  is pressurized with inert gas through delivery port  55 . The lower chamber  19  is also pressurized via port  18 . The reagent dispensed into the top of reaction vessel  17  will not flow out of the bottom of reaction vessel  17  because the pressure on the reaction vessel  17  inlets and the reaction vessel  17  outlets is equal. Flow will result only from the liquid pressure head exerted by the liquid dispensed into the open tops of the reaction vessels which is negligent given flow restrictions found within common use reaction vessels. 
         [0053]    With reference to  FIG. 11 , the hollow drain shaft  27  is moved in the direction of arrow  28 . The flow channel in the center of the horizontal shafts  27  is connected to port  18  and sealed by seal  25 . One of the two way valves  36  is opened by the system controller, allowing gas to pass into tube  30 , through manifold  31 , into one of calibration tubes  32  and into one of tubes  37  and to waste  38 . This creates a pressure differential between upper chamber  3  and lower chamber  19  for that bank of reaction vessels  17 . The system controller will select the appropriate two way valve  36  to open based on the flow rate that is desired for the particular reaction vessel being used in a given bank of reaction vessels. Fluid will flow through the reaction vessel for a set time required for the reaction protocol. In the illustrated embodiment, either one of the two valves could open, or both valves could open. This provides three flow rates. More flow passages could be added to allow a greater number of flow rates. Alternatively, a different type of valve could be used to regulate flow rate. 
         [0054]    With reference to  FIG. 13 , flow to the variable flow waste system is stopped by closing valves  36  and activating pneumatic cylinder  29  to retract the shaft  27  such that port  18  does not connect to the hollow interior of shaft  27 . This will prevent gas from flowing out through the variable flow waste system. At the time in the process when it is desired to quickly empty the reaction vessels  17  in a given bank Hollow shaft  34  is moved in the direction of arrow  33 . This opens the ball check valve as discussed with respect to  FIG. 8 . The gas and liquid contents of the lower chamber  19  are drained into hollow shaft  34 , into tube  39  when valve  41  is opened. The gas and liquid will then flow into tube  40  and go to waste. 
         [0055]    With reference to  FIGS. 14 and 15 , the bowl  5  includes means to capture and divert waste that escapes to normal waste disposal system. This includes a gearbox/motor mount  35  that includes an annular trough  43  machined into the surface. Any spilled waste from the rotor will flow into annular trough  43 . Four drain holes  44  are machined into motor mount  35 . The drain holes  44  are connected to a waste container  47  through tubes  46  as shown in  FIG. 15 .