Classifications

• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00277—Apparatus
  • B01J2219/00279—Features relating to reactor vessels
  • B01J2219/00281—Individual reactor vessels
  • B01J2219/00286—Reactor vessels with top and bottom openings
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00277—Apparatus
  • B01J2219/00279—Features relating to reactor vessels
  • B01J2219/00306—Reactor vessels in a multiple arrangement
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00277—Apparatus
  • B01J2219/00351—Means for dispensing and evacuation of reagents
  • B01J2219/00389—Feeding through valves
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00277—Apparatus
  • B01J2219/00351—Means for dispensing and evacuation of reagents
  • B01J2219/00418—Means for dispensing and evacuation of reagents using pressure
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00277—Apparatus
  • B01J2219/00479—Means for mixing reactants or products in the reaction vessels
  • B01J2219/00488—Means for mixing reactants or products in the reaction vessels by rotation of the reaction vessels
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00277—Apparatus
  • B01J2219/00497—Features relating to the solid phase supports
  • B01J2219/005—Beads
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/0059—Sequential processes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00583—Features relative to the processes being carried out
  • B01J2219/00596—Solid-phase processes
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/0068—Means for controlling the apparatus of the process
  • B01J2219/00686—Automatic
  • B01J2219/00689—Automatic using computers
• • B—PERFORMING OPERATIONS; TRANSPORTING
  • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
  • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
  • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
  • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
  • B01J2219/00718—Type of compounds synthesised
  • B01J2219/0072—Organic compounds
  • B01J2219/00725—Peptides
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B40/00—Libraries per se, e.g. arrays, mixtures
  • C40B40/04—Libraries containing only organic compounds
  • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C40—COMBINATORIAL TECHNOLOGY
  • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
  • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
  • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

• the present invention relates to a method and apparatus for automated synthesis of peptides on insoluble supports.
• the reagent is then delivered to the reaction vessel by activating a valve.
• the disadvantage to this system is that the premetering flask must have input conduits from many reagent reservoirs. This common
• OMPI connection can allow contamination of not only the solvent being delivered, but also of the reagent reser ⁇ voirs themselves by vapors or liquid from other reagent reservoirs.
• SPPS solid phase peptide synthesizer
• the present invention contemplates an apparatus and method not only capable of performing the functions of currently marketed SPPS models, but which also possess several advantageous improvements as set forth below.
• the inventive apparatus and method provide for synthesis of more than one peptide to be carried out under the same condition(s) simultaneously, but with only one set of reagents being required.
• Two methods are contemplated. In a single path method a multi- chambered vessel or multicolumns are used. In a multi- path method each synthesis is carried out in an individual vessel or column. Simultaneous synthesis of more than one peptide at a time has the following advantages. a) Cost saving (labor, initial investment in the number of apparatus, space and other related types of expense) . b) Time saving.
• the inventive method and apparatus also facilitate synthesis of peptides of higher quality with more rapid coupling and synthesis than those synthesized on prior machines known to me because of the following reasons:
• the inventive apparatus and method also are particu ⁇ larly flexible in enabling easily and precisely controllable synthesis of a wide variety of peptides and are adapted for control manually, by hard wired circuitry and by a microprocessor having a master program contain ⁇ ing from one to over 200 files. Individual synthetic procedures can be stored in each file. The .output from the controller can be connected to a printer for a permanent record of the synthesis in progress. In each file any reagent in any order one wishes can be chosen. This flexibility provides the following advantages. a) Number of reagents is not limited. b) Order of reagents is- not restricted. c) Type of solid supports is not limited. d) Number of amino acids is not limited. e) No limitation on new types of protecting groups, or coupling methods. f) None need to write a program or load a pro-? gram. g) A permanent record of the synthesis is pro ⁇ quiz.
• a method and apparatus for peptide synthesis in which a plurality of supply valves are arranged in series to define a line for supplying successive reagents to a reaction vessel in which a peptide is to be synthesized.
• a corresponding plurality of containers contain respective reagents and each, by actuation of its corresponding valve, is connectable through down ⁇ stream ones of the series of valves to supply its respective reagent to the reaction vessel.
• further valving permits draining of the reaction vessel to waste and, between applications of successive reagents to the reaction .vessel, cleaning of the line of valves by flushing with a cleaning reagent sent from the upstream end of the series of valves through the line to waste.
• the plurality of reagent containers and series line of valves supplies reagents to multiple reaction vessel chambers in such a way that the multiple chambers may simultaneously be occupied by the same reagent in one peptide synthesis step and by several differing reagents, delivered thereto in sequence, in a differing peptide synthesis step, to enable simultaneous production of similar but different peptide analogs.
• Figure 1 schematically discloses major units of a solid-phase peptide synthesizer embodying the invention.
• Figure 2 diagrammatically discloses an embodiment of a peptide synthesizer including a top emptying reaction vessel.
• Figure 2A discloses an apparatus similar to that of Figure 2 but modified to provide a bottom emptying reaction vessel.
• Figure 2B is an elevational view of the agitator and 180° rotation device of Figure 2 detached from the reaction vessel and looking toward the left in Figure 2.
• Figure 3 diagrammatically discloses an embodiment similar to that of Figure 2 but modified to have a porous wall multi-chamber vessel.
• Figure 3A shows an alternate position of the reaction vessel of Figure 3.
• Figure 4 discloses a central cross-sectional view in more detail of the Figure 3 reaction vessel.
• Figure 5 is a cross-sectional view substantially taken on the line V-V of Figure 4.
• Figure 6 diagrammatically discloses a further peptide synthesizer using plural column-type reaction vessels with a Figure 2 type reagent selection assembly.
• Figure 7 diagrammatically discloses a reagent reservoir filling control apparatus usable with the peptide synthesizers disclosed herein.
• Figure 8 diagrammatically discloses a further peptide synthesizer modified to provide a pump-type reagent supply assembly feeding a recirculating column- type reaction vessel assembly.
• Figure 9 diagrammatically discloses an embodiment similar to that of Figure 3, wherein the multi-chamber reaction vessel has a solid dividing wall.
• Figure 10 diagrammatically discloses an embodiment similar to Figure 2 but utilizing plural independent reaction vessels.
• Figure 11 diagrammatically discloses a further embodiment similar to Figure 10 but employing a flip-flop valve for supply reagent to separate reaction vessels.
• Figure 12 is a diagrammatic view similar to Figure 1 and disclosing control of an accessory device by the control unit of the peptide synthesizer.
• Figure 13 diagrammatically discloses a modification of the Figure 2 reagent selection assembly which provides for preactivation of an amino acid with a coupling reagent prior to application to the reaction vessel.
• DETAILED DESCRIPTION Figure 1 discloses, in very general form, the interrelation of major units of a solid phase peptide synthesizer in which a plurality of reagents from a reagent unit 201 are supplied in desired sequence by
• Control unit 206 may comprise manual actuators for each of the valves in valve unit 203, a hard wired or otherwise fixedly preprogrammed unit for automatically actuating the valves in the unit 203 in desired sequence, or, in the example shown in Figure 1, a programmable microprocessor 207 acting through a valve controller 208 connected to the valves of the unit 203 to actuate same in desired sequence.
• the microprocessor 207 and controller 208 may be conventional, it being
• Figure 2 discloses a preferred embodiment of a peptide synthesis apparatus according to the present invention.
• a plurality of containers for reagents as in Figure 1 reagents unit 201) , for example containers S, through S for solvents, containers B, through B for bases, containers A.
• the reagent containers S, through C are sealed except for connections hereafter described.
• a three-way pressurizing and venting valve 2 connects through a gas line 3 (such as a conduit, hose or the like and hereafter referred to merely as a "line") with the upper part of the reagent container S, .
• the valve 2 in its solid line position connects with the gas pressure line 1 to pressurize container S, and in its dotted line position connects with a conventional gas drying tower, or dryer,- D for alternately venting the container S, to the atmosphere without danger of contaminating the reagent in the container with atmospheric moisture.
• a further three-way, reagent-gas supply valve 5 connects through a line 4 with the lower portion of the reagent container.
• the remaining reagent containers S, through C are similarly provided with corresponding pressurizing and venting three-way valves 7, 22, 27...45, lines 9, 24...46 leading therefrom to the corresponding reagent container, drying " tubes D, reagent-gas supply valves 11, 21...48 and flow restricters R disposed in lines 10, 25...47.
• the flow restricters R may for example be needle valves and each is adjustable to compensate for differences in flow resistance caused by differences in flow path length and number of reagent-gas supply valves 5, 11...48 encoun ⁇ tered by reagents and differences in viscosity of reagents flowing to the reaction vessel 35 from the various reagent containers S....C .
• the flow restricters R each can be adjusted so that the rate of flow into the reaction vessel 35 will be the same regardless of which reaction vessel S,...C is supplying the reagent liquid.
• adjustment of the flow restricters R allows timed delivery of accurate volumes of different viscosities and for reagent containers at different distances from the reaction vessel 35. It has been found that without the flow restricters R, the error in delivery volume is typically only 0-10% and so the restricters R are preferably employed when greater accuracy is required.
• reagent selection assembly 212 upstream of the reaction vessel 35, may be referred to as the reagent selection assembly 212 and may be thought of as comprising an array of parallel reagent selectors 213, each comprising a series array of pressuring and venting valve, pressurizing line, reagent container, reagent
• reagent-gas supply valves 5, 11...48 of the several reagent selectors 213 are interposed in series in a line 6 leading from the two-way nitrogen gas control valve 51, rightwardly toward line 54 which takes a selected reagent from the reagent selection assembly 2-12 toward the reaction vessel 35 as hereafter more fully described.
• each pressuring and venting valve for example 2
• each reagent-gas supply valve for example 5
• the "oDen" positions of the valves of a given reagent selector 213 are those in which they supply reagent from their particular reagent container to the reaction vessel 35.
• valves 5, 11 48 when all in their "closed” positions, continue the line 6 from the nitrogen valve 51 to the assembly output line 54 so that opening of the two-way nitrogen control valve 51 causes nitrogen gas flow along such line 6 and line 54 toward the reaction vessel 35.
• reagent selection assembly 212 In the normal or rest condition of the Figure 2 reagent selection assembly 212 all three-way valves therein are in their "closed" position (reagent-gas supply valves 5...48 are in their horizontal position and all pressurizing-venting valves 2...45 are angled to their drying tubes D) and the two-way nitrogen supply valve 51 is off so that no flow is applied to the line 54.
• the common line 6 is open from the closed nitrogen control valve 51 to line 54 but neither nitrogen gas nor a reagent is supplied to it.
• valves 2 and 5 of the reagent selector 213 furthest from the reaction vessel 35 are "opened” (to their Figure 2 solid line position) , nitrogen gas from the line 1 will pres- surize the reagent container S, and reagent liquid therefrom will flow through the valve 5 and line 6 (as completed by the "closed" condition of the several three-way supply valves 11. -.48 shown in solid line in Figure 2) to the line 54 leading to the reaction vessel 35.
• valves of a reagent selector 213 in the middle of the reagent selection assembly 212 are "open", for example if valves 27 and 31 are "opened” to their dotted line position, reagent (acid A,) from the corresponding container A, will be driven by the nitrogen gas pressure in line 1 only to the right along line 6 to line 54 leading to the reaction vessel 35 and no acid A, can pass leftwardly along the portion of the line 6 prior to (to the left of) the now dotted line positioned valve 31 and such a leftward flow is blocked by the dotted line position of the valve 31. Thus, cross-contamination is prevented.
• the solvents for flushing (washing) the system are placed at__ the end of the line 6 furthest from the reaction vessel 35, namely the most leftward end thereof, at containers S,...S .
• Next along line 6 are basic reagent containers B,...B preceding (being to the left of) acid reagent containers A,...A .
• Next along line 6 are protected amino acid containers AAi, ...AAn and closest to the reaction vessel 35, the coupling reagent containers C1- . . .Cn.
• a reaction vessel assembly 218 is supplied by the line 54 of the reagent selection assembly 212 and includes the reaction vessel 35.
• the reaction vessel 35 is a single chambered reaction vessel or single column-type reaction vessel and may itself be of conventional type.
• the reaction vessel 35 is provided with conventional fluid passing, resin particle blocking filters 32 and 33 at the bottom and top ends thereof in a conventional manner.
• the reaction vessel 35 is equipped with means for agitating the contents thereof, which means may take the form of a stirring stick, an internal stirrer powered by an external rotating magnet, a vibrator or any other conventional means.
• the agitator is a conventional rocker agitator 221 for supporting and rocking back and forth through a small arc the reaction vessel 35 (for example an arc of 10° to 30°) .
• Two-way valves 13 and 14 connect to the top T and bottom B of the reaction vessel 35 and are closable to prevent unintended escape of liquid from the reaction vessel 35, particularly during agitation, but are openable to permit loading of a quantity of liquid reagent into the reaction vessel 35 and emptying of the latter.
• the outlet end of the line 54 connects to a further two-position, three-way valve 20 which in its solid line position delivers reagent liquid from line 54 through inlet two-way valve 13 to reaction vessel 35.
• the alternate dotted line position of valve 20 connects line 54 to the waste container W.
• a further three-way, two-position valve 15 in its soli line position connects the reaction vessel (through open valve 14) to a further drying tube D.
• the alternate dotted line position of the valve 15 connects the upper end of the reaction vessel 35 through a line 17 to the waste container W.
• the particular reaction vessel, assembly 218 shown in Figure 2 is arranged as a bottom loading, top emptying reaction vessel.
• the reaction vessel 35 is here provided with a conventional 180° rotation device (inverting device) 223.
• the 180° rotation device supports the agitator 221 which in turn supports the reaction vessel 35.
• the reaction vessel top and bottom connect to the two-way valves 14 and 13 through flexible tubes 226 and 227.
• the reaction vessel 35 can thus be loaded with a liquid reagent in its upright position shown in Figure 2.
• same can then be rotated through 180° so that its top T is lowermost and nitrogen gas under pressure applied through line 54 and valves 20 and 13 to the now upper end B thereof drives residual liquid out through the valve 14 and the
• All the valves in the assemblies 212 and 218 are preferably electrically controlled, for example conven ⁇ tional solenoid controlled valves.
• P-AA,-AA Q -resin Procedure for the synthesis of the generalized peptide (P-AA,-AA Q -resin) where P is a protecting group, AA is an amino acid, and resin may be e.g. chloro- methylated polystyrene divinylbenzene (Merrifield peptide resin) or benzhydrylamine resin, or any other polymeric resin.
• the protected starting component is prepared using a standard procedure according to the type of resin and protecting group on AA Q . For example, if the protecting group on the N-terminal of the amino acid is a t-Butyloxycarbonyl group (t-Boc) , then the following method steps are used:
• valve 2 is opened so nitrogen pressurizes wash solvent container S, through line 1, 3.
• valve 5 is opened and cleaning solvent S, travels through the line 4 and then through valves 5...48 interposed in line 6, 54.
• valve 20 With valve 20 shifted to its dotted line, waste position the cleaning solvent S, sweeps any residue in its path through line 6, 54 to waste W.
• the nitrogen valve 51 is opened and nitrogen transfers the liquid left behind in the line 6, 54 to waste W, to empty the line 6, 54. This step may be repeated for as long and with as many different cleaning reagents (in addition to S, and not shown) as required to clean the line 6, 54 from valve 5 to line 18 and waste container W.
• Resi wash Depending on the resin initially loaded in the reaction vessel 35, the appropriate type of resin washing solvent can be selected, e.g. S-. Venting-emptying valve 15 is normally in its solid line venting position shown. Waste valve 20 is returned to its solid line position shown.
• S- the appropriate type of resin washing solvent
• Valve 7 is then opened and nitrogen pres- surizes the reagent container S 2 « The valves 11, 13, 14 are then opened so reagent S 2 transfers through line 10 and through valves 11...48 interposed in line 6, 54 and through valves 20 and 13 to the reaction vessel 35. Then after a time sufficient to admit a desired amount of solvent S 2 to vessel 35, valves 7 and 11 are "closed” to shut off flow of solvent S 2 to vessel 35.
• Nitrogen valve 51 is opened and nitrogen transfers the liquid left behind in the line 6, 54 to reaction vessel 35 the same way as in above subsection (a) except solvent container S 2 is vented at D.
• the pressurized gas performs premixing and line drying functions in that the pressurized gas violently propels the solvent S 2 from lines 6, 54 into the reaction vessel 35 to enhance mixing of the solvent S 2 with the existing material in reaction vessel 35 and the gas empties the lines 6, 54.
• valves 51, 13 and 14 are closed. Then the reagents mixed, as by mechanical arm twist (shaker 221 here shown) , or other means not shown such as further injection of inert gas from a separate inlet or stirring.
• OMPI 51, 13 and 14 are opened and the venting valve 15 is opened to waste W.
• Vessel 35 empties of leftover liquid through line 17 to waste W due to nitrogen pressure through valve 51.
• Steps 2, 3, 4 and 5 are repeated several times (e.g. 4 or 5 times) .
• Valve 27 is opened so that nitrogen from line 1 pressurizes corresponding acid container A,. Then valves 31, 13, 14, 15 are opened and acid A, transfers through lines 30, 6 and 54 to the reaction vessel 35. Then valves 27, 31 are closed. Then step 2b is repeated.
• Step 3 is repeated, usually mixing for 3 to 15 minutes in a first cycle and in second cycle or third cycle mixing for 20 to 30 minutes each time.
• Step 1 is repeated.
• Neutralization Valve 22 is opened and pressurizes the base container B.
• Valves 26, 13, 14, 15 are opened so that base liquid B, transfers to the reaction vessel 35 by way of lines 25, 6, 54. Valves 22, 26 are closed and step 2b is repeated.
• the Boc-AA will couple to AA Q -resin already in the reaction vessel 35 and form Boc AA,-AA Q -resin plus side products such as dicyclohexyl urea.
• the valve 41 is opened and nitrogen pressurizes the amino acid container AA, containing Boc-AA,.
• the valves 44, 13, 14 and 15 are opened. Amino acid solution from container AA, will transfer through lines 43, 6, 54 to reaction vessel 35. Then valves 44 and 41 are closed and valve 51 is opened to push residual amino acid in the lines 6, 54 into the vessel 35.
• a second reagent (coupling reagent) C. for example dicyclohexylcarbodiimide (DCC)
• DCC dicyclohexylcarbodiimide
• Step 3 is repeated. Mixing is continued for 30 minutes to 24 hours depending on the specific amino acid and coupling reagent used. 19. Empty and restore. Steps 4 and 5 are repeated. 20. Resin wash. Steps 2, 3, 4, 5, and 6 are repeated, which removes nonreacted portions of the reagents .AA. and C, . 21. Further resin wash. Steps 2, 3, 4, 5 and 6 are repeated with a different solvent S. (such as ethyl alcohol) to remove side products, e.g. like dicyclohexyl urea.
• solvent S such as ethyl alcohol
• Example 1 The foregoing method steps result in formation in the reaction vessel 35 of the generalized peptide Boc-AA,-AA-.-resin. More complex peptides, and final products, can similarly be formed.
• the foregoing method of Example 1 can be used to synthesize a specific peptide, a simple but repre ⁇ sentative example being the following.
• Boc-Gly-C-O-Merrifield peptide resin polystyrene divinylbenzene resin
• the Boc-Gly-Resin starting material is placed in reaction vessel 35 in a conventional manner.
• Step 2 is carried out with S, and S, is CH 2 C1 2 ( ethylene chloride) .
• Steps 3, 4 and 5 are carried out with mixing step 3 timed at 1 minute each time.
• Step 6 is carried out 4 or 5 times.
• Step 8 is carried out wherein A ⁇ ⁇ is 40% trifluro-acetic acid in CH 2 C1 2 .
• a first mixing cycle lasts 5 minutes and a second cycle lasts 30 minutes.
• Step 11 like Step 1, uses
• Step 12 uses CH 2 C1 2 for 1 minute " each time for 5 times.
• Step 14 is carried out for 2 minutes 2 times.
• Step 15 washes the base from the resin using CH 2 C1 2 for 1 minute each time for 3 times.
• Step 17 is carried out except AA, is Boc-Phe and the coupling reagent is C 2 , namely 1-Hydroxybenzotriazole (and hereafter HoBt) .
• the valve 45 is opened and nitrogen pressurizes the C 2 (HoBt) con ⁇ tainer.
• valves 48, 13, 14, 15 are opened and the HoBt transfers to reaction vessel 35 by way of lines 6, 54 through valves 48, 20 and 13.
• valves 45 and 48 are closed.
• step 17 is repeated using DCC.
• the time for coupling may be 15 minutes - 2 hours.
• Step 20 is carried out using as the solvent CH 2 C1 2 for 1 minute each time for 3 times.
• Step 21 is carried out using ethyl alcohol.
• the reaction is completed (for example as indicated in testing conventionally the contents of .reaction vessel 35) , then the preparation of the Boc-Phe-Gly-Resin has been completed. If, on the other hand, the reaction is found not complete or if one wants further assurance of completion, a double coupling sequence can now be performed. Thus, double coupling is normally carried out if the reaction is not completed or as a safety precaution, e.g. if the reaction was carried out overnight or under other unsupervised conditions.
• the lines 6, 54 may be emptied (by flushing with pressurized inert gas) after flowing each solvent or other reagent to the reaction vessel 35. Also, before adding the next reagent to the reaction vessel 35, the lines 6, 54 may be flushed with an appropriate solvent S,, S 2 , etc., located at or adjacent the leftward (remote from reaction vessel 35) end of lines 6, 54, which line flush liquid is then directed to waste container W, to eliminate from the line 6, 54 any residual reagent (e.g. base, acid, amino acid, coupling reagent, etc.) left behind in the line 6, 54.
• an appropriate solvent S, S 2 , etc. located at or adjacent the leftward (remote from reaction vessel 35) end of lines 6, 54, which line flush liquid is then directed to waste container W, to eliminate from the line 6, 54 any residual reagent (e.g. base, acid, amino acid, coupling reagent, etc.) left behind in the line 6, 54.
• Figure 2A discloses an embodiment modified to empty the reaction vessel 35 from the bottom B thereof, rather than from the top T as in above-described Figure 2.
• the Figure 2A embodiment is similar to the Figure 2 embodi ⁇ ment except in having a reaction vessel assembly 218A modified as follows.
• reaction vessel venting valve 15 to waste W is eliminated and instead such normally closed port is connected to a branch of the pressurized nitrogen line 1.
• a further three-way waste valve 49 is added and has a solid line, normally open path interposed between the valves 20 and 13 and a broken line, normally closed path interposed between the valve 13 and waste W.
• the 180° rotation device. 223 is unnecessary since the reaction vessel 35 is arranged in Figure 2A to empty the waste through its bottom end.
• FIGS 3 and 3A disclose a further modified embodiment incorporating a modified reaction vessel assembly 218B, to which is connected the reagent selec ⁇ tion assembly 212 of Figure 2.
• the modified reaction vessel assembly 218B includes a modified reaction vessel 35B shown in more detail in Figures 4 and 5.
• the modified reaction vessel 35B is a multi-chamber reaction vessel. For illustration, a two-chamber vessel 35B is shown, but the reaction vessel may include more than two
• the vessel 35B is here divided into two parallel upstanding chambers 235 and 235' by a two-part divider 237, 238.
• the top portion 237 of the divider is nonporous and, with the peripheral wall 241 and top and bottom end walls 242 and 243 is preferably of substantially transparent glass to maximize visibility of contents during use and minimize interreaction of reagents with the material of the reaction vessel 35B.
• the top part 237 of the divider ' (preferably about two-thirds the length of the divider) is nonporous and may be of single or (as here shown) double-wall construc ⁇ tion.
• the bottom part 238 of the divider is constructed of a porous material (e.g.
• each chamber 235 and 235' is a corresponding fluid inlet-outlet fitting 246 and 246* for fluid entry and exit with respect to the chamber and which is conventionally provided with a resin particle blocking, glass frit filter 33 and 33'.
• the bottoms B of the two chambers 235 and 235* communicate through a common fluid inlet-outlet fitting 247 with tube 226 and a further conventional glass frit filter 32 blocks exit of recin particles P through the fitting 247.
• particle inlet-outlet fittings 248 and 248' communicate through the peripheral wall 241 with such chambers 235 and 235' and are normally closed by removable plugs 249 and 249'.
• the interior surfaces of the reaction vessel are pref ⁇ erably silicone coated, as generally indicated at 251 to prevent the resin particles P from adhering to the reaction vessel walls.
• Anti-splash fingers 253 protrude into the chambers 235 and 235' at the center portion of the reaction vessel 35B and are spaced from the ends thereof.
• the fingers 253 are arranged to prevent, when the reaction vessel 35B is agitated in its inverted Figure 3A position, splashing of reagent liquid therepast and above the nonporous part of the divider 237 so as to avoid contami ⁇ nation of the reagent liquids (which may be two different reagents) in the inverted (hence divided) chambers 235 and 235' of Figure 3A.
• the reagent liquid level is normally well below the level of the anti-splash fingers 253, for example as shown as LL in Figure 4.
• the fingers 253 are configured and spaced so as not to interfere with dropping of the resin particles from one end of the chambers 235 and 235' to the other upon 180° inversion of the vessel 35B from its Figure 3 position to its Figure 3A position, or vice versa. This assures that the resin particles always drop by gravity to the lower end of the vessel 35B.
• the path of reagent liquid or pres ⁇ surized gas on line 6, 54 from reagent selection assem ⁇ bly 212 to the bottom B of the vessel 35B is through a series path comprising a three-way valve 256, line 257, above-described three-way waste valves 20 and 49, a line 257', a further three-way valve 256*, a line 261, a further three-way venting valve (equipped with a drying tube D) , and the above-described two-way valve 13 and flexible conduit 226.
• This path can be used to apply both reagent and a premixing quantity of pressurized gas from the line 54 to the bottom B of reaction vessel 35B in the manner above described with respect to reaction vessel 35 of Figure 2.
• the venting path from the top T of chamber 235 through flexible conduit 227, two-way valve 14 and venting valve 15 to dryer D (as above described with respect to Figure 2) is duplicated at the top of chamber 235' at 227', 14' and 15'.
• valves 256, 15 and 49 are shifted to their dotted line positions, valves 14 and 13 are open as shown (valve 14' being closed) and the reagent selection assembly 212 is actuated in the manner above described with respect to Figure 2 to pressurize the line 54 with nitrogen gas, which then passes through valve 256, line 264, and valves 15 and 14 into the top of vessel 35B to drive leftover liquid reagent from the bottom B of the vessel down through valves 13 and 262 ' , through line 261 and valves 256' and 49 to waste W.
• Valve 14' in its closed position blocks escape of the incoming gas through its dryer D.
• the vessel 35B can have both its chambers 235 and 235' filled with the same reagent and then emptied.
• the Figure 3 apparatus with the reaction vessel 35B in its upright position shown permits all of the method steps, above described with respect to Figure 2, to be carried out therein.
• the modified reaction vessel assembly 218B of Figure 3 permits substantially simultaneous synthesis of two similar but different peptides (peptide analogs) , for example peptide analogs which are identi ⁇ cal peptides except for having differing amino acids at one or more locations in the chain of amino acids defining the peptides.
• the portions of the amino acid chain which are the same in the two analogs are syn ⁇ thesized in the Figure 3 position of the vessel 35B.
• the vessel 35B is invertable through 180° to its Figure 3A position to permit application of differ ⁇ ent amino acids (for example AA. and AA, ) on the resin particles P in the respective chambers 235 and 235'.
• reagent selection assembly 212 can be actuated as above described to apply a desired reagent (for example .AA.) above described through line 54, the Figure 3 dotted line position of valves 256 and 15, valve 14 ( Figure 3A) , flexible conduit 227, and into the now depending top T of leftward reaction chamber 235.
• the line 6, 54 is then preferably cleaned (e.g. as in Example 1, Step 1 above) by flushing through valve 20 to waste W with a suitable solvent, for example solvent S, to wa&te.
• line 6, 54 may be blown dry with pressurized gas N 2 through the same path.
• a differing reagent for example AA, ) may be supplied from reagent selection assembly 212 through line 54, the solid line position of valves 256, 20 and 49, the dotted line position of valve 256', line 264', valve 15' in its dotted line position, valve 14', flexible conduit 227' and thence into the now downwardly facing top T of the
• the Figure 3A positioned reaction vessel 35B can be restored, by 180° rotation of device 223, to its Figure 3 upright position, dropping all of the carrier resin particles P onto the filter 32 at the now depending bottom B of the reaction vessel.
• Inert gas N shall can then be supplied by reagent selection assembly 212 through line 54, dotted line position of valves 256 and 15 (valve 15* being in its dotted line position and valve 256' in its solid line position to seal the top end T of chamber 235'), valve 14 and flexible line 227 to pressurize the vessel 35B.
• the vessel 35B remains in its Figure 3 position, being inverted to its Figure 3A position only for processing by different reagents in the two chambers 235 and 235'.
• most of the steps required to prepare two differing peptide- analogs will be carried out precisely simultaneously in the Figure 3 position of the two chambers 235 and 235* , the remaining steps being carried out one after the other for the two chambers in the inverted Figure 3A position.
• two peptide analogs are prepared in very little more time than one peptide analog, using Applicant's Figure 3, 3A and 4 apparatus.
• the lines 227, 264 and 257 from the top of chamber 235 to waste 20 be as short as possible and this applies also to the lines 227', 264* and 257', as well as to the other through lines 226, 261 and 257'. Since, in the embodiment shown, these lines are not subject to line washing (as in Step 1 of Example 1 above) or blowing dry to clear same of all reagent from a prior method step, maintaining these lines short minimizes the amount of potential contami ⁇ nating material therein.
• the line 6, 54 is considerably longer and therefore is washed and, if desired, blown dry by gas to minimize cross-contamination between successively used reagents.
• Figure 9 discloses a further modified multi-chamber reaction vessel 35C provided as part of the modified reaction vessel assembly 218C also for synthesis of more than one peptide at a time.
• the reaction vessel assembly 218C is similar to the reaction vessel assembly 218B of Figures 3, 3A and 4 except for the following differences.
• the bottom portion 238C of the reaction vessel divider is a solid, nonporous, downward continua ⁇ tion of the upper part 237C of the divider and the divider has no porous part. This further avoids the possibility of cross-contamination when different reagents are present in the two chambers 235C and 235C* with the reaction vessel 35C in its upside-down position not shown (analogous to the Figure 3A position) .
• connection of the top T of the chambers 235C and 235' to the reagent selection assembly 212 may be identical to the corresponding connections described above with respect to Figures 3 and 3A.
• Figure 9 shows a further modification in that connection which may be used in the Figure 3, 3A embodiment as well.
• an additional pair of three-way valves 266 and 266* have their dotted line position interposed between corre ⁇ sponding valves 14 and 15 and corresponding valves 14' and 15' and it is the solid line position of such additional valves 266 and 266' which connects the corresponding two-way valves 14 and 14' to the corre ⁇ sponding line valves 256 and 256' above described with respect to Figure 3.
• a corresponding change is that an extension IC of the connection gas supply line 1 is connectable through the dotted line position of the valves 15 and 15' to the valves 266 and 266*.
• valve arrangement Operation of this Figure 9 valve arrangement is the same as discussed above with respect to Figure 3 and 3A except for the following.
• the pressurized gas from the extension IC is passed by switching the valves 15, 15', 266, 266' to their dotted line positions such that pressurized gas flows therethrough and through valves 14 and 14' into the chambers 235C and 235C to drive leftover liquid reagent downward through valves 13 and 262, line 261, the solid line position of the valve 256' and the dotted line position of valve 49 to waste W.
• a further difference is that with vessel 35C in its 180° inverted position (not shown in Figure 9 but analogous to the position of the vessel in Figure 3A) , reagents and premixing nitrogen gas from line 54 pass to the then downward facing ends T of chambers 235C and 235C ' (individually at different times as above described) not through the valves 15 and 15' but rather through the additional valves 266 and 266". Similarly, upon returning the vessel 35C to its upright Figure 9 position, it is the valves 266 and 266', not the valves 15 and 15', which are in the path for transfer of leftover reagent liquid from valves 14 and 14' through lines 264 and 264', valves 256 and 256', and valves 20 and 49 to waste W. It should be noted that the Figure 9 additional valves 266 and 266' make it possible, in the Figure 9 upright position, for nitrogen gas from extension line IC to be simultaneously applied to the tops of both chambers 235C and 235C.
• Figure 10 discloses a further modified reaction vessel assembly 218D for substantially simultaneous synthesis of several peptide analogs. Rather than using a double-chamber single vessel, either with a partially porous divider 238 as in Figure 3 or a nonporous divider
• each chamber 235D and 235D 1 has individual fluid connections via flexible conduits 227 and 227' and two-way valves 14 and 14* at the top T and flexible conduits 226 and 226' and two-way valves 13 and 13' at the bottom B thereof. Fluid connections can be made to these two-way valves as in either Figure 2 or Figure 2A.
• reaction vessel assembly 218D is generally of the Figure 2A type in which the multi-chamber vessel 35D is mounted by a fixedly supported agitator 221 (or otherwise provided with agitation or stirring means) and 180° rotation of the vessel 35D is thus not needed.
• the top two-way valves 14 and 14' connect to a three-way vent valve 15.
• the valve 15 normally vents the chambers 235D and 235D' to the drying tube D but is actuable to its dotted line position to pressurize such chambers at the top thereof from an extension ID of the pressurized nitrogen line 1 for emptying the leftover liquid contents of the chambers.
• a reagent or premix nitrogen gas from line 54 enters leftward chamber 235D substantially as in Figure 2A, namely through the solid line positions of waste valves 20 and 49 and two-way valve 13, and leftover process liquid is driven from the leftward chamber 235D also as in Figure 2A, namely through two-way valve 13 and the dotted line position of waste valve 49 into waste W.
• FIG. 10 there is added in Figure 10 a further three-way waste valve 49' having a solid line position permitting a reagent or premixing gas flow from line 54 to enter chamber 235D' through valve 13' and a dotted line position for emptying chamber 235D' through valve 13' to waste W.
• an extra pair of two-way valves 271 and 271' are interposed between the line 54 and waste valves 20 and 49'. Opening of valves 271, 20, 49' and 13* to their solid line positions shown admits flow from line 54 into chamber 235D and opening of valves 271, 20', 49' and 13' to their solid line positions shown admits flow from line 54 to chamber 235D'.
• the same reagent or gas flow can be simultaneously admitted to both chambers 235D and 235D'.
• a particular reagent from line 54 can be admitted only to one of the chambers 235D and 235D' and after appropriate line washing steps a different reagent can be admitted from line 54 to the other of such chambers.
• the line washing solvent e.g. S, passes through line - 6, 54, valves 271 and 271' and the dotted line position of valves 20 and 20' to waste W.
• two peptide analogs can be simul ⁇ taneously prepared, which analogs differ by one or more amino acids in the series making up such peptides but are similar in another amino acid or amino acids such series.
• the paths between the chambers 235D and 235D' and the waste valves 49 and 49', including lines 12 and 12', valves 13 and 13' and resilient lines 226 and 226', are preferably made as short as possible to minimize the possibility of cross-contamination therein.
• valves 271 and 271' permit different amounts of reagent to be added to the respective chambers 235D and
• Synthesis of more than one peptide analog at a time can also be carried out using the further modified reagent vessel assembly 218E of Figure 11, in which two separate reaction vessels, either mounted together as in Figure 10 or separately (e.g. with separate external agitators 221 and 221' if desired), are provided as here shown.
• the vessels 35E and 35E' of Figure 11 may be fed with liquid reagent or premixing inert gas through line 54 through a valve arrangement similar to that above described with respect to Figure 10. However, in Figure 11 this is done through a modified valve circuit inter- posted between line 54 and the two-way valves 13 and 13' and here comprising a symmetrical "T" connection at 276 which connects individual lines 277 and 277' from valves 13 and .13' to a single line 281 which is connected through the solid line positions of the waste valves 49 and 20 and valve 284 to the supply line 54.
• Reagent or premixing gas from line 54 thus passes through the solid line positions of valves 284, 20 and 49 through the "T" 276 and into whichever of the vessels 35E and 35E* that is selected by the valve 283.
• Residual liquid reagent is expelled from the reaction vessels alternately by applying nitrogen gas through line extension IE and the dotted line position of valve 15 to blow out the contents of the reaction vessels upon opening of their valves 13 and 13', shifting of waste valve 49 to its dotted line waste position and switching the position of the valve 283 to the desired one of the vessels 35E and 35E', whereafter the other said vessel can be emptied by reversing the position of the flip-flop valve 283.
• cleaning or drying of the line 54 is accomplished by cleaning reagent or nitrogen gas flowing through line 54 through valve 20 open to its dotted line waste position and hence to waste W.
• the two-position valve 283 it may be desired to add the two-position three-way valve 284 at the end of line 54 and a further two-position valve 285 connected between valves 283 and 13 in its solid line position shown and having a dotted line position connect ⁇ ing valve 284 with valve 13.
• a first reagent can be loaded from line 54 through valves 284, 20, 49, 283 and 13' into vessel 35E*, the path through lines 6, 54 and valves 284 and 20 can be cleaned by flushing to waste W as above generally described with respect to Figure 2, and a further reagent can be loaded (without contamination by the first reagent) into vessel 35E through the valves 284, 283 and 13.
• Figure 8 which discloses a reagent selection assembly 212F modified for use of a mechanical pump to move liquids through the system, rather than nitrogen gas as in the reagent selection assembly 212 of Figure 2.
• the Figure 8 modified reagent selection assembly 212F differs from Figure 2 assembly 212 in that its drying tubes D directly connect by lines 3F...46F to the upper portion of the corresponding reagent containers S,...C and the Figure 2 nitrogen gas line 1 and pressurization valves 2...45 are eliminated.
• a modified reaction vessel assembly 218F includes a pump 301 for drawing preselected amounts of liquid from the reagent selection assembly 212F.
• the pump may be periodically switched on and off.
• the pump 301 is preferably of the continuous run type capable of moving liquid at a preselected rate therethrough except when a valve downstream thereof is closed, in which case the pump 301 maintains liquid pressure thereagainst.
• valve 315 To supply a reagent from assembly 212F to assembly 218F, valve 315 is placed in its above-discussed solid line position with the pump 301 running and the reagent supply valve 5...48 associated with the particular desired one of the reagents S,...C is shifted from its normally closed (solid line) to its open (dotted line) position so that the pump draws that reagent.
• the pump 301 draws the selected reagent from the line 54 through the solid line position of a three-way valve 315 and propels it rightward through the solid line position of a waste valve 20 and a pressurized nitrogen supply valv-e 303, a line 304, two-way valve 13, resilient tube 226, a reaction column 307, flexible tube 227, two-way valve 14, a line 309 and the solid line position of a three-way valve 310 to the top of an overflow reservoir 311 to fill the reservoir 311 to the desired level.
• the top of the overflow reservoir 311 is vented by a drying tube D.
• a pickup tube 314 extends down into the reservoir 311 to near the bottom thereof and is connected through a- line 313 and the broken line position of three-way valve 315 back to the input end (leftward end) of the pump 301, such that the valves 20, 303, 13 and 14 in their solid line position and the valve 315 in its broken line position provide a continuous loop for liquid recirculation by the pump 301 through the column 307 and overflow reservoir 311.
• valve 315 is shifted to its broken line, recirculating position whereupon continuing operation of the pump 301 recircu ⁇ lates the reagent liquid in the overflow reservoir 311 back through the column 307.
• the column 307 is a conventional peptide synthesis column containing resin particles with starter amino acid (e.g. amino acid AA Q ) much as above described with respect to reaction vessels 35-35E' above.
• valve 310 When the time of treatment by this reagent is completed, the valve 310 is shifted to its dotted line waste position connecting to waste W through a line 308.
• the pump 301 then empties the recirculation loop by pumping through the path 311, 314, 313, 315, 301, 20, 303, 304, 13, 14, 309, 310, 308 into waste W.
• the overflow container 311 and tube 314 are arranged to pump the former substantially dry by means of the pump 301.
• any remaining unused reagent liquid droplets in the path 304, 307, 310 can be blown to waste W by shifting gas supply valve 303 to its dotted line positi ⁇ n whereupon nitrogen gas from pressurized line IF moves through the latter path to waste.
• a cleaning reagent for example solvent S
• a cleaning reagent for example solvent S
• the Figure 2 nitrogen two-way valve 51 and solvent S,, reagent-gas supply valve 5 can be "opened" to the line 6, 54 apparatus, as in Figure 2, the running pump 301 permit ⁇ ting the drying gas and residual droplets of reagent (if any) to go to waste W.
• the flexible tubes 226 and 227 and the two-way valves 13 and 14 can be omitted as can any agitating or vibrating means, such as the agitator 221, if sufficient interaction between the reagent and the material in the column 307 is provided in the column 307 by recirculation by pump 301 of each reagent liquid.
• Figure 7 merely indicates the possibility of controlling the amount of liquid drawn from any one of the reagent containers S,...C by shutting off the pump 301, or a valve in series therewith such as valve 315 of Figure 8, by using a level sensing device such as a photocell PC ( Figure 7) to sense the amount of liquid pumped into a reservoir 321 in a reaction vessel assembly like those at 218-218F.
• a suitable control device e.g. microprocessor 207, receives a photocell signal over electric signal line 326 when the reagent liquid in reservoir 321 rises to its level, whereupon the micropro ⁇ cessor 207 acts through electric power line 327 to turn off pump 301 or valve 315 to prevent further flow into reservoir 321.
• the measured amount of liquid in reser ⁇ voir 321 may then be passed through an extension 54' of line 54 to the corresponding assembly 218-218F for use as above set forth.
• a simple control relay can be substituted to turn off the pump 301 or valve 315.
• Figure 6 discloses a still further apparatus for substantially simultaneous synthesis of different peptide analogs, wherein a plurality of separate reaction vessels (here in the form of conventional reaction columns) are alternately connectable in parallel and in series for, in any given synthesis step, supplying the several reaction columns with either the same or differ ⁇ ing reagents.
• a plurality of separate reaction vessels here in the form of conventional reaction columns
• reaction vessel assembly 218GG comprises a plurality, here three, of preferably identical reaction vessel assemblies 218G, 218G' and 218G' ' defining respective loops comprising respective pumps 301-301' ', lines
• the Figure 6 assembly 218GG provides for the following alternatives in loading and circulating reagents:
• any two reservoirs e.g. reservoirs
• the assemblies 218G-218G' ' may be identical to the reaction vessel assembly 218F of Figure 8, which utilizes pump 301 not only to recirculate reagent from reservoir 311 back through the column 307, but also to initially supply reagent from the nonpressurized reagent container assembly 212F.
• the reaction vessel assemblies 218G-218G* * instead utilize their pumps 301-301'' solely
• valves 20-20' ' supply fresh reagent from line 54 to the respective reservoirs 311-311 • ', rather than routing fresh reagent directly into the columns 307-307''.
• a given reservoir e.g. 311
• line 54 can be cleaned, with the cleaning solvent (e.g. S.) going through the solid line position of the series valves 20-20* ' to waste W.
• more reagent can be supplied to a different reservoir (e.g. 311' or 311") via line 54 and the dotted line position of the corresponding valve 20* or 20*'.
• the particularly series valving 20-20' • shown does not permit simultaneous filling of the several reservoirs 311-311*'. This is not usually a disadvantage because usually the same reagent will not be loaded into all reservoirs 311-311" in a given synthesis step. Restated, foregoing alternate 4 will not normally be used, alternate 3 normally being more efficient for a single reagent step.
• the pump 301 is connectable in loop to draw reagent from near the bottom of its reservoir 311 and pump it through line 304, its column 307 and the solid line position of waste valve 310 back into its reservoir 311 to complete the recirculation loop through its reservoir and column.
• the correspond ⁇ ing waste valve 310 can be shifted to its dotted line position and the pump 301 draws all the leftover reagent liquid from the reservoir 311 and drives same through column 307, valve 310 and line 308 to waste W.
• Each recirculation loop (single or multiple column) is emptied in a corresponding manner.
• the pump 301 is preferably of a type (for example a peristaltic pump) capable of propelling all such reagent liquid there ⁇ through and then pumping a plug of air to move the last
• the Figure 6 assembly 218GG includes valved paths permitting a particular reagent in one of reservoirs 311-311'* to circulate through more than one of the columns 307-307'' to implement alternatives 1 and 2 above.
• reagent liquid circulated from reservoir 311 by pump 301 through column 307 and valve 310 may then be routed through the dotted line position of a valve 331, the solid line position of a three-way valve 332 and the dotted line position of a further three-way valve 334 to the column 307' and thence through the dotted line position of a return three-way valve 338 and a return line 339 back to the reservoir 311.
• liquid from reservoir 311 circulates through both columns 307 and 307'.
• a different reagent may simultaneously circulate within the loop 301", 307", 311", which includes the solid line positions of further three-way valves 354 and 331".
• valve 331 in its solid line position " completes the loop portion between waste valve 310 and reservoir 311.
• the valve 334 in its solid line position completes the loop portion between the adjacent pump 301* and column 307' and when in its dotted line position blocks passing of a liquid by pump 301*.
• valve 332 may instead be thrown into its dotted line position to cause reagent liquid pumped from reservoir 311, column 307 and the dotted line position of valve 331 to instead pass through a line 341 and the dotted line position of a valve 343, the column 307", the solid line position of
• ⁇ OMPI .,.- -tf lPO valve 310 the broken line position of valve 331", a return line 344, the solid line position of a valve 346 and a line 347 back to the top of reservoir 311.
• liquid from reservoir 311 is circulated through columns 307 and 307* '.
• a different reagent liquid may be circulated through the loop 301', 307', 311', which includes the solid line position of a further three-way valves 351.
• the liquid in reservoir 311' may be circulated beyond its column 307', through the dotted line position of valve 351, a line 352, the broken line position of a valve 354, line 304* ', the solid line position of valve 343, column 307'*, the solid line position of valve 310", the dotted line position of valve 331", a line 344 and the dotted line position of a valve 346 back to reservoir 311'.
• a different reagent may circulate in the loop 301, 307, 311.
• each column is isolated or a specific column is separated from the rest.
• the specific reagents are delivered to each loop separately without cross- contamination.
• the individualized reagents can be simultaneously circulated in their corresponding pump-column loops.
• Figure 13 discloses a reagent selection assembly 212K similar to assembly 212 above described with respect to Figure 2, but modified by addition of preactivatmg structure 420 to permit preactivating of a given amino acid (for example AA,) with the desired coupling reagent (for example C, ) , such that the amino acid has already been reacted with its coupling reagent to form an active ester (a liquid) and a side product (a solid) prior to application through line 54 to the reaction vessel.
• the Figure 13 modified apparatus thus permits a modification of coupling step 17 of the method of Example 1 above, such that an active ester, rather than the amino acid and corresponding coupling reagent, individually in sequence, would be applied to the reaction vessel.
• the Figure 13 modification permits bringing reacting of an amino acid and its coupling reagent substantially earlier than in Example 1, Step 17, for example between earlier steps of Example 1 and, if . desired, even prior to Step 1 of Example 1, if substan ⁇ tial time is needed to interact the amino acid with its corresponding coupling reagent.
• the time required to add each successive amino acid to the peptide chain in the reaction vessel may be reduced and correspondingly the overall time required to prepare a multi amino acid peptide can be correspondingly reduced.
• such reacting in the container 422 prevents reaction of the coupling reagent with an amino acid side chain on the resin in the reaction vessel.
• such reacting in the container 422 prevents removal of a protecting group on an amino acid side chain on the resin in the reaction vessel.
• the resulting preactivated amino acid (active ester) from the container 422 will react very quickly with the end terminus of the peptide chain in the reaction vessel 35.
• the Figure 13 modification will speed synthesis of a peptide, especially a long chain peptide, and produce a higher quality peptide product.
• the preactivating structure 420 includes a preactivating container 422.
• Three-way valves 424 and 426 are interposed between the corresponding reagent containers AA,, C, and their respective three-way supply valves 44, 40. The dotted line position of the interposed valves
• valves 424 and 426 permits connection of the reagent containers .AA,, C, directly to their corresponding three-way supply valves 44, 40 as in Figure 2. However, the valves 424, 426 are shiftable to their solid line positions for pumpiny of reagents AA, and C, through respective lines
• the container 422 is vented at the top thereof through the solid line position of a further three-way valve 431 to a drying tube D to permit entry of such reagents thereinto.
• the reagent containers AA. and C are pressurized with nitrogen gas from the line 1 in the manner above described with respect to Figure 2, in order to drive the corresponding liquid reagents through the valves 424 and 426 to the preactivating container 422.
• suitable pumps may be interposed in the lines 425 and 427.
• the containers AA, and C are vented to the atmosphere by shifting to the solid line positions of their corresponding pressurization and venting valves 41 and 37, and shifting to their dotted line positions the valves 424 and 426, such that the reagent vessels AA, and C, are now isolated from the preactivating container 422. Thereafter, the reagents within container 422 are interreacted.
• the pressurized source N 2 of inert gas connects through an extension 1H of above-described line 1, the dotted line position of a further three-way valve 433 and a line 434 into the bottom of the container 422.
• a normally closed two-way valve 436 may connect the line 434 to the bottom of the container 422. With the valve 433 in its dotted line position and the two-way valve 436 open, nitrogen gas on line 1H can enter and bubble up through the reagent liquids .AA, and C, in the container 422 to hasten mixing thereof. Excess gas in container 422 is vented through valve 431 to dryer D.
• a filter 438 such as a glass frit filter, is provided at the bottom of container 422 adjacent the opening to valve 436 and line 434 to permit draining of active ester liquid downward from container 422 but blocking passage therethrough of unwanted side products, which are of solid or semi-solid form and result from interacting of the amino acid with its coupling reagent.
• the preactivating unit 420 further includes a three-way valve 442 which in its normal solid line position of Figure 13 continues the line 6H through the various supply valves 5...44...40...48 to the line 54.
• the valves 431, 436, 433 and 442 are shifted to positions which shut off the pressurized gas flow to line 434 and the venting of container 422 through dryer D, and which instead permit pressurized gas to flow through line 1H and the dotted line position of valve 431 into the top of container 422 to drive the active
• preactivating structures 420 can be provided for each desired amino acid-coupling reagent combination. Alternately, by addition of suitable valving for cleaning and switching of reagents, a single preactivating container 422 can serve where not more than one amino acid-coupling reagent at a time is to be interacted.
• control unit 206 ( Figure 1) is of micro ⁇ processor type
• the major cost of the solid phase peptide synthesizer may be the programmed microprocessor. It is contemplated, however, that the microprocessor cost can be more reasonably justified by spreading it over several related systems.
• the microprocessor 207 could be employed simultaneously to control other peptide chemistry systems, such as a low pressure chromatography system 401 ( Figure 12) for preparative purification of peptide.
• the low pressure chromatography system 401 is conventional and includes, as shown in Figure 12, a reagent supply 403, a valve 404 controlled by unit 206, a pump 406, a chromatography column 408, a low pressure chromatography detector 409, fraction collector 411 and data recorder 412, wherein the valve 404, under control of the unit 206, supplies reagent from source 403 to pump 406 which sends same through the column 408, fractions from the column being detected at 409, detector 409 being monitored by recorder 412, so that desired fractions can be collected at 411.
• the disclosed apparatus particularly of Figures 3, 6, and 9-12, exemplify the important capability of the present invention to provide for simultaneous synthesis of several differing peptide analogs from a common reagent supply and with only a minimum number of extra parts being added to the apparatus for each additional peptide analog involved in the simultaneous synthesis of multiple peptide analogs.
• the disclosed apparatus may be equipped to constantly monitor both temperature and effective acidity or basicity ("pH") throughout the course of a synthesis. This xeature is particularly useful when large-scale reactions are being performed (i.e. pilot plant applica ⁇ tions) .
• a sensitive (for example plus or minus 0.05°C) temperature probe, or temperature sensor, TS can be provided on each reaction vessel 35.
• a separate connection from this probe to the control unit 206 allows for either a permanent recorded record of temperature and/or an automatic alarm AL actuation in case the temperature should fall outside of preprogrammed allowed limits.
• an automatic temperature traction can be effected by providing the reaction vessel with a conventional heating-cooling -means HC (e.g. refrigeration coils, a heating tape, a hot or cold water supply, etc.).
• the acidity or basicity of the contents of the reaction vessel 35 can constantly be monitored by the control unit 206 by means of a conductivity probe or sensor provided on " the reaction vessel 35.
• pH control is most appropriate for large scale reactions, but can prove useful as a failsafe synthesis check in smaller scale syntheses.
• a written record of pH changes can be provided via a strip chart recorder RE.
• the program for microprocessor 207 can be correlated to the anticipated conductivity values expected for each step of the synthesis and the synthesis can be automatically inter- rupted if these pH values do not correspond to the previously programmed expectations. If this should occur, the operator can be alarmed by an alarm at .AL.
• control unit 206 can be arranged to incorporate a number of capabilities and such may be readily implemented by means of a programmed microprocessor.
• Contemplated possible capabilities include preprogramming for single or multiple variation of solid phase peptide syntheses, programming of simul ⁇ taneous or multiple syntheses, constant interaction of various monitored systems (such as pH, temperature, ultraviolet monitors) with safety checks and automatic correction and/or interrupt of synthesis at a given step, recording synthesis steps by means of an auxiliary recording or printout device, alarms for additional potential problems such as low reagent level in reagent containers or need to prepare fresh reagents, control of automatic preparation of preactivated amino acids with appropriate temperature control, control of automatic low pressure preparative chromatography (LPC) as in Figure 12, operator training capabilities including strategy aids for the novice operator, and built-in self-diagnostics.
• LPC low pressure preparative chromatography
• monitoring and safety check provisions can be included, as above generally discussed with respect to Figure 1.
• the pump 301 of Figure 8 is preferably a conven- tional, fast, self-priming pump. Such pump may be more precise in measuring fluid supplied to the column 307 than the Figure 2 gas pressurized, timeable flow delivery.

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