WO1985001224A1

WO1985001224A1 - Solid phase peptide synthesizer

Info

Publication number: WO1985001224A1
Application number: PCT/US1984/001486
Authority: WO
Inventors: Hossain H. Saneii
Original assignee: Saneii Hossain H
Priority date: 1983-09-22
Filing date: 1984-09-20
Publication date: 1985-03-28
Also published as: EP0156875A4; EP0156875A1

Abstract

A method and apparatus for peptide synthesis in which a plurality of supply valves (5, 11, 21, 26, 48) are arranged in series to define a line (6, 45) for supplying successive reagents to a reaction vessel (35) in which a peptide is to be synthesized. A plurality of containers (S1, S2, Sn, C1, Cn) contain respective reagents and each, by actuation of its corresponding valve, is connectable through downstream ones of the series of valves (5, 11, 21, 26... 48) to supply its respective reagent to the reaction vessel (35). For cleaning or reacting with peptide starting material therein valving (12, 13, 14, 15) permits draining of the reaction vessel (35) to waste (w) and cleaning of the line of valves by flushing with a cleaning reagent sent from the upstream end of the series of valves (5, 11, 21, 26, 48) through the line (6, 54) to waste.

Description

-1-

SOLID PHASE PEPTIDE SYNTHESIZER

FIELD OF THE INVENTION The present invention relates to a method and apparatus for automated synthesis of peptides on insoluble supports.

BACKGROUND OF THE INVENTION In practice, peptides have been synthesized in quantities ranging from milligrams to less than 10 grams. Existing machines have proven more reliable in the 0.1-1 gram scale. Syntheses can be carried out by

10 stepwise addition of amino acids or by fragment coupling reactions under inert gas or dry atmospht-ric conditions. Reagents can be transferred by inert gas pressure, by pump or by vacuum. Reagent delivery in the two currently marketed SPPS instruments (namely, Model 990 of Beckman located in California and Model 250 of Vega Biochemical located at Tucson, Arizona) is accomplished by trans¬ ferring reagent from a reservoir to a premetering flas (s). The volume of reagent is measured by monitoring with a photocell. From the premetering

20 flas (s) 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. Currently available solid phase peptide synthesizer (hereafter SPPS) machines known to me have up to 12 amino acid reservoirs and up to 11 solvent and other reagent reservoirs are capable of synthesizing only one peptide at a time.

Currently available SPPS machines of which I am aware thus have certain disadvantages which the method and apparatus of the present invention is intended to overcome.

Accordingly, 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.

OM c) Assurance that if two similar peptides are synthesized simultaneously the analogs will be essen¬ tially identical, except for the intended differences.

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:

A) Less chance of cross contamination because of the following: a) The manner in which reagents are transferred to the reaction vessel. b) Flushing of the lines after each use. c) Minimizing the amount of acid or base left behind in the reaction vessel, which reduces the side products and/or racemization. d) Preactivation of protected amino acids before addition to peptides on the resin, which reduces contact time and possible side reaction of the peptide with reagents and other additives. e) Addition of preactivated amino acids to increase the rate of coupling reaction and decrease the side products.

B) Rapid transfer of reagents to the reaction vessel (s) or draining from the reaction vessel (s), which is especially advantageous for large scale production because of uniform treatment of the resin in the reaction vessel.

C) Adaptability to control of temperature and pH, which should also help purity, increase rates of reaction, and otherwise improve the synthesis.

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) Never need to write a program or load a pro-? gram. g) A permanent record of the synthesis is pro¬ duced.

The objects and purposes of the invention are met by providing 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. For cleaning or reacting with peptide starting material therein ^■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. In modified embodiments, 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. Other objects and purposes of this invention will be apparent to persons acquainted with apparatus of this general type upon reading the following specification and inspecting the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

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

OM valves of a valve unit 203 to a reaction vessel or vessels 35 in which actual peptide synthesis takes place. Waste products removed from the fluid transfer lines connecting the reagent containers and valves of the units 201 and 203 and the reaction vessel 35 are routed by further valves of the valve unit 203 to a liquid waste ^"unit W. It is primarily by manipulation in appropriate sequence of the valves of the valve unit 203 that synthesis of a desired peptide is accomplished and

10 the valves of such valve unit 203 are controllable by a control unit 206. 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

20 known to utilize a microprocessor or other control to control valves in a peptide synthesizing apparatus, for example in Verlander et al U.S. Patent No. 4 362 699. TOP EMPTYING REACTION VESSEL - FIGURE 2 Figure 2 discloses a preferred embodiment of a peptide synthesis apparatus according to the present invention. In Figure 2 are provided 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.

30 through A for acids, containers AA, through AA for amino acids (selected ones of which are to be coupled in the reaction vessel 35 to form the desired peptide) , and containers C, through C for coupling reagents, such as

OMPI -8-

N_fN'-dicyclohexylcarbodiimide (DCC) and 1-hydroxybenzotriazole (HoBt) . The reagent containers S, through C are sealed except for connections hereafter described. A pressurized inert gas supply N., for example of nitrogen gas, supplies an inert gas pressure line 1.

Associated with each of the reagent containers S, through C are a pair of two-position, three-way (three-port) valves having alternate positions respec- tively shown in solid line and dotted line. For example, 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 valve 5, in its solid line position, connects the interior of container S, through the solid line position of a series of similar valves 11, 21, 26...48 for supplying reagent from container S. (hereafter simply reagent S,) to reaction vessel 35 and in its dotted line position closes its connection to the container S. and instead supplies inert gas from source N- via an opened two-way (two-state, two-port) valve 51 through the series of valves 11, 21, 26...48 to supply inert gas under pressure to the reaction vessel 35. Interposed in the line 4 between the container S, and

O its reagent-gas supply valve 5 is an adjustable flow restricter R.

In a similar manner, 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 . In other words, 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. Thus, 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.

The above-described portion of Figure 2, 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

OMP

' outflow line, reagent-gas supply valve, and associated dryer tube and restricter, for example as at 2-5, with associated elements D and R. It will be seen that the 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. The two-position, three-way valves 5...48 and

2...45 of the reagent selection assembly 212 may for convenience have their alternate positions defined as an "open" position and a "closed" position. In its "open" position each pressuring and venting valve (for example 2) opens the nitrogen supply line 1 to its corresponding reagent container (for example S,) to pressurize the latter. In its "open" position each reagent-gas supply valve (for example 5) connects the corresponding thus- pressurized reagent container (for example S,) to the line 6, 54 and the reaction vessel 35. In other words, 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. Correspondingly, in their "closed" positions, such valves block reagent supply from their particular reagent container to the line 6, 54. In effect then, the reagent-gas supply 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.

OM Accordingly, depending on the condition of the valves in the reagent selection assembly 212, same will provide, on the line 54, a flow of one of the reagents S....C or a flow of nitrogen gas under pressure, or the line 54 will be quiescent, substantially at atmospheric pressure.

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. Thus, 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. If then the 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. Alternatively, if the 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. In the Figure 2 embodiment, 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. In the particular embodiment shown, 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. At the top T of the reaction vessel, 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. As to the latter, the particular reaction vessel, assembly 218 shown in Figure 2 is arranged as a bottom loading, top emptying reaction vessel.

To facilitate emptying, the reaction vessel 35 is here provided with a conventional 180° rotation device (inverting device) 223. Here the 180° rotation device supports the agitator 221 which in turn supports the reaction vessel 35. To permit such agitation and 180° rotation, 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. To empty the reaction vessel, 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

OMPI alternative dotted line position of the valve 15 into the waste container W.

All the valves in the assemblies 212 and 218 are preferably electrically controlled, for example conven¬ tional solenoid controlled valves.

The operation of the Figure 2 embodiment has been generally indicated above but can be further illustrated by the following example of a procedure for synthesis of a relatively simple generalized peptide. EX.AMPLE 1

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:

1. Prewash the line 6, 54. To do this, the valve 2 is opened so nitrogen pressurizes wash solvent container S, through line 1, 3. Then valve 5 is opened and cleaning solvent S, travels through the line 4 and then through valves 5...48 interposed in line 6, 54. 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. Then 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.

2. 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.

(a) Valve 7 is then opened and nitrogen pres- surizes the reagent container S₂« The valves 11, 13, 14 are then opened so reagent S₂ 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 S₂ to vessel 35, valves 7 and 11 are "closed" to shut off flow of solvent S₂ to vessel 35.

(b) 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 S₂ is vented at D. The pressurized gas performs premixing and line drying functions in that the pressurized gas violently propels the solvent S₂ from lines 6, 54 into the reaction vessel 35 to enhance mixing of the solvent S₂ with the existing material in reaction vessel 35 and the gas empties the lines 6, 54.

3. Mixing. First, 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.

4. Empty reaction vessel 35. The inverting device 223 is actuated so that vessel 35 inverts 180°. Valves

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.

5. Restore reaction vessel 35. Inverting device 223 is reverse actuated and the reaction vessel 35 inverts back to its original upright position. Valves 51, 13, 14, 15 are -closed (closed valve 15 venting the top of vessel 35 to the drying tube D) . 6. Steps 2, 3, 4 and 5 are repeated several times (e.g. 4 or 5 times) .

7. Line wash. Step 1 is repeated.

8. Deprotection. (Removing protecting group, in the present example the t-Boc group, making the NH of amino acid available for coupling to a next amino acid .AA, to be supplied hereafter.) 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.

9. Mixing. 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.

10. Empty and restore. Steps 4 and 5 are repeated.

11. Line wash. Step 1 is repeated.

12. Resin wash. Steps 2, 3, 4, 5, and 6 are repeated.

13. Line wash. Step 1 is repeated. 14. 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.

15. Base wash. Steps 2, 3, 4, 5 and 6 are repeated. Some amino acids will require an additional step at this time before coupling step 17 hereafter, and for these repeat steps 2, 3, 4, 5, and 6 using an appropriate different solvent, e.g. S .

16. Line wash. Step 1 is repeated.

17. Coupling. At this step 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) , is transferred to reaction vessel 35 after valve 37 is opened and nitrogen pressurizes container C, , and valve 40 is then opened to transfer the DCC to the reaction vessel 35 by way of lines 39, 6 and 54 through valves 40, 48, 20 and 13. Then valves 37 and 40 are closed. Then step 2b is repeated.

18. Mixing. 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.

22. Resin wash. Step 20 is repeated.

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.

EX.AMPLE 2 Procedure for the synthesis of the specific peptide Boc-Phe-Gly-Resin wherein the final product is Phe-Gly, namely phenylalanylglycine, wherein Phe represents phenylalanine, and Gly represents glycine.

0 11 As a preliminary step, Boc-Gly-C-O-Merrifield peptide resin (polystyrene divinylbenzene resin) is prepared by the well known standard method.

Then the Figure 2 apparatus is sequenced through the steps of Example 1 with the following specifics or modifications.

I. The Boc-Gly-Resin starting material is placed in reaction vessel 35 in a conventional manner.

II. Resin wash. Step 2 is carried out with S, and S, is CH₂C1₂ ( ethylene chloride) .

III. Mixing, empty and restore. Steps 3, 4 and 5 are carried out with mixing step 3 timed at 1 minute each time.

IV. Repeating. Step 6 is carried out 4 or 5 times. V. Deprotection. Step 8 is carried out wherein A^_^ is 40% trifluro-acetic acid in CH₂C1₂. A first mixing cycle lasts 5 minutes and a second cycle lasts 30 minutes.

VI. Line washing. Step 11, like Step 1, uses

CHnClaa, .

VII. Resin wash. Step 12 uses CH₂C1₂ for 1 minute ^"each time for 5 times.

VIII. Neutralization. Step 14 is carried out for 2 minutes 2 times.

IX. Base wash. Step 15 washes the base from the resin using CH₂C1₂ for 1 minute each time for 3 times.

X. Further resin wash step to wash the resin to remove CH₂C1₂, using diraethylformanide (DMF) . (This repeats step 2 except using reagent container of DMF - see note at end of step 15.)

XI. Coupling. Step 17 is carried out except AA, is Boc-Phe and the coupling reagent is C₂, namely 1-Hydroxybenzotriazole (and hereafter HoBt) . The valve 45 is opened and nitrogen pressurizes the C₂ (HoBt) con¬ tainer. Then 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. Then, valves 45 and 48 are closed. Then step 17 is repeated using DCC. The time for coupling may be 15 minutes - 2 hours.

XII. Resin wash. Step 20 is carried out using as the solvent CH₂C1₂ for 1 minute each time for 3 times.

XIII. Further resin wash (washing the side product) Step 21 is carried out using ethyl alcohol. At this point, if 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. At this point, for double coupling the latter above- listed steps of Example 2 will be repeated from step VI of preparation of Boc-Phe-Gly-Resin to step XIII. In the general and specific Examples 1 and 2 immediately preceding, it will be noted that 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,, S₂, 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.

BOTTOM EMPTYING REACTION VESSEL - FIGURE 2A 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. The line 17 of Figure 2 connecting the upper

(normally closed) port of 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.

Operation in Figure 2A is like that above described with respect to Figure 2 except that fluid (gas and liquid) from line 6, 54 passes additionally through the valve 49 to enter the bottom of reaction vessel 35. Also, to empty excess reagent liquid from the reaction vessel 35, the valves 15 and 49 are shifted to their dotted line positions so that pressurized inert gas from line 1 passes downwardly therethrough and through open valve 14, reaction vessel 35, open valve 13 and through the second waste valve 49 so as to drive residual liquid from the reaction vessel 35 through valves 13 and 49 into waste receptacle W. The latter takes place with the first waste valve 20 in its solid line position shown. Therefore, waste fluid from the reaction vessel 35 cannot pass through first waste valve 20 to contaminate reagent supply line 6, 54.

POROUS MULTI-CHAMBER VESSEL - FIGURE 3 Figures 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

OMPI chambers, such chambers preferably being identical. More particularly, 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. glass frit) which allows gas and liquid to pass freely therethrough, but blocks passage of particles (in this embodiment, particles larger than, for example, 0.060 mm) , to maintain separated in the chambers 235 and 235' the resin particles P above described, on which respective peptides are to be formed. When the reaction vessel 35B is inverted (top T down and bottom B up as in Figure 3A) the nonporous part 237 of the divider prevents flow of both particles and fluids between the two chambers 235 and 235^» .

At the top T of 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. For initial loading of resin particles carrying an initial amino acid AA_Q into the chambers 235 and 235*, and for subsequent removal from such chambers of the peptide synthesized on such resin particles, 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.

In Figure 3, 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'.

To drain the vessel 35B in its upright Figure 3 position, 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. In this manner, 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.

OMPI In addition, 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. However, 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'.

To this end, such inversion of vessel 35B to its Figure 3A position drops the resin particles P onto the filters 33 and .33' at the now depending tops T of the chambers. Then, 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. Optionally, line 6, 54 may be blown dry with pressurized gas N₂ through the same path. Then, 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

OM e λ , IP rightward chamber 235'. In this way, two entirely different reagents can be added nearly simultaneously (one after the other) in the same process step, and hence in the same amino acid location in the peptide chain to simultaneously form similar but nonidentical peptides.

To expel leftover reagent from the chambers 235 and 235', 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„ 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. This drives the excess reagent liquid downward out of the bottom B of the vessel 35B through line 226, valves 13 and 262, line 261, solid line positioned valve 256' and dotted line positioned valve 249 to waste W.

Thus, for most of the synthesis of a pair of peptide analogs, 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'. Thus, 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. Thus, instead of two peptide analogs requiring twice the number of days to prepare with one reagent selection assembly 212, two peptide analogs are prepared in very little more time than one peptide analog, using Applicant's Figure 3, 3A and 4 apparatus.

As above discussed with respect to line 12 in Figure 2, it is preferred that 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.

SOLID WALL MULTI-CH.AMBER VESSEL - FIGURE 9 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. In Figure 9, 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) . With

OMPI i/Λfi.. WIPO the reaction vessel 35C in its upright position shown in Figure 9, reagent entering the bottom B of the vessel through valves 262 and 13 will divide equally between the two chambers 235C and 235C due to the identical cross-section of the two chambers and the identical nature of the portions of the filter 32 at the bottom of each such chamber.

The connections of the top T of the chambers 235C and 235' to the reagent selection assembly 212 (shown in Figure 2) may be identical to the corresponding connections described above with respect to Figures 3 and 3A. However, Figure 9 shows a further modification in that connection which may be used in the Figure 3, 3A embodiment as well. More particularly, in Figure 9, 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*.

Operation of this Figure 9 valve arrangement is the same as discussed above with respect to Figure 3 and 3A except for the following. To empty the reaction vessel 35C in its Figure 9 upright position, 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.

In both Figures 3 and 9 line cleaning with solvent

(e.g. solvent S,) and inert gas blow dry of line 6, 54 exhaust through the solid line position of valve 256, line 257, the dotted line position of valve 20, and thence through line 18 to waste W.

INDIVIDUAL VESSELS - COMMON REAGENT SUPPLY - FIGURE 10

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

OMPI as at 238C in Figure 9, the Figure 10 modification employs a multi-chamber peptide reaction vessel 35D which may be considered a plurality (here for example two) of isolated individual reaction vessels 35D and 35D* of single-chamber type, in which the individual chambers 235D and 235D' are devoid of any internal connection to each other. Instead, each chamber 235D and 235D¹ 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. In the embodiment shown, the 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. As in Figure 2, 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. However, unlike in Figure 2A, 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. As a further difference, 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'. Thus, depending on the settings of the valves 271 and 271', the same reagent or gas flow can be simultaneously admitted to both chambers 235D and 235D'. Alternately, by opening just one of valves 271 and 271' 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. For line washing, 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.

Thus, again 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. Again, 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.

/^' OMPI

^ _f

•_* '

^■m-:^' ϊ->'r⁴ 7 The valves 271 and 271' permit different amounts of reagent to be added to the respective chambers 235D and

235D', if desired.

INDIVIDUAL VESSELS - FLIP-FLOP CONNECTION - FIGURE 11

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. As in the above-discussed figures, 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.

If some unaccounted for asymmetry in the path from line 54 into each vessel 35E and 35E* should result in different amounts of reagent liquid thereinto at a given time period, such asymmetry can be compensated for by feeding reagent to vessel 35E for a first time period and then to the vessel 35E' for a second time period, with the time period lengths being adjusted to provide the compensation and assure entry of equal amounts of reagent into both vessels. This may be_. ccomplished by the three-way "flip-flop" valve 283 at the "T" 276 as shown in Figure 11. The same effect can be accomplished by omitting the flip-flop valve 283 and opening the valves 13 and 13' for nonoverlapping timed intervals. If the two-position valve 283 is present, 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. Thus, 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. MECHANICALLY PUMPED SUPPLY AND RECIRCULATION - FIGURE 8

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. In Figure 8 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. However, 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.

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. Thus, after such filling of reservoir 311 to the desired level, 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.

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.

If desired, 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.

To wash the line 54 free of such reagent, a cleaning reagent, for example solvent S,, can be pumped through line 6, 54, solid line positioned valve 315, pump 301 and dotted line positioned valve 20 into waste W. In general, it would not be necessary to blow dry the line 6, 54 thereafter, but if such is desired, 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.

In Figure 8, 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. RESERVOIR LEVEL SENSING - FIGURE 7

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. Instead of a microprocessor 207 in Figure 7, a simple control relay can be substituted to turn off the pump 301 or valve 315.

PLURAL COLUMNS - COMMON SUPPLY - FIGURE 6

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.

In a particular embodiment shown in Figure 6, the 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

304-304'', columns 307-307^•', waste valves 310-310'', and reservoirs 311-311^•'.

The Figure 6 assembly 218GG provides for the following alternatives in loading and circulating reagents:

1. Sequentially load three separate reagents (e.g. AA , AA and AA ) into respective reservoirs 311-311' and then simultaneously circulate them through respective columns 307-307" ' . 2. Sequentially load two different reagents (e.g.

.AAx and AAy) into any two reservoirs (e.g. reservoirs

311 and 311'') and then simultaneously circulate one reagent AA in reservoir 311 through columns 307 and

'$TF 1Λ

OMPI ^ WIPO 307' while the different reagent AA in reservoir 311' * circulates through its own column 307' '.

3. Load only one reagent (e.g. AA ) into only one reservoir (e.g. reservoir 311) and circulate it through all columns 307-307'* in series through its own column 307 and one other (307* or 307'') or through only its own column 307.

4. Load only one reagent into all reservoirs 311-311' and simultaneously circulate the contents of

10 each reservoir only through its own column.

If desired, 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. However, in contrast to Figure 8, and for purposes of illustration of a further modification, the reaction vessel assemblies 218G-218G* * instead utilize their pumps 301-301'' solely

20. for recirculation of reagent through the columns 307-307* ' and reservoirs 311-311' * and a reagent is supplied via line 54 by nitrogen gas pressurizing of line 1 in assembly 212 as described above in connection with Figure 2.

Also in contrast with Figure 8, the Figure 6 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''. To supply a given reservoir (e.g. 311) with a

30 reagent (or nitrogen gas) from line 54, such fluid is routed through the solid line position of any upstream supply valves (here 20' ' and 20^•) through the dotted line position of the corresponding one (e.g. 20) of the

-gΪ E

OMP series valves 20-20^{• •} into such reservoir 311. Then 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. Then more reagent (different or the same) 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*'. Thus, 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.

In each assembly 218G-218G* ' (e.g. 218G) of Figure 6 , 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.

To empty such a recirculation loop 301, 307, 311 of leftover reagent after a synthesis step, 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

- TJ E

OMPI m λr--_*. WIPO of such liquid through the column 307, waste valve 310, and waste line 308 to waste W.

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.

First, consider alternative 2. For example, 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. Thus, 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".

The 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*.

As a further example, 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. Thus, liquid from reservoir 311 is circulated through columns 307 and 307* '. Simultaneously therewith, 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.

As a further example, 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'. Simul¬ taneously, a different reagent may circulate in the loop 301, 307, 311.

Under alternative 1, liquid from reservoir 311 is circulated by pump 301 through column 307 and also through columns 307' and 307" in series back to reservoir 311. Reagent circulation would thus be from reservoir 311 through pump 301, column 307, dotted line positioned valve 331, solid line positioned valve 332, dotted line positioned valve 334, column 307, dotted line positioned valves 351 and 354, solid line positioned valve 343, column 307*', dotted line positioned valve 331", solid line positioned valve 346 and a line 347 back to reservoir 311. Thus, Figure 6 provides for simultaneous prepara¬ tion of three identical peptide batches or up to three different peptide analogs in which some steps require application of the same reagent to all columns and other steps require application of different reagents to different columns.

Thus, at any point in the synthesis where the peptide analogs being formed in columns 307-307" are to be treated with individualized (differing) reagents, either 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. By providing one pump column, the individualized reagents can be simultaneously circulated in their corresponding pump-column loops.

PREACTIVATION OF AMINO ACID WITH COUPLING REAGENT PRIOR TO REACTION VESSEL - FIGURE 13

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. Thus, by reacting of a given amino acid and coupling reagent simultaneously with earlier steps of the generalized method set forth in Example 1, 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.

Also, such reacting of the amino acid and its coupling reagent in the container 422 permits close ' temperature control during the reaction by controlling (in conventional manner) the temperature of container 422.

Also, 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.

Also, 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.

Also, 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.

Accordingly, the Figure 13 modification will speed synthesis of a peptide, especially a long chain peptide, and produce a higher quality peptide product.

It will be understood that the Figure 13 modifica- tion is described in terms of an amino acid A. and coupling agent C, by way of example only and that various amino acids and coupling reagents may be utilized In the particular example shown in Figure 13, 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

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

425 and 427 into the top of preactivating container 422. 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.

In the embodiment shown, 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. If the reagent containers .AA, and C, are not of pressurizable type, but instead are to be mechanically pumped, as above discussed with respect to Figure 8, suitable pumps (not shown) may be interposed in the lines 425 and 427. Once the desired amount of reagents AA, and C, has been supplied to the container 422, 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. In the embodiment shown, the pressurized source N₂ 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. If desired, and to prevent premature draining of the container 422 into the line 434, 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. To supply the active ester in container 422 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

OMPI W WIIPPOO ftN l ester therein downward through filter 438, valve 436, line 434. valve 433, line 441 and the valve 442 (in its dotted line position) , into the portion of line 6H downstream (to the right) of valve 442 and thence through line 54 toward the reaction vessel 35.

Further 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.

Where the 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. For example, in addition to controlling the peptide synthesis, 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. Although operation of the above-described apparatus has been discussed, by way of example, in terms of synthesis of one or more peptides or proteins on insoluble polymeric supports, the same apparatus could be used for other purposes, such as a nucleotide synthesizer, and amino acid analyzer, a protein sequencer, or for other types of chemical processes. The sequential arrangement of liquid containers S,...C in assemblies 212 and 212F, which reduces cross-contamination, and the arrangement of assemblies 212 and 212F in combination with the downstream assemblies 218-218G which provides the capability for draining and flushing the entire liquid system, further reduces cross-contamination and is particularly suited for automatic control of peptide synthesis. 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.

Further as to Figure 1, it is contemplated that 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) . To monitor the temperature, 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. Alternately, 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. As with temperature control, 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.

It is contemplated that the 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. The above-described embodiments have considerable advantageous characteristics in terms of liquid transfer. All of the foregoing embodiments provide for periodic delivery of discrete quantities of reagents to the reaction vessel (S) as above described with respect to Figures 2 , 3, etc., which permit control of valves in various ways, including by microprocessor control, wherein self-cleaning of fluid lines is provided and wherein is provided the ability to perform repeated couplings, multiple cycles, and even simultaneous multiple syntheses and wherein the number of reagents, for example amino acids, is virtually unlimited. Further, and particularly as demonstrated by the Figure 6 embodiment, it is possible to provide continuous pump-driven recirculation of reagents through a loop including a reaction vessel and reagent replenishment reservoir. In each of the above-disclosed embodiments, 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.

ΓRIX

OMPI

«fc WIPO $NATl Although particular preferred embodiments of the invention have been disclosed in detail for illustrative purposes, it will be recognized that variations or modifications of the disclosed apparatus, including the rearrangement of parts , lie within the scope of the present invention.

Claims

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. Apparatus usable for peptide synthesis and the like, comprising: a reaction vessel; a plurality of supply valves connected in series in a supply line, said valves each being positionable in a first position for fluid flow therethrough from one end of the line to the other; a plurality of reagent containers for containing respective reagents, said supply valves each having an alternate second position in which each connects a corresponding reagent container through the portion of said supply valve line downstream thereof and dis¬ connects said downstream portion of said line from the- portion of said line upstream thereof; means for connecting the downstream end of said line to said reaction vessel; means responsive to shifting of a said supply valve to its second position for moving reagent liquid from the corresponding said reagent container through the portion of said supply valve line downstream of said second positioned valve toward said reaction vessel; whereby ones of said reagents in sequence can be moved from their respective containers through their respective supply valves downstream along -the line of supply valves toward said reaction vessel for entry into said reaction vessel.

2. The apparatus of Claim 1 including waste outlet means, said means connecting the downstream end of said

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OMPI line to said reaction vessel including means alternately connecting the downstream end of said line to said waste outlet means and flushing means adjacent the upstream end of said line of supply valves for flushing leftover reagent liquid from said line to said waste outlet means so as to avoid contamination, by said leftover reagent, of another reagent subsequently passed through said line of supply valves to said reaction vessel.

3. The apparatus of Claim 2 in which said flushing means comprises a source of inert gas under pressure and an on/off valve actuable for connecting said gas source to the upstream end of said supply valve line for- emptying said line, with said supply valves in their first position, to said outlet means.

4. The apparatus of Claim 1 including a plurality of pressurizing and venting valves each having a first position for venting a corresponding said reagent container and an alternate second position for pressurizing the corresponding said reagent container from said pressurized gas source, such -that with a said supply valve and the corresponding pressurizing and venting valve in their second positions, reagent liquid from the corresponding reagent container is supplied through the downstream portion of said supply valve line and the downstream ones of said supply valves in their first positions to flow toward said reaction vessel.

5. The apparatus of Claim 1 including a pump interposed in series with said supply valve line and reaction vessel and responsive to shifting of a given said supply valve to its second position for pumping

OMP reagent from the corresponding reagent container through the downstream ones of said supply valves in said supply valve line and thereby supplying said reagent to said reaction vessel.

6. The apparatus of Claim 5 in which said reaction vessel is a column, and including an overflow reservoir and means connecting said pump, column and overflow reservoir in loop for drawing of reagent liquid from said reservoir through said pump and column and back into said reservoir for circulation by said pump through said column for reacting of said reagent with material in said column.

7. The apparatus of Claim 3 in which said reaction vessel is connected as a top emptying reaction vessel, said means connecting the downstream end of said supply valve line to said reaction vessel comprising waste valve means shiftable to connect said supply valve line alternately to the bottom of said reaction vessel or to said waste outlet means, means mounting said reaction vessel for inverting to place the top thereof downwardly, valve means alternately shiftable to vent the top of said reaction vessel through a drying tube during reagent flow into the bottom of said vessel, the latter valve means being alternatively shiftable to a position connecting the top of the reaction vessel with the waste outlet means for emptying the inverted reaction vessel, said supply valves all being shiftable to their first position and said on/off valve being openable for flowing of pressurized gas through said supply valve line and into the bottom of said reaction vessel to

OMPI WIPO drive residual reagent liquid therefrom to said waste outlet means.

8. The apparatus of Claim 3 in which said reaction vessel is a bottom opening reaction vessel, said means connecting the downstream end of said supply valve line ^"to said reaction vessel comprising waste valve means alternately positionable for forwarding reagent liquid into the bottom of said reaction vessel or flushing said supply valve line to said waste outlet means to clean and ready same for a new reagent and to drain leftover contents of said reaction vessel to said waste outlet means, venting valve means alternately actuable for venting the top of said reaction vessel or connecting the top of said reaction vessel to receive pressurized gas for blowing leftover reagent liquid in said reaction vessel through said waste valve means to said waste outlet means.

9. The apparatus of Claim 1 including a plurality of said chambers a said reaction vessel having at least one said chamber, said means connecting the downstream end of said line to said reaction vessel comprising valve means actuable for connecting several of said plurality of chambers at least alternately to a single said supply valve line, said valve means being actuable to at least alternately supply reagents to said chambers.

10. The apparatus of Claim 1 in which the order of arrangement of reagent containers from the upstream end of said supply valve line to the downstream end of said supply valve line adjacent said reaction vessel is cleaning reagents, bases, acids, amino acids and coupling reagents.

11. The apparatus of Claim 1 in which said reagent containers include at least one amino acid container and at least one coupling reagent container, a preactivating container, transfer valve means for applying a first amino acid and coupling reagent from corresponding reagent containers to said preactivating container, means actuable for reacting said first amino acid and first coupling reagent in said preactivating container, said plural supply valves including at least one activated amino acid supply valve in series in said supply valve line, and outlet means actuable for supply¬ ing thus activated amino acid from said preactivating container through said last-mentioned valve to said supply valve line and hence to said reaction vessel, filter means associated with said outlet means for blocking escape from said preactivating container of solid size products resulting from said reacting.

12. The apparatus of Claim 1 in which said connecting means include filling-emptying valves for alternately connecting said reaction vessel with said supply line for filling and with waste receiving means for emptying, and control means for sequencing said supply valves and filling-emptying valves and thereby for applying reagents from said reagent containers in a desired sequence to said reaction vessel, said valves being free of mechanical interconnection, said supply valves and reagent containers being in one-to-one relation, such that without changing the structure of said supply valves or their location along the supply valve line said supply valves can be sequenced in any desired one of a number of different operating sequences so as to apply the corresponding reagents from corre¬ sponding reagent containers to the reaction vessel in the desired one of a number of possible sequences, including a sequence in which a given supply valve is actuated to apply its corresponding reagent to the reaction vessel several times in interspersed relation with single applications of other reagents.

13. The apparatus of Claim 12 including means for controlling the amount of reagent delivered to the reaction vessel and the temperature of such reaction vessel and means for determining the pH of the contents of said reaction vessel.

14. The apparatus of Claim 12 in which said apparatus is a peptide synthesis apparatus and including a low pressure chromatography apparatus ancillary to the production of peptides, said chromatography apparatus including a valve for passing reagents through a chromatography column and means for operating said valve from said control means.

15. Apparatus usable for peptide synthesis or the like comprising: reaction vessel means including a plurality of reaction chambers; waste receiving means for receiving leftover liquids from said reaction chambers; a source of reagents and a supply line for supply¬ ing reagents from said source to said plural reaction chambers; valve means actuable for connecting said supply line to (1) said plurality of reaction chambers for supplying reagent liquid thereto and (2) said waste receiving means for flushing residual contents of said supply line to waste, said valve means including individual valve paths to each said reaction chamber and being actuable for supplying a first reagent from said supply line to a first said reaction chamber and there¬ after supplying a second reagent from said supply line to a second said reaction chamber and alternately actuable for supplying the same reagent from the supply line to said first reaction chamber and to said second reaction chamber.

16. The apparatus of Claim 15 in which each reaction chamber has a top and a bottom, means fcr inverting said reaction chambers to place the top thereof downward, said reaction chambers having a common port at the bottom thereof and individual ports at the top thereof, each said port being connected to said supply line through a said individual valve path for supplying a common reagent liquid simultaneously to the bottom of both said chambers with said chambers upright and said bottom downward, a source of pressurized inert gas and means actuable for supplying said gas along a said individual valved path to the port at the top of at least one of said chambers for discharging residual liquid from the bottom of said reaction chambers through the individual valve path thereof to said waste receiving means, said valve paths- to the ports at the top of said chambers being actuable for supplying reagent liquid from said supply line sequentially to said chambers with said chambers in their inverted position such that their tops are downward, said valved paths to the tops of said chambers being alternately actuable for supplying, in succession, different reagents from said supply line to said chamber tops to enable synthesizing of differing peptide analogs in said chambers.

17. The apparatus of Claim 16 in which said chambers are part of a single reaction vessel, the reaction vessel including means dividing same into said chambers, said dividing means being solid and impervious to liquid transfer therethrough at least at the top portion of said reaction vessel and including porous means permitting liquid communication between the bottom portions of said chambers but precluding transfer of solids therebetween.

18. The apparatus of Claim 16 in which said reaction chambers are provided in corresponding reaction vessels and do not directly communicate, a source of

• inert gas under pressure, a said valve path being alternately actuable to communicate the top of said chambers with chamber venting means and with said source of gas under pressure, further said valve paths being interposed between the bottoms of said reaction vessels and said waste receiving means and supply line and being actuable for coupling said supply line to said reaction chambers simultaneously for supplying the same reagent thereto or to individual chambers in sequence for supplying different reagents thereto.

19. The apparatus of Claim 18 in which said valve paths interposed between said supply line and the bottoms of said reaction vessels include a common flip-flop valve actuable for supplying reagent alter¬ nately to said chambers.

20. The apparatus of Claim 15 in which each reaction vessel comprises a column, a reservoir asso¬ ciated with each column, a pump connected in loop with each column and reservoir for circulating reagent liquid from said reservoir through said column and back to said reservoir, said valve means being actuable for supplying reagent liquid from said reagent source through said supply line to a selected said reservoir, said valve means including waste valves interposed in said loops and actuable for draining said loops selectively to said waste receiving means, transfer valves interposed in said loops and actuable for connecting the columns of first and second ones of said loops in series with the pump and reservoir of said first loop for simultaneously circulating a single reagent through several of said columns, said transfer valves being alternately shiftable for limiting circulation of reagent liquid by a given • pump and from a given reservoir to the loop containing said given reservoir and pump.

21. A method for sequentially applying reagents to a reaction vessel and usable for synthesis of peptides, comprising: arranging a plurality of supply valves in series in a supply line connectable at its downstream end to a reaction vessel with its supply valves each having a selectable first position connecting therethrough the portions of said supply line upstream and downstream thereof and a second position connecting a respective reagent container therethrough only to the portion of the supply line downstream thereof, said plurality of supply valves being associated with respective ones of a plurality of reagent containers;

OMP loading said reagent containers with respective differing reagents; loading the reaction vessel with starting material; washing the supply valve line by flushing with a cleaning reagent liquid, including (1) moving the cleaning reagent from its corresponding said container through a said supply valve adjacent the upstream end of said line and downstream through said line to a waste outlet and (2) emptying said line of residual cleaning reagent by applying pressurized inert gas from the upstream end of said line through said line to said waste outlet, the supply valve associated with said cleaning reagent container being in a second position to move cleaning reagent to said supply line and returned to a first position to supply pressurized gas to said supply line; adding a selected reagent liquid to said reaction- vessel by transferring said selected reagent from its container through its corresponding supply valve and the portion of said line downstream of the latter said supply valve to said reaction vessel with the latter supply valve in its second position, and shifting the latter supply valve back to its first position and supplying pressurized inert gas therethrough and through the portion of the line downstream therefrom to drive residual selected reagent in said line into said . reaction vessel and thereby substantially empty said line of leftover selected reagent; mixing the selected reagent thus added to the reaction vessel with said material therein; emptying the reaction vessel of leftover liquid by opening the reaction vessel to the waste outlet and

OMPI IPO cau^'sing residual liquid therein to drain to said waste outlet; repeating the foregoing steps of line washing and emptying, adding reagent liquid to said reaction vessel, mixing and emptying the reaction vessel for several cycles with any desired one of said reagent containers supplying the reagent for a given cycle.