JPS61205298A - Automating polypeptide synthesization appliance - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION Field of the Invention The present invention relates to an apparatus for automatic synthesis of polypeptides, and in particular for automatically preforming activated species of (alpha-amino protected) amino acids immediately before introduction into a solid phase synthesis reaction. Regarding the device.

Since its inception in 1962, R.B. Merrifield's concept of solid phase peptide synthesis has seen numerous improvements and is now an established technology. Hundreds of works have been published describing the chemical details of this method [see, e.g., Merrifield + R, B-5cie.
nce 150.178 (1965): Merrif
ield.

LB: Set, Amer, 218, 56
(1968); Stewart.

J.M., Young, J.D. : 5o
lid Phase Peptide Synthesis
s, San FranciscoLaco, Califo
rnia: Freeman 1969: and Er1
ckson, B.W.

Merrifield, L.B.: The P.
roteins (editor, Neurath, R,L
, Hill) r th edition, Volume 2, 255-527
Page, New York: Academic Pre
ss 1976)].

Typically, solid-phase peptide synthesis involves insoluble resin beads (typically 25 to 300 micron diameter) begins: [F] = removable protecting group resin = synthetic resin The general cycle of synthesis then begins with resin binding - deprotection of alpha-amino groups, washing (and optional reaction with some carboxyl-activated form of the next (alpha-amino protected) amino acid gives: Repeating this cycle up to the nth amino acid gives: At the end of the synthesis, the bond of the peptide to its polymeric support is cleaved and the dissolved peptide is separated from the insoluble resin and purified.

Although this process is simple in principle, in practice it is extremely difficult to obtain peptides longer than about 30 amino acids with any substantial purity.

The reason for this is that the average process yield has a large effect on the product peptide, as exemplified by the values for the synthesis of 30 amino acid peptides in the following table.

Table 30 Amino acid peptide average process yield (a) Product purity (a) 95,0
21 99.5 86 99.7 91 These results become even more problematic for longer peptides. For example, for a target peptide with 101 residues, a process yield of 99.0% gives only 36% pure product.

In all cases, the by-products of peptide synthesis consist of a complex mixture of molecules chemically similar to the target peptide. Chromatographic purification becomes very difficult and time consuming once the relative amount of by-product molecules begins to exceed about 25 molecules.

The efficiency of the process yield depends on many factors, such as the nature and quality of the protected amino acid, the purity of the solvent, the chemical integrity of the resin, the chemical nature of the organic linker, the form of the activated carboxyl of the amino acid, the efficiency of the washing process, It will vary depending on the synthetic experimental design and the identity of the amino acids linked to the particular sequence segment to which it is added, as the case may be.

Each of the above factors, if not optimally controlled, will make a significant contribution to yield reduction in each ligation step. At present, the complexity of these factors is such that the average step yield in solid-phase peptide synthesis is typically 93-97% for both manual and automated methods. For the development of substrates and inhibitors, hormones, vaccines, and diagnostic reagents, etc., such low process yields considerably increase manufacturing costs and often require direct solid-phase synthesis of such peptides. make it impractical.

Prior art peptide synthesizers automate the tedious liquid operations of deprotection, coupling agent addition, and washing.
It essentially operates as a "cleaning machine". In any case, existing commercial peptide synthesizers do not form activated amino acid species outside or independently of the reaction vessel. Typically, the protected amino acid and DCC are added to the reaction vessel containing the resin-bound initial peptide chain such that activation of the amino acid occurs in the presence of the deprotected alpha-amino group.

This method limits the possibility or feasibility of optimizing the activation conditions for individual amino acids and any modification of the activation conditions in the presence of the deprotected alpha-amino group and the growing resin-bound peptide chain. need something to be done. This fact makes optimization of activation parameters by analyzing the formation rates and relative thermal and solvent stabilities of individual activated amino acid species difficult, if not impossible. Furthermore, various thermal inputs during the activation process can only be used in the presence of peptide chains.

9 Beans 1 According to a preferred embodiment of the present invention, an apparatus is provided for automatically constructing highly pure polypeptides up to 50 amino acids in length using only a single linkage. The device includes an activation system for receiving one protected amino acid at a time and for activating each amino acid in the order in which it is received to form an array of aliquots of the activated species for each amino acid. ), each aliquot containing one type of amino acid, and the sequence of the aliquots of each type of amino acid in the desired order within the peptide. Also included are reaction vessels for containing resins used in solid phase peptide synthesis to attach peptide chains. Also provided is a transfer system operated under computer control for transferring active species from the activation system to the reaction vessel and for transferring amino acids, reagents, gases and solvents from one part of the apparatus to another. be done. This activation system also allows the activation solvent used in the activation vessel to be substituted with the coupling solvent to generate the activated species of the amino acid, thereby allowing the coupling of the activated species to the peptide chain in the reaction vessel. It also includes a temperature-controlled concentration vessel. This substitution is carried out by adding the coupling solvent together with the activated species and the activated solvent into a concentration vessel, and purging the resulting vessel with gas to selectively evaporate the activation solvent. This is achieved within a short period of time (typically less than 30 minutes) before introduction into the reaction vessel, and the activation solvent is chosen to have a boiling point lower than that of the coupling solvent. The condenser is heated as necessary to replace heat lost through evaporation.

This synthesizer system includes a vortex stirrer that affects the total cleaning of the materials in the reaction vessel and the reaction vessel itself, an automatic peptide resin sampling system, and an automatic peptide resin sampling system for providing individual containers of amino acids to the synthesizer in the desired order of the peptide sequence. This includes automatic transportation systems. Also, special containers are provided for use in automatic transport systems with a V-shaped bottom to allow extraction of as many amino acids as possible allowing for precise control over the stoichiometry. Also included is a liquid sensor system for monitoring transitions between gas and liquid in special tubes in the synthesizer to provide input signals to the computer system for control purposes.

Computer system that controls the operation of the synthesizer 7
The software is organized according to a series of menus that allow the user of this system to select individual operating cycles for each container in the synthesizer. Additionally, each cycle can be determined by the user during a series of functions, each of which corresponds to a combination of standard instructions for individual valves and other switch systems associated with the synthesizer. This menu system also allows the user to determine alternative individual functions.

In addition to this menu-driven control system, algorithms were developed that involved organizing the computer software into individual cycles for each container. Operating the synthesizer according to this algorithm provides optimal efficiency of peptide production for any given cycle selection.

DETAILED DESCRIPTION OF THE INVENTION Before describing the details of the apparatus of the present invention, it is useful to first understand the chemistry of solid phase peptide synthesis that the apparatus is designed to optimize.

For the purposes of this invention, the preferred mode of synthesis occurs primarily in three phases. The first or activity ratio phase involves the production of (alpha-amino protected) amino acid symmetric anhydrides as acylated strains: O R = various amino acid side chain symmetric anhydrides whose coupling occurs in the absence of base. Virtually no racemization and quantitative for most amino acid additions with the exception of asparagine, glutamine, and arginine, each of which is more effectively coupled by the alternating activation method described below. The activated carboxyl form of the amino acid is highly effective since a single coupling is usually ensured. The generally quantitative nature of couplings with symmetric anhydrides makes them most useful where automated synthesis precludes convenient stepwise quantitative follow-up. The apparatus described in Section 5 automatically synthesizes a symmetrical anhydride immediately before introduction into a peptide chain. Due to the limited stability of symmetrical anhydrides and the difficulty of isolating them in pure form, their use in the past has been limited to manual preformation followed by introduction into automatic synthesis machines.

The preformed symmetric anhydride (PSA) synthesis procedure involved 0.5 equivalents of dicyclohexylcarbodiimide (DCC) and 1.0 equivalents of dicyclohexylcarbodiimide (DCC).
An equivalent amount of the protected amino acid was added to dichloromethane (D
dichloromethane is used especially when the alpha-amino protecting group P is t-
When a butyloxycarbonyl group (t-noc) is present, it is the best solvent for the synthesis of PSA.

However, the DCU formed in the reaction is highly insoluble in dichloromethane and precipitates during the PSA reaction. After the reaction is complete, the PSA/DCM solution is separated from the DCU precipitate and a second or concentration step is initiated. In the concentration step, DCM is removed and replaced with a polar neutral solvent, preferably N,N-dimethylformamide (DMF), to increase the coupling efficiency during the subsequent solid phase reaction. The general reaction scheme described in the background of the invention is then followed by a third or reaction step, whereby an additional amino acid is attached to the sequence where the carboxyl terminus of the PSA is reacted with the alpha-amino deprotected resin-bound peptide chain. .

Apparatus In accordance with a preferred embodiment of the present invention, the apparatus for accomplishing the above synthesis is illustrated in FIGS. 1A, 1B, 2 and 3, each containing various amino acids, reagents and solvents. , and a liquid transport system for transporting gases into the device,
A computer system to automatically control the valves, sensors, and numerous switches that control the temperature of certain containers and motors within the device, and to deliver the protected amino acids to the device in the desired order in the peptide sequence. The figure shows an automatic transportation system.

The liquid transport system includes three primary containers. That is, P.S.A.
an activation container 11 in which a PSA from the activation container is formed;
a concentration vessel 13 into which the /DCM solution is transferred such that DCM is replaced by DMF to enhance coupling, and a reaction vessel 15 containing the growing resin-bound chain.

Activation vessel 11 is typically about 40-volume cylindrical and preferably constructed of glass to allow the operator of the apparatus to visually monitor the progress of the reaction or wash cycle. At the bottom of the activation vessel is a coarse diameter glass frit 17 that is used to filter the DCU precipitate from the solution when transferring the PSA/DCM solution to the concentration vessel 13. Activation vessel 11 also contains an overhead nozzle 19 pointing upwards to achieve total cleaning of the head space and walls after each amino acid is transferred out of the vessel. Activator 11
No. 4,008,736 (issued February 22, 1977, Title: Valve Arrangement for Liquid Dispensing, Wi.
valve block 23, which is an assembly of zero dead volume valves as described in ttman-Liebold et al., as well as an automated conveyance system and various gases and reagents as shown via any and all valve blocks in the system. connected. Valve block 23, like all other valve blocks and gas blocks in the system, is operated under computer system control. Activator 1
1 controls the flow of methanol and DCM into the container to dissolve and clean the DCU precipitate through the nozzle 19, and controls the pressure in the container to allow substances to enter and exit from the block valve 23. It is connected to another valve block 25. Transfers from the bottom of the container take place via translucent tubes 29, typically made of Teflon, and these transfers occur through tubes 29 between gas and liquid.
A liquid sensor 27 that detects transitions in the fluid is monitored by the computer system. Typically tube 2
9, and other tubes in the system to which similar liquid sensors are attached, have a roughly calibrated flow resistance and operate at a constant, known pressure upon displacement, resulting in the time required for displacement. The length of corresponds directly to the volume of material transferred. DC requiring special capacity
For C and HOBT, calibrated transport loops are used to achieve a higher degree of accuracy. Thus, the computer system can accurately monitor all flows into and out of the activation vessel. Details of the liquid sensor will be discussed later, but it is important to emphasize that the use of a liquid sensor is not required for operation of the device.

However, they are useful for achieving better system control.

The next important part of the liquid transport system is the concentrator vessel 13. Its construction is substantially the same as the activation vessel 11, including an overhead nozzle 31 and a glass filter frit 35 at the bottom.

However, in addition the concentration vessel is also P S A/D 0M
It has an attached band heater 37 which is used to control the temperature inside the vessel using a thermistor when the DCM in the solution is replaced with DMF.

The concentrator 13 includes a valve block 33 and a liquid sensor 4.
A translucent moving tube 39 is attached which is monitored by 0. The movement through the tube 39 is the valve block 4
1 is controlled by a computer system.

The next main part of the liquid transport system is the reaction vessel 15,
In a preferred embodiment, it is a vertically oriented perfect circular cylinder and is constructed of a machined fluorocarbon polymer such as TeflOn or KEL-F. This container has a valve block 4 at the top.
3 and at the bottom by valve blocks 45, each valve being separated by a filter such as membrane 47 and membrane 49, which membranes are typically
TEX (manufactured by Chemplast, New Chassis)
glass frit can also be used. The reaction vessel is designed to be conveniently opened for both initial loading of loaded resin and periodic removal of sample aliquots. This is accomplished by threading the top and bottom of the cylinder of the reaction vessel to fit the threaded cap. Threaded caps are also used to hold the membranes in place, each cap being configured to receive a tube for transferring liquid in and out of the reaction vessel. The typical capacity of this reaction vessel can vary widely depending on the length of the peptide chain being synthesized and the weight of the synthetic resin and amino acid loading.

For example, 0 for chains up to 50 amino acid units long.
If starting with 65t of resin (0.5 mmol of amino acids), the preferred size is 40-.

Those skilled in the art of solid phase synthesis will recognize that solvent uptake during synthesis causes the resin to swell by a factor of 3-5.

The increase in mass as a result of peptide chain growth can increase the occupied volume of the reaction vessel from an initial 10% to as much as 80 cm at the end of the synthesis, depending on the length of the peptide.

It is important to agitate the reaction vessel at various stages of the reaction cycle to promote efficient coupling and avoid clumping of the resin beads.

It is also particularly important that all internal surfaces of reaction vessel 15 are thoroughly rinsed during each wash cycle during the addition of PSA from the concentrator vessel. To achieve this agitation, the center of the top of the reaction vessel is held substantially fixed, and the bottom of the reaction vessel is controlled by a motor 48 (pulley) under computer-based control, while the vessel itself is prevented from rotating. connected to an eccentric on the bottom of the reaction vessel) around its central axis at about 150 rpm.
It is moved on a circle with a radius of 3 inches (approximately o, 24z). The result is a conical rotational movement around the longitudinal axis of the liquid resin mixture within the reaction vessel with the appearance of a vortex.

This "vortex" agitation method allows the use of very small incremental volumes of wash solvent for all wash operations, thus distributing spent reagents and reagent by-products from synthetic resins sequentially during removal of these materials. is much more efficient than single extraction, greatly improving its removal efficiency.

The ability to use small volume increments is a result of the angular momentum of the liquid resin mixture imparted by the conical motion. this"
The "vortex" action creates a liquid distribution in the container as shown in Figure 4, and in (2) the liquid in the reaction container reacts to a very small incremental volume of solvent by selecting an appropriate rotation speed. All internal surfaces of the container can be contacted. As a result, the resin can be washed more efficiently with less expensive solvent.

Furthermore, this stirring system prevents resin agglomeration and allows full liquid-resin interaction without the use of impeller type mechanical stirring. With mechanical agitation, the shear and resin abrasion caused by the impeller will break the resin beads into progressively smaller particle sizes, eventually clogging filters such as membranes 47 and 49, forcing an interruption of the synthesis process. There are things to do. Such interruptions can have a negative effect on the synthesis, e.g. by causing flow restriction (out of the reaction vessel) during the acid deprotection step and thus leading to degradation of the resin-bound peptide due to too long acid exposure. Become. There is no impeller type shear or abrasion effect on the resin beads when using a vortex stirrer. In the preferred embodiment, the reaction vessel was configured to have a perfect circular cylindrical shape, but other shapes may be used as long as they do not interfere with relatively smooth swirling of the solution within the vessel. will be understandable to those skilled in the art. For example, a wine glass shape appears to have some desirable properties for cleaning cycles. Also, for maximum efficiency, whereas the apparatus is typically equipped with three reaction vessels 15, only one concentrator 13 and activator 11 are used, the latter two of which Liquid distribution from the two vessels is suitably valved to operate with each of the three reaction vessels and their corresponding valve blocks 43 and 45.

However, each of these reaction vessels corresponds to a sequential process of producing peptides, i.e. a first peptide is formed in a first reaction vessel, then a second peptide is formed in a second reaction vessel. It should be understood that the third peptide is then formed in the third container.

A resin sampler system 51 is provided to track the progress of the synthesis within the reaction vessel. The sampler includes a tube 53 connected into the side of the reaction vessel for withdrawing material from the reaction vessel, and the flow in this tube is controlled by a computer-controlled valve 55, typically a solenoid-operated pinch valve. controlled by. tube 5
3 extends into the bottom of the resin sampler reservoir 5T with a membrane 59 typically located at the top. Further, a tube 61 connected to the valve block 45 is connected to the top of the reservoir 5T on the opposite side of the membrane 59. Valve 67, typically a solenoid operated pinch valve, for collecting fractions from the reservoir from the bottom of the reservoir.
Extending is another tube 65 which is controlled by (to achieve zero dead capacity).

The gas distribution system to achieve the desired movement within the device is
Illustrated in both Figure A and Figure 1B are three manifolds (C1 manifold)'CC.

and a manifold D and an attached controller for controlling the distribution from the bottle and through the valve block. Also included are two smaller manifolds: manifold 70, which distributes DCM into the apparatus, and manifold 71, which distributes DMF into the activation and concentration vessels. It is controlled by a computer system using a side light if' a fluorocarbon bulb (manufactured by Ang ar) and the valve block already described.

FIG. 2 shows a schematic diagram of a computer system consisting of a microprocessor-based microcomputer 81, which includes an arithmetic unit 87, a mass storage device 84 such as a floppy disk, and a high-speed random access memory (RAM). ) 83, a hard copy output device 85, such as a printer, and, in a preferred embodiment, a touch screen 82 which acts as the only input device directly available to the user.

This system operates a switching device 89, which is the basic on/off device used by the operator to control all valves, vortex stirrer, automatic conveyance system 90, and heater 37. Typically, there is one switch between devices in the system so that each device corresponds to a particular switch number.
There is a one-to-one correspondence. For example, switches 0 to 63 refer to valve numbers O to 63, the heater is controlled by switch 64, and so on. In order to provide the necessary means for the other elements of automatic control, the microcomputer 81 also receives input signals from the liquid sensor 9 to determine the number of gas-liquid and liquid-gas transitions, and the amino acid input signals entering the synthesizer. information from a barcode reader 108 located on the automated transport system to cross-check the identity of the carrier.

FIG. 3 shows a plan view of the automatic transport system.

The system has a guideway 101 which serves as a track to maintain and control the direction of movement of a series of cartridges such as cartridge 105. Each of the series of cartridges contains individual protected amino acids, placed in rows in the desired sequence into the peptide to be constructed. For convenience, this guideway is left open to visually monitor the column, mimicking typical methods for peptides shown in the literature and comparing the sequence in the loaded guideway with that of the desired peptide. It is positioned to correspond to a peptide with the carboxyl terminus on the right side to facilitate Steel recovery tape 110t-equipped spring reel 107 typically passed on pulley 109 to hold the row of cartridges
The last cartridge in the row is pressed by the pressure block 103.

This allows movement of the pressure block while exerting a substantially constant force on the column of υ cartridges, allowing it to accommodate columns of different lengths corresponding to different lengths of peptides. Can be done. Furthermore, more than one peptide can be synthesized from a single cartridge. Pressure block 10
At the end of the guideway opposite 3 is an ejector driven by an air cylinder 113 for holding the cartridge in a sampling position 117 as indicated by cartridge number 2 and ejecting the cartridge once the amino acids in the cartridge have been extracted. -There is a system. Cartridge number 1
111 illustrates the protruding position of ejector system 115 from which spent cartridges fall into a chute (not shown) for disposal. When the ejector returns to its normal position after being ejected, the pressure block 10
A constant force spring 107 acting through 3 forces the next cartridge into the transport position.

The automated transport system also includes a barcode reader 108. In a preferred embodiment, each cartridge is labeled with a barcode unique to the type of amino acid it contains. When the cartridge moves down the guide path to the barcode reader, the reader reads the barcode label and sends the information to a computer system. If the computer is preconfigured for a particular polypeptide, it performs a consistency check to ensure that the cartridge is in the correct position in sequence for that polypeptide. If the computer was not preconfigured for a particular polypeptide, the system runs an open loop and the computer transfers the information from the barcode into the cartridge to determine which synthetic experiment to use for that particular amino acid. Used to call up a draft plan and record its used amino acids. Also, each Car1-IJ azalea contains stoichiometrically accurate amounts of amino acids.

To extract the amino acids from the cartridge, the system contains (
Two injection needles (shown in FIG. 1) are provided, needle 121
The needle 123 is for gas pressure supply and exhaust, and the needle 123 is for supplying DCMf to the cartridge to dissolve the amino acids, and for extracting the dissolved amino acids and DCM.

These two needles are typically loaded vertically and removed by moving the needles up when the new car is moved into position.

and connected to an air cylinder (not shown) for inserting these needles through the top of the cartridge for mixing and brewing operations.

Figures 5&, 5b, and 5c show details of a typical cartridge 105 used in an automated delivery system. In a preferred manner, the container is constructed of blow-molded high density polyethylene and has a body portion 130 of substantially rectangular cross-section capable of holding approximately 7 rnt. It has a neck portion 133'e having an integral septum 13 which serves to provide a positive seal to the neck of the cap.
A serum-finishing cap 135 with 7 is attached. As shown in Figures 4 & 4b, dimension DI (approximately 0.5 inches and approximately 1.27 crn) is equal to dimension D2 (approximately 1.27 crn).
120 inches = approximately 2.84 crn), so that a relatively large number of cartridges (
up to 50) can be used. The cartridge also has two substantially flat surfaces 134 and 136 on each side to facilitate proper placement in the row.

The bottom of the cartridge is shaped such that two surfaces 140 and 141 intersect at an angle forming a V-shaped gutter. When the cartridge is in the sampling position within the automated transport system, the needle 123 descends very close to the intersection line 143 of the two planes that have the lowest height within the cartridge, i.e. correspond to the point at the bottom of the cartridge. Ru. The needle located near the lowest point within the cartridge helps ensure that all substances within the cartridge are extracted, thereby allowing careful control of stoichiometry. Seven langes 145 extend across the bottom of the cartridge in a direction perpendicular to the line of intersection 143 for the purpose of stabilizing it at certain times. This V-shape and flange 145 allows the cartridge to stand on its own in a stable upright position. The cartridge also includes a recess 147 around its periphery to facilitate accurate placement of the barcode label to ensure accuracy of the barcode reader 108 when reading the label.

Figure 6 is a table listing various dimensions of the bottle.

Figures 7a and 7b illustrate cutaway views of typical liquid sensors used in synthesizers. In the top view of Figure 7b, the device is symmetrical about the centerline CL, so that the top half of Figure 7b corresponds to a bottom view of the top half of the device, and the bottom half of Figure 7b corresponds to a bottom view of the top half of the device. Corresponds to the plan view of the lower half of . The device comprises a clothespin-like tube holder compartment having a top portion 222 and a bottom portion 220, each of which is typically constructed of glass-filled nylon or plastic, each of which includes a translucent tube. They have grooves 229 with double curvature extending in their thickness direction to accommodate. The double curvature is provided to allow the top and bottom portions to positively engage two different tube diameters, which in the preferred embodiment typically have external diameters of IA inches or 1. /16 inches) and is made of Teflon. Top and bottom portions 222 and 22G are attached by nails 212, 213, 214 and 215, respectively, to two substrates 209 and 211, typically constructed of printed circuit board material (phenolic resin). At opposite ends of the tube holder these substrates are fitted with two rivets 240 and 2 of substantially the same length.
41 at a constant distance υ. By providing a thickness of the tube holder compartment that is greater than the length of these rivets, these substrates provide a force for the springs to hold the top and bottom of the compartment together, thereby firmly holding the tube in the groove 229. do. Also, to ensure accurate alignment of the top and bottom sections, holes 227 (not shown due to cuts in Figure 7b) and 228 located in the bottom section 220 fit into holes 228 on each side of the top section. A keying device is provided having keys 225 and 226 which are connected to each other. 7th &
In the side view shown, the bottom portion 220 has been cut away to reveal the hole 250 in which the photodiode 270 is installed. A photodetector 2 is located on the opposite side of the hole 250 beyond the groove 229.
An identical hole 251 is provided in the top portion 222 to fit the 71, and this photodetector can be inserted into the tube where the interface between the liquid and the gas or between the gas and the liquid is held in the groove 229. It is used to detect changes in the intensity of the light received from the photodiode as it moves to the surface of the photodiode, where the changes in intensity are due to differences in the focusing of the light rays due to differences in the refractive index properties of the liquid and gas. Also, a cavity 252 is provided in the top portion 222 to accommodate a holder for a photodetector, and a similar cavity 253 is provided in the bottom portion 222 to accommodate a holder for a photodiode. Similarly, conduits 260 through the bottom portion 222 and conduits 261 through the top portion provide electrical conductor passage from the diodes and detectors to solder pads 230 and 231 located at the ends of wires 233 and 234, respectively; The exterior usually provides passage for the detector signal conductors. The photodiode and detector are powered via input terminals 235 and 236. terminal 237
and 238 provide a common ground for both the photodiode and detector.

Synthesis of synthesizer-engineered peptides typically begins by first filling a reaction vessel with a resin to which the first amino acid in the sequence is attached and entering the desired amino acid sequence into a computer. The operator then places the amino acid carp (IJ) in a linear sequence or chain corresponding to the amino acid sequence of the desired peptide into an automated delivery system.

The activation cycle begins when needles 121 and 123 rupture the septum of the first cartridge, and needle 123 injects a calibrated amount of DCM. Mix and dissolve the protected amino acid in the DCM using gas sparge from needle 121. The dissolved temporarily protected amino acid is removed and transferred to the activator. A second (and possibly third) DCM is added to the cartridge to ensure total transfer of protected amino acids and then transferred to the activator. Then DCM based on amino acids
Transfer 0.5 equivalents of DCC to the activation vessel and mix the solution by periodically bubbling gas such as argon or nitrogen through the solution.

After a predetermined period of time sufficient to completely convert the amino acid to its symmetrical anhydride, the gas passage in valve block 25 is opened and the PS
The DCM solution of A is pressurized into the condensation vessel through valve block 23. The frit 17 at the bottom of the activation vessel collects all DCU formed as a by-product in the activation reaction.
Retain the precipitate. Using software controls, the PS reaction number is individually adjusted for each amino acid to optimize PSA formation and to obtain maximum precipitation of DCU.

After the PSA/DCM solution is transferred to the concentration vessel, a volume of DMF is added. The exhaust valve on valve block 33 is then opened and inert gas sparge is initiated through valve block 41 to volatilize the DCM. This is done without further reducing the initial volume of DMF, which has a significantly higher boiling point than DCM. Heat is supplied from band heater 37 to compensate for the heat lost due to evaporation of DCM. The different maximum internal temperatures during this solvent displacement operation can be automatically adjusted to the unique thermal stability of the various protected amino acids PSA using a thermistor. Simultaneously with the solvent replacement operation in the condenser, the DCU precipitate in the activator is removed via the top valve (valve block 23) and overhead wash nostle 19 by sequential washing with an alcohol/DCM mixture. The activator is finally cleaned with dichloromethane in preparation for the next PSA reaction.

In the concentration container after dichloromethane has been removed,
The PSA/DMF solution is pumped from the concentration vessel to the reaction vessel containing the resin-bound alpha-amino deprotected growing peptide chain.

PSA/DMF brought into the reaction vessel from the concentration vessel
reacts with the deprotected alpha-amino functionality of the resin-bound peptide for a time sufficient to complete the reaction (typically greater than 99%), and then the used reagents and solvent are washed out with successive solvent washes using vortexing. It will be done.

In order to start a new synthetic cycle in the reaction vessel, it is first necessary to remove the alpha-amino protection of the last amino acid attached to the chain. In the special case of 1-BOC protected amino acids, trifluoroacetic acid (TFA) and D
A solution of CM, typically 65% TFA, is transferred under pressure to the reaction vessel and vortexing is applied periodically for effective mixing. After a period sufficient to remove all of the t-BOC-alpha-amino protecting group (typically about 15 minutes), the fluid is pressurized to waste through valve block 45 and discharged. The resin is then quickly washed with a small amount of DCM introduced through the top or bottom valve with vortexing. Neutralization is carried out by introducing mustard apylethylamine (DIEA) and DMF or DCM without vortexing and then pressurized to waste. Neutralization is usually repeated once.

While the resin is then swirled (vortexing may be continuous or intermittent), the top valve (valve block 43) is opened to the waste liquid, and the valve block 45 is
A small amount of DCM or DMF is successively added to wash the solution. After thorough washing of the amino-unlinked Ii resin, remove the amino acid P.
The SA/DMF mixture is transferred from the concentration vessel to the reaction vessel under pressure.

To sample the resin during synthesis or upon completion of the peptide, valve 55 is first opened, and line 61 is opened via valve block 45 leading to waste while valve 6T is closed. The reaction vessel is then pressurized from the top while being swirled to force the resin and reaction solution into the sample reservoir 5T. This forces the resin onto [59].

Valve 55 is then closed, valve 67 is opened, and the waste line of valve block 45 is closed. DCM is passed through line 61 from valve block 45 to sweep the resin from the membrane 1 and deposit the resin/DCM mixture into fraction collector 64 . The sample reservoir and its associated tubing are then closed by closing valve 67, draining the reaction vessel and passing DCM into the reaction vessel through line 61 from valve block 45.
Clean by transferring through valve 55.

This integrated system allows simultaneous operation in the reaction vessel and the activator and concentrator. For example, deprotection, neutralization, coupling, and washing operations can be performed in the reaction vessel at the same time that the next amino acid PSA is formed in the activation vessel. The concentrator vessel can be cleaned at the same time that activation is taking place within the activation vessel, and the activation vessel can be cleaned at the same time as the concentrator vessel is undergoing solvent replacement. This simultaneity of operation allows for significant economic advantages in cycle time. Appendix Table A explains this synchronicity in more detail by illustrating the operating time of each vessel during a complete run to couple a single amino acid.

This system also allows the use of various synthetic methods. The above method was performed for peptide synthesis using t-BOC-amino acid PEA, but for example, F-MO
Alternative methods using protected amino acids such as PSA can also be easily implemented. Similarly, other activated carboxyl species such as mixed anhydrides, active esters, acid chlorides, etc. can be used to pre-form the activated carboxyl species immediately prior to introduction into the reaction vessel using activation and concentration vessels. It is possible to perform the synthesis by eliminating the need for a reservoir of activated amino acid species that is maintained during the time frame of peptide synthesis in a similar manner.

As mentioned above, special coupling operations are required for asparagine, glutamine, and arginine, which can be initiated in the activation and concentration vessels.

In these cases, equimolar amounts of HOBT and DCC in DMF or DMF/DCM are pre-equilibrated and then hydroxybenzotriazole (HOB) is combined for reaction in a reaction vessel with an equivalent amount of protected amino acid.
Dual coupling with T) and DCC is generally required.

To achieve this result, one technique is HOBT/D
One equivalent each of MF and DCC/DCM is first transferred to the concentrator vessel through valve blocks 23 and 41.

Two equivalents of protected amino acids from the amino acid cartridge are then transferred into an activation vessel in a suitable solvent (DMF/DCM). Half of the substance in the activation vessel is then transferred to a concentration vessel (pre-equilibrated HOBT/DC) by time-pressure control.
(containing C/DMF/DCM) for activation, and then the activation mixture is transferred to a mixing vessel. Near the end of the first coupling cycle, the concentrator vessel is refilled with a second equimolar mixture of HOBT/DCC and then a second equimolar amount of amino acid is added from the activation vessel for a similar activation. Start for the second coupling.

Computer Software Systems At the most basic level, the software control of a device involves turning on and off valves or other switching devices at appropriate times to direct the desired flow of various substances from one container to another. It is about achieving. At the same time, each step in solid phase synthesis is extremely repetitive and the number of steps is not abnormally large. Rather than requiring the operator to instruct the operation of individual valves in detail to achieve a desired result, the various situations are useful in guiding the operator to more complex control concepts that phase in functional control. It is. For example, an operator will very often instruct the system to transfer the contents of an activation vessel to a concentrator vessel, rather than assemble a more detailed set of instructions such as: (1) the concentrate vessel is ready for receiving; (2) Open the outlet of the valve block 33. (3) Open the valve blocks 23 and 41 at the connection of the transfer line between containers.
4) Open the gas valve to pressurize the activation container.
(5) Close the valve after the signal from liquid sensor 39 indicates that fluid has been transferred. In order to achieve this type of user-friendly approach while still maintaining the ability to have completely independent control over each element of the device, the software control system is implemented via a touch screen using a series of menus. These menus serve to give the operator a wide range of possibilities, from one extreme of using the system in a fully automated manner to the other extreme of operating the system by switching individual valves. It is.

The control concept involved is that the coupling of individual amino acids to the peptide chain corresponds to three complete cycles: one cycle in the activation vessel, one cycle in the condensation vessel and one cycle in the reaction vessel. It is. Each of these cycles consists of an ordered and timed set of individual steps, each of which corresponds to a certain function that may occur within the vessel. As a general definition, a function can be thought of as corresponding to a named set of switches that are turned on at the same time (the normal position of each switch is the off state). In practice, it is advantageous to number the various functions and separate them for each container. One function is e.g. DCM-To ACTI
VATOR). Such functionality requires the integration of a special release valve for the DCM to be delivered to the activator. In the activator cycle, this function may be activated several times, e.g. after the symmetrical anhydride is transferred to the concentration vessel, the activator and DCU precipitate may be washed several times with DCM to remove the remaining symmetrical anhydride. It is advantageous to remove the target anhydride. Each time this function occurs, it corresponds to a different step in the reaction cycle occurring within the activation vessel, as well as other functions required in each cycle. The net result is that each cycle in a container is a series of steps, each step corresponding to one function associated with that container. A table of typical functions is given in Appendix B. The individual control menus are described below to better illustrate this concept.

FIG. 8 shows the general main menu that appears on the touch screen, which corresponds to the bottom of the various other more detailed menu trees (shown in FIG. 17).

Each rotated block corresponds to an area on the touch screen from which a series of menus in that family are approached. For example, scree y (D@PEPTIDE SEQ, E
The block labeled DITOR' starts another display listing all the amino acids synthesized by simply selecting the block containing the name of the desired amino acid in the desired sequence. allows selection and display of the order of N- to C-terminal amino acids appearing in the desired peptide.

When the main menu is displayed, @PEPTIDE CH 5TRY EDITOR on the screen
” PEPT in Figure 10.
The I DECHEMISTRY EDITOR menu is brought up, which allows the operator to create or edit either a cycle specification or a motion file specification in a particular container. - For example, if you choose to create or edit a new activator cycle, the CYCLE ED corresponding to the activation container.
The ITOR menu will appear on the screen (see Figure 11). This menu allows the operator to change the order of functions included in each cycle of the activator, and similarly for menus corresponding to other vessels.

Generally, a given vehicle has three time fields associated with it: the required main time "TIME", the time based on the detector measurements 'MIN TIME' and '''
ERRMODE ', and the additional time added to a special set of steps in the reaction vessel ``''ADD TIME
'is. TIME has several purposes. If liquid detection is not specified for a process, TIME is the total time for that process. If liquid detection is specified,
The main time is the time -out allowed before continuing whether or not the appropriate transition is detected by the liquid sensor.
'That is, the maximum time. Additionally, if liquid sensing is specified, the user can set the minimum time MIN before the appropriate transition (liquid to gas or gas to liquid) is registered.
TIME must be specified. It is important to note another TIME effect when detection is specified.

If the sensor indicates that a valid transition has occurred after the specified minimum time, the indicated function ends. That is, the function is switched off. However, the next step is not started until the main time has elapsed.

This is necessary to ensure proper cycle consistency within each vessel to obtain optimal throughput. ERRM
ODE is used when detection is indicated and the specified transition is not seen before the main time has elapsed, such as when a valve is blocked or a special reservoir is empty. This mode can be used to issue an alarm or, in some cases, terminate the synthesis in a non-hazardous manner. ADD TIME specifically refers only to the reaction vessel and corresponds to the amount of time (expressed in seconds) to be added as a function of the number of amino acids in the sequence of the peptide to be synthesized in each of the three aforementioned fields. do. Since the volume occupied within the reaction vessel increases with each additional amino acid coupling, the process time within the reaction vessel also increases. For example, if the value 10 is entered in this field, 1 second (1%
0 seconds) is coupled in the peptide sequence.
is added to the principal time for the amino acid. For the third amino acid, the time is increased by 2 seconds, and so on.

If you want to edit RUN FILE, enter PEPTID
EDIT in ECHEMISTRY EDITOR
OLD is selected and the RUN FIL shown in Figure 12
E The EDITOR menu will be displayed. The table displayed in it is static operation file A/ (5tatic ru
n file ). This file specifies three cycles for each of the 26 amino acid possibilities (one cycle each for the activation vessel, concentration vessel, and reaction vessel), with independent concentrator temperatures for each amino acid. It provides adjustment. The 26 amino acid possibilities offered are more than the 20 standard amino acids and an additional 4 amino acids for specialized uses desired by the operator (J), the BEGINEND cycle allows for independent control of these points in the synthesis. enable.

This static operation file is for each '11 CYC that has already been explained.
This is the result of specifying the chemistry for each cycle via the LE EDITOR. Additionally, all cycles specified in a particular run file must reside on disks currently installed in the system. Because RUN FILE
This is because EDITOR only allows selection from a list of existing cycles. Also, each of the three reaction vessels can be synthesized from different (or the same) run files. This is because at the time the operation is set, the operation file is REACTION VESSELM.
This is because it is specified in a special container of ONITOR (described later). Typically, an operator can create, edit, and store numerous operational files on a single disk. Also, although the system is designed to allow different cycles for each amino acid potential, it turns out that in practice not that many cycles are needed to give efficient out-of-control operations. .

The menu explained next is VESSEL FUNCTI.
It is ONEDITOR. This menu is accessed from the main menu and operates at the most basic level of the synthesizer. It contains the specific instructions necessary to accomplish a particular function within a particular container. For example, if it is desired to change the definition of the functions occurring within the activation container or to add or delete functions, the F corresponding to the activation container shown in FIG.
UNCT I 0NEDITOR is called. You can explore and change the entire set of functions for the activation container in the bush.

As mentioned above, a function is a set of named switches that are turned on at the same time. These features normally form chemical flow paths through the system.

For example, the @DCCTOACTIVATOR' O function is activated by switches 114 (DCC conveying valve), 119 (DCC
bottle pressurization), and 125 (opening the activator to waste). Also, some functions may be described as mechanical or electrical actions, such as 1 heater on' (1 switch).

The system has three types of functions designated as types 0, 1, and 2, respectively: 0N10FF, TOGGIJ ON.

and organized by TOGOLE OFF. ON10
The FF function turns on a switch for a given process only in a certain cycle. At the end of the process, all switches for that function are cleared (turned off) before the next function is activated. TOG
The GLE ON function instructs the machine to turn on certain switches and leave them on until the corresponding TOGOLE OFF function turns them off. Subsequent functions do not affect the state of the switch turned on by the TOGGLHON function. Therefore, when creating and editing functions, all TOGOLE ON functions are
It is important to have an OGOLE OFF function. T
A specific example of the OGGLE ON function is "Heater on" for a concentration vessel or "Heater on" for a reaction vessel.
Examples include VORTEX ON'.

REACTION VESSEL MO from Lord Me-nee
Invoking NITOR brings up the screen shown in Figure 14, which allows the operator to set up and track the composition. Two response areas allow the operator to select two modes of operation, the first mode being PEPTID
The second modality is to automatically control the device based on input to the ESEQ, EDITOR and a preprogrammed set of cycles called a dynamic run file. (amino acid type) is loaded into the automated delivery system and operated only based on a preprogrammed set of cycles from a specified static operating file. If the first mode is selected, the computer issues a question as to whether the operator wishes to change the dynamic run file. If so, the operator responds in the affirmative and the screen shown in FIG. 15 corresponding to the edits to the dynamic run file is displayed. This file allows the operator to input the desired peptide sequence into PEPTIDE SEQ.

It is first generated internally when entering the EDITOR.

It is simply a table listing the sequences of amino acids in the order they are to be constructed, and the designated cycles for each amino acid are those already programmed from the designated static run file. This compilation is designed to allow operators to vary the cycles used for particular amino acids within the sequence depending on their placement in the peptide chain, so that the operator is position dependent on chemistry. Can control sex.

Unlike static running files, dynamic running files are not stored on disk.

Following the mode selection in the REACTION VESSEL MONITOR, a series of questions are then asked to confirm that the device is properly configured to begin operation. For example, a check is made as to whether the reaction vessel has been filled and whether the automatic filler has the desired number and sequence of amino acids.

The final question that follows the series of questions is whether to start or stop the operation. R.E.
As part of the ACTION VESSEL MONITORO function, the status of the reaction vessel is displayed, including the name of the activated amino acid currently undergoing the reaction,
This includes the time when peptide synthesis was started and the time when synthesis was completed. Additionally, the current coupling is displayed and the array of already completed couplings can be displayed and viewed by moving the screen back and forth.

Invoking CYCLE MONITOR from the main menu brings up the screen shown in FIG.

5, the current status of each container is displayed in real time by several parameters. The first line lists, for each container, the particular number of amino acids in the peptide sequence currently active in that container, along with the abbreviation of the name of the particular amino acid.

The second line lists the special cycle of salt currently in progress in each container. The third row lists which step in the sequence of steps in a particular cycle is currently occurring within each container. The fourth line lists the function number and function name corresponding to the special process listed on the container. Also listed is the time elapsed for each cycle as well as the status of each of the liquid sensors.

MACHINE/5TATUS 5ELECTION8
You can also select several other menus from the main menu under the general category of. These include: RESEE LVOIR 5 TATUS - This allows the device to track the volume used from each reservoir in each cycle, so that when the amount in the reservoir is low,
It is related to the fact that an alarm is raised and the operator is informed of this problem; INSTRUMENT C
0NFIQ - This is used to display and set the configuration of the machine itself, e.g. time of day, power line - 120v at 60Hz, etc.; SYSTEM 5ELF TES
T-This tests all electronic functions to a practical extent; C0NTR0L A″NDTEST-This allows for easy debugging and manual interruption of the synthesis if necessary. Allows manual control of the equipment to the extent that the operator can operate each valve and switch one at a time: and DISKU
TILITIES - This allows normal disk functions necessary for computer system operation such as setting up files, erasing files, and renaming files.

Another feature of this software controlled device is the simultaneity of operations in the reaction vessel, concentration vessel and activation vessel. By noting the various times required to perform each step of a cycle in each container,
An automatic optimization mechanism, hereinafter referred to as a cycle compiler, has been developed to provide maximum efficiency in the synthesis of each particular peptide given a particular chemistry.

To achieve this efficiency, certain events (functions) within the time of each cycle for each container determine when a transfer can occur and when the container is ready to begin the process of another transfer. It is important to recognize that the

For activated containers, these functions include initiation of movement, B
OTA, end of movement, corresponds to EOTA.

Similarly, for the concentrator cycle, key functions include determining when a container is ready for receipt, RC, and the start of transfer, % BOTC, and end of transfer EOTC. For reaction vessels, these functions are expressed as RR, when the vessel is ready for receipt. Using these concepts, a graphical representation of the cycles occurring within each container can be shown as illustrated in FIG.

From this figure, the first standard of operation is T = T and TBOTC = TRRBOT^
RC, i.e. the condensation vessel must be ready for reception at the time transfer from the activation vessel begins, and the reaction vessel must be ready for reception at the time transfer from the concentration vessel begins. must be kept in order. The next step is to determine in which vessel the operation should be started first for process optimization.

This is achieved by calculating the left side delay (from Figure 18) for each container.
% and “DLR” These left side delays relate to the waiting time allowed before preparing a particular container for the function that occurs therein, e.g. for an activation container, the amino acids are transferred to the container and the symmetric Some waiting time is allowed before the anhydride is formed and the reaction vessel is resin-free.
Some waiting time can also be tolerated before beginning deprotection of the bound peptide chain. Once these left-hand delays have been calculated, the shortest delay (ie, the longest warm-up time) corresponds to the cycle to be started first, and the master time should be equal to zero for that cycle. These various delays can be calculated using the following equations. First, let's make the following definition.

TMA = (Ti1X)'rA-"BOTA)+("B
OTC-TRC)' and Tiltt, = MAX (
TBOTA% TRC1”RX) However, TRX 3T
RR-”MA (See Figure 18 for physical interpretation of TMA and TML) Next, TDL,=TML-TBoTA.

TDLC""ML-TRC・andTDLJl"TML"' TRX 1'For example,
At the start of the cycle shown in Figure 18, if the master hour is set to zero, 'DLA=0, DLC'.
5, and a delay time such as "DLR=3, i.e. the activation vessel starts first at T=O, then the reaction vessel starts 3 minutes later, and 17kk'!4 vessels are at T=O.
Start at 5 seconds.

The next chain build for optimal throughput involves calculating the minimum separation of these three cycles using the next three cycles of the coupling sequence. To calculate this minimum value, for the first cycle the right delay 't
It is important to tF value: ”DRA=TBOX””TDLA-TAT:TD
RC=TBOX-”DLC-[-TCT”(TEOTA
−TBOTA) ] : and TDRR:TBOX −TDu −(TRT +(”E
OTC-TBOTC) -'However, TBOX=MAXC
(TAT-TDLA), (TCT”(TKOTA-”B)
OX) 1TDLC), (TRT” (”goTc-T
BOTC) ” TDLR) ): In the above formula, TA
T% TCA% and TRT correspond to the total time of the activator cycle, concentrator cycle, and reactor cycle, respectively.

These right side delays are then combined with the various left side delays of the next cycle to arrive at a minimum separation of three cycles. That is, TS(2-cl = MIN((TDRA+TDL)
)-(TDRC+TDLC2)・(TDRR+TDLR2') ] In the formula, Ts is the first cycle and the second cycle c
2-cl, where TDLA2, TDLC2, and DLR2 are the left-hand delays of the second cycle (calculated by the same technique as for the left-hand delays in the first cycle). .

This minimum separation value is then translated into the waiting time required to initiate the second cycle in each of the vessels. That is, TWAi? = TDLA2 +TDRA -TS(!
2-(!l\”WO2” TDLC2” TDRC-T
S(!f!-C1% and TWR2"TDLR2+"D
"MA2, TRC in 0 formula which is RR""T8Q2-C1
2 and "WB2" are the waiting times from the last step of the first cycle to the first step of the second cycle for the activation vessel, enrichment vessel, and reaction vessel, respectively.To achieve optimal efficiency, TMA2, T 1 and TWR2 must be zero as in the case of the C2 left-hand delay in the first cycle.

FIG. 19 shows the results of the above calculations for a series of three cycles.

Utility of the Invention The ability to deliver a symmetrical anhydride of an amino acid in a predetermined, intact state to a reaction vessel allows most coupling reactions to proceed in greater than 99% yield using only a single coupling. enable. Because of the extremely high yields in each step, it is possible to synthesize peptides completely automatically with extremely high overall efficiency. The combination of activation vessel and thermally coated condensation vessel individually has a stoichiometry, D
We aimed to explore and develop optimal conditions for maximal symmetric anhydride formation for individual protected amino acids by modifying the reaction time in CMK, the temperature during DMF substitution of DCM, and the time frame in which the solvent substitution operation is performed. enable. In this way it is possible to automatically implement conditions that reproducibly deliver the maximum amount of symmetrical anhydride to the reaction vessel containing the peptide resin for each amino acid type.

In prior art techniques using peptide synthesizers with the ability to form preactivated amino acids, the user had to stop the reaction at each reaction cycle to track the extent of coupling. A second or third coupling may then be performed if the first coupling reaction was relatively inefficient in order to improve the yield. As a result,
It was just a single cycle synthesis automation that eliminated cycle-to-cycle automation. Alternatively, existing synthesizers can be used automatically from cycle to cycle using multiple couplings with in situ activation within a reaction vessel containing the peptide resin gold. The result is that this activation method is not uniquely optimized for each individual amino acid in the presence of the reactive amino groups of the growing peptide chain, resulting in relatively inefficient coupling.

The results shown in Figure 20 are based on the claimed apparatus Acyl Carri@rPro for the synthesis of decapeptides.
tein (d5-74) is exemplified. The top line is a graphical representation of the individual cycle yields for each amino acid addition during peptide chain assembly performed on the apparatus of the invention.

All amino acid additions except for glutamine (designated Gin) were accomplished by fully automated sequence single coupling. Glutamine requires a well-known special chemical formulation consisting of a double coupling via HOBT (hydroxybenzotriazole) activation. All other couplings are Boa-
It was carried out using amino acid symmetrical anhydrides.

The bottom line was pre-formed by hand outside the instrument and used with Beclanan 990P using nine Boa-amino acid symmetric anhydrides.
Results of the synthesis of the same peptide performed on the eptide 5 synthesizer. Literature: Reza A
r++had7+Er1e Atherton + D
erek 01ive and Robert C+5hep
pard J, Chem, Soc, Perkl
n Trans 1 .

(1981) 529-537 These results demonstrate that fully automated synthesis is precluded by the need to manually preform symmetric anhydrides; Indicates that it is not optimally formed.

Certain peptide amino acid sequences, regardless of the activated form of the amino acid, are unique in that the second and third couplings typically do not give a coupling yield of greater than 9996 in DCM. Illustrating sequence-specific coupling problems.

(W, S, Hancock, D, J, Pr
eacott, P, R.

vagelog + and G, R, Marshall
1, J. Org, 3B (1973) 774). However, those skilled in the art are aware that sequence-dependent incomplete couplings performed in poor solvents such as DCM are greatly improved if performed exclusively in more polar solvents, e.g. DMF'C. S, Mo1st
er, S, B, H.

Kent r Peptides, Structure and Function” Bulletin of the 8th American Peptide Symposium, pp. 103-106 (P.
eptides, 5lcture & Fun
ction.

Proceedings of the 8th Ame
rican Peptide Symposium,
ppm, (103-106)”].

Acyl Carrucir Protein (d5~
74) The decapeptide is such a known problem sequence and the excellent results achieved with the claimed device by Boe-amino acid symmetric anhydride coupling in DMF are one of the main advantages of this device. One example shows the optimal formation of activated amino acids in DCM, but the coupling of activated amino acids in DMF. This automatically implemented method provides a general solution to the construction of the peptide sequence in question.

In addition, Appendix Tables A and B are provided below to illustrate this synchronicity in more detail by illustrating the operating time of each vessel during the complete operation to couple the single amino acids described above.

Appendix Table A Examples of Preformed Symmetrical Anhydride Cycles of Typical Amino Acids GLN, ASN, and ARG Each line illustrated in the book for all amino acids outside of tJ is a separate type of function.

Some functions open special valves for a given operation.

DCM with dichloromethane, DMF=N, N-dimethylformamide, DCC=N,
N-dicyclohexylcarbodiimide, TFA = trifluoroacetic acid, DIEA = diimpropylethylamine Note: Vortex the RV wash solution at the beginning of each 1o-0 wash solvent addition. This helps break up resin agglomerates. Optional operation: Clean from the top or bottom of the RV.

The valve block is cleaned with DCM and sparged with nitrogen after transfer and delivery of certain reagents.

A remolar loading resin (~1 mmol amino acid/?polystyrene) is used.

can do.

Ru.

Appendix Table B List of possible functions for the peptide synthesizer 1i11: T = Top valve block B = Bottom valve block 0 = Off 1 = On Reaction vessel function DCM to waste σ) Excess to waste (1) Gas VB ke) RV discharge) RV pressurization (1) DCMf7f: RV Ker) DCM as waste liquid (B) DMF as waste liquid (B) DIEA as waste liquid (B) DIEA pressurized TFA as waste liquid (B) Gas VB (B) RVO liquid drain (d) Blow back into TFA bottle TFA bottle waste TFA pressurization manifold Pressure compensating agent 1 Pressure neutralization side transport RV discharge (81 RV pressurization (B) To DCMIRV (B) DMF to RV (BI DIEA t-RV (B) TFA to Hv (B) Gas to RV (Bl Resin sampler (RV function) Sample removal DCM to RV DCM to fraction collector DMFt-RVIC DMF to both collectors, gas to RV, gas to fraction collector, DIEA to RV, miscellaneous RV function simple stirring (0,1) Gokai I Yusuke Haya #Tuna Jun! Im device [) MF to waste liquid ('r) DCM to waste liquid σ) Gas V B (Tl Concentrator pressurization σ) Exhaust σ) Liquid removal DCM to concentrator (1) DMF to concentrator σ) DMF to waste liquid ( B) Surplus to waste liquid (B) Gas VB (B) Concentrator pressurization (B) Exhaust (B) DMF to concentrator (B) Surplus to concentrator (B) From manifold pressurized transfer concentrator DMF transfer line surplus to RV Transfer line Gas transfer line miscellaneous condenser function heater (0,1) Activator MeOH to waste liquid σ) DCM to waste liquid α) Gas VB ('rl Addition of activator Pressure (T) Draining the liquid from the activator Exhausting the activator (1) DCM to the activator (T) MeOH to the activator (TI DCM to the waste liquid (B) Extra-1 to the waste liquid (B) ) DMF as waste liquid (B) Extra-2 as waste liquid (B) AA as waste liquid (B) Gas VB (BI DCC measurement HOBT measurement Pressurization of manifold pressure activation device (B) Accumulate gas (B) DCM as activator (B) DMF as activator DCC as activator HOBT as activator Amino acid as activator Extra-1 as activator Extra -2' t” Cartridge transport system DCM to cartridge to activator 1B
MF to Extra-1t-cartridge to Extra-11 Cartridge pressurize the cartridge (sample needle) Pressurize the cartridge (exhaust needle) Mix the cartridge Exhaust the cartridge (sample needle) Exhaust the cartridge (exhaust needle) Move activation DCM transfer line from device to RA device DMF transfer line Extra-1 transfer line Extra-2 transfer line Gas transfer line Miscellaneous activator function needle upper and lower (0,1) Cartridge protrusion 1

[Brief explanation of drawings]

FIG. 1A is a diagram showing the whole of FIGS. 1a and 1b, FIG. 1a and FIG. 1b are diagrams showing a liquid transport system of the device according to the present invention, and FIG. Figure 2 is a diagram showing the computer system used to control the apparatus; Figure 3 is a plan view of the automatic transport system for feeding individual containers to the synthesizer; Figure 4 is a diagram showing the gas distribution system of the apparatus; The figure is a cross-sectional view of a reaction vessel according to the invention, showing the result of swirling of the fluid contained therein. Figures 5&, 5b and 5a are partially cutaway explanatory diagrams of containers used in the automatic transport system, Figure 6 is a diagram showing the dimensions of the container used in the automatic transport system, and Figures 7 & 7B are 2 of the liquid senna used in the device
Diagram showing the surface. Figures 8 to 16 are diagrams showing each menu used in the computer system to determine the six operations for operating the synthesizer device, and Figure 17 is the entirety of Figure 171 and Figure 17b. 17a and 1
Figure 7b is a flowchart of the menu. Figure 18 is an explanatory diagram for providing the definitions used in the calculation formula for optimizing peptide production, and Figure 19 is a calculation for optimizing peptide production that requires three cycles of coupling. It is a figure showing an example. FIGS. 20A and 20B are diagrams showing a comparison of synthesized peptide purity between the prior art and the devices of the present invention. FIG, 8 FIG, 10 FIG, 12 FIG, 13 FIG, 14 FIG, 15 FIG, +6 FIG, +7b FIG, old ^, amazing if. , 耀, 绅客路TAT -Tc7- TRT 9
20a Congregation 950 Congregation I Trode 201 California 91105 615I California 94022 Los Al-16 Sea North San Antonio Loro California 94619
Oakland Avenue 4601

Claims (1)

[Scope of Claims] 1. An automated peptide synthesizer comprising the following means: receiving one protected amino acid at a time, and activating each of the amino acids in the order in which they are received to generate each activated species of the amino acid. activation means providing a common container for forming a sequence of aliquots of each type of amino acid, each aliquot containing one type of amino acid, said sequence of aliquots of each type of amino acid in the desired order in said peptide; a means for containing a resin used to attach a peptide chain thereto in solid phase peptide synthesis; a means for transferring each of said activated species of said amino acids from said activation means to said reaction vessel means; a transfer means for transferring and controlling said transfer means to transfer said activated species of each amino acid formed within said activation means, one amino acid at a time, in the order in which said activated species are received by said activated species; computer means for moving. 2. said transfer means is also for transferring a deprotection medium into said reaction vessel means, said transfer of said deprotection medium being controlled by said computer means to transfer another amino acid in said sequence; 2. A synthesizer according to claim 1, in which a subsequent activation occurs for each of said activated species. 3. A synthesizer according to claim 1, wherein said reaction vessel means includes stirring means to prevent agglomeration of resin during each reaction of said activated species of amino acid. 4. A synthesizer according to claim 1, wherein said stirring means moves the substance contained in said reaction vessel means by swirling action. 5. A synthesizer according to claim 4, wherein the reaction vessel means comprises a vessel having an axis of rotational symmetry. 6. The synthesizer according to claim 5, wherein said stirring means includes rotation means for causing one end of said container to perform a circular motion around said axis of rotational symmetry while statically holding the other end. 7. A synthesizer according to claim 1, wherein said activation means comprises an activation container receiving one amino acid at a time, said sequence of individual amino acids and an activator solvent. 8. The transfer means is for moving an activation medium into the activation vessel to form each activated species of amino acid a short time before being transferred to the reaction vessel. 7. The synthesizer according to item 7. 9. The synthesizer of claim 8, wherein said activation means includes an activation solvent and a concentration vessel for receiving each of said activated species of amino acids one amino acid at a time from said activation vessel. 10. The transfer means also removes the activated solvent medium from the concentrating vessel and transfers a coupling medium with each of the activated species into the concentrating vessel to remove the activated species of the amino acids from the resin or 10. The synthesizer according to claim 9, which enhances coupling to the peptide chain. 11. The synthesizer of claim 10, further comprising heating means under control of the computer for supplying heat to the concentrating vessel. 12. The transfer means also includes an automatic delivery means under the control of the computer for supplying the amino acids one at a time to the activation means in the desired order in the peptide. Apparatus described in section. 13. The apparatus of claim 12, wherein said automatic transport means includes holding means for holding individual containers of amino acids in a linear array of the desired order in said peptide. 14. The apparatus of claim 13, wherein said automated conveying means includes extraction means for extracting the contents of said individual containers one container at a time. 15. The apparatus of claim 14, wherein said extraction means includes needle means for supplying solvent to each of said containers and for extracting said solvent from said containers together with the amino acids. 16. The apparatus of claim 15, wherein said automatic transport means includes a transport means for moving said container into position of said needle means. 17. The apparatus of claim 13 further comprising said linear array of individual containers within said holding means. 18. The apparatus of claim 17, wherein each of the containers has a bottom internally in the form of a gutter. 19. The device of claim 18, wherein the bottom of the container externally fits into the bottle and stands upright without external restraint. 20. An automated peptide synthesizer, comprising: an activation container for receiving an amino acid, wherein the amino acid is converted to a more active species of the amino acid; a concentrating vessel in which is combined with a coupling agent, a concentrating vessel containing a resin-bound peptide chain coupled to the concentrating vessel;
and a reaction vessel for receiving the more active species and the coupling agent for coupling the amino acid to the resin-bound chain. 21, an activation container, a concentration container connected to the activation container, a reaction container connected to the concentration container, and a reaction container connected to the activation container, the concentration container, and the reaction container to transfer amino acids to the activation container. and a transfer means for transferring the amino acid from the concentrator to the reaction vessel. 22. The apparatus of claim 21, wherein the concentrating vessel includes heating means for heating the substance contained in the concentrating vessel. 23, further for presenting events in the activation vessel, the concentration vessel and the reaction vessel, and movement by the movement means according to a preprogrammed schedule;
23. Apparatus according to claim 22, comprising computer means for controlling and for controlling the heating by said heating means according to a preprogrammed schedule. 24, the moving means having a first tube between the activation vessel and the concentration vessel and a first tube controlled by the computer for controlling flow from the activation vessel to the first tube; 24. The apparatus of claim 23, including valve blocking means for controlling flow from said first tube to said concentrator container and second valve blocking means controlled by said computer. 25. the moving means also includes a second tube between the concentration vessel and the reaction vessel and a third valve block means controlled by the computer for controlling flow to the reaction vessel; The second valve block means also comprises:
25. The apparatus of claim 24 for controlling flow from said concentrator to said second tube. 26. The apparatus of claim 25 further comprising resin sampler means controlled by said computer for extracting material from said reaction vessel. 27. The apparatus of claim 25 further comprising liquid sensor means for detecting transitions between gas and liquid flowing within the first tube. 28. The apparatus of claim 23, wherein said computer means includes menu means for classifying physical and scientific operations within said apparatus to enable control of said operations by an operator. 29. The method according to claim 28, wherein said classification is separated into a set of cycles, each cycle having the characteristics of operating one complete set of said activation vessel, said concentration vessel and said reaction vessel. Device. 30, wherein said computer means is configured to maximize the production rate of polypeptide for any given particular sequence of cycles in each of said activation vessel, said concentration vessel, and said reaction vessel; 30. Apparatus as claimed in claim 29, including storage means for storing algorithms for calculating operating times of the second and third valve blocking means. 31. Claim 30, comprising means for the computer to calculate the operating time, and switch means for operating the first, second and third valve block means in accordance with the operating time. Apparatus described in section. 32. A resin sampler comprising the following elements: a reservoir; a membrane disposed within the reservoir for stopping the flow of material exceeding a certain size past the location of the membrane within the reservoir; a first connected to the reservoir on one side of the membrane;
a connecting tube, a first valve for controlling flow through the connecting tube, a second connecting tube connected to the reservoir on the same side of the membrane as the first connecting tube, a second valve connected to the reservoir on the same side of the membrane as the first connecting tube; a second valve for controlling flow through the connecting tube and a third connecting tube connected to the reservoir on the opposite side of the membrane. 33. Peptide synthesis method comprising the following steps: (a
) adding an activation medium to an activation vessel containing an alpha-amino-protected amino acid and a first solvent having a first boiling point to activate the amino acid; (b) the activated amino acid and the first solvent; (c) adding a coupling solvent having a second boiling point higher than the first boiling point to the concentrator; (d) adding the first coupling solvent to the concentrator;
(e) sparging a gas to selectively evaporate the first solvent through a concentration vessel containing the activated amino acid and the coupling solvent; Transferring from the concentration vessel to the reaction vessel containing the resin for solid phase peptide synthesis. 34, a liquid sensor for detecting transitions between a gas and a liquid in a translucent tube, comprising the following means: spaced apart a distance substantially opposite each other near a first end; two substantially flat substrates of elastic material held together; a holding means placed opposite the first end and between the substrates holding the tube; the thickness of the retaining means together exceeds the predetermined distance; an optical means carried by the retaining means for imparting radiant energy from one side of the tube; Sensing means held by the holding means on the opposite side of the tube from the optical means for receiving the transmitted radiant energy and for detecting changes in the intensity of the energy transmitted through the tube. 35. A container for holding a fluid comprising the following elements: two other side ends symmetrically placed opposite each other and connected to the two substantially parallel side ends to form a box; a bottom portion having an internal configuration of a V-shaped gutter connected to the side ends, the length of the gutter being perpendicular to the parallel side ends, and the bottom portion forming a substantially a bottom portion having an external portion at the center of the gutter that is flat; a flat surface connected between the bottom portion and the balanced end, perpendicular to the length of the gutter and at the center of the gutter; a flange having a flat surface substantially coextensive with the flange; and a top portion having a diaphragm therein and connected to the side ends to form a closed volume. 36. Automated transport system for an automated peptide synthesizer for transporting pre-measured amounts of amino acids to a synthesizer, comprising the following means: holding containers of individual amino acids in a row and directing the movement of the cartridge; a track for pushing the row of cartridges in a direction along the track; located opposite the forcing means and facing the forcing means;
and an ejector means for holding the column fixed and ejecting the cartridge from the column when the amino acid is extracted from the cartridge; and an ejector means for extracting the amino acid from the cartridge. Extraction means installed near the truck.