WO1990007975A1

WO1990007975A1 - A method and an apparatus for dissolving a solid substance in a solvent

Info

Publication number: WO1990007975A1
Application number: PCT/DK1990/000015
Authority: WO
Inventors: Arne Holm; Morten Peter Meldal; Ole Buchardt
Original assignee: Arne Holm; Morten Peter Meldal; Ole Buchardt
Priority date: 1989-01-18
Filing date: 1990-01-17
Publication date: 1990-07-26
Also published as: AU4966590A; DK21389D0

Abstract

A method for dissolving a solid substance in a solvent comprises the steps of: arranging the solid substance (11) within a chamber defined in a substance-receiving body (10); establishing a closed fluid-flow circuit (18) partly defined by said chamber; and circulating the solvent in said circuit so as to at least partly dissolve the solid substance (11) in the solvent. The apparatus for performing the method comprises: a body (10) defining a chamber for receiving the solid substance (11) therein; fluid-flow circuit means; connecting means (12, 13) for connecting said circuit means to said body (10), so as to form a closed fluid-flow circuit (18) partly defined by said chamber; and means (20) for circulating the solvent in the fluid-flow circuit. The method and apparatus described are advantageously applicable for performing a fully automated peptide synthesis.

Description

A METHOD AND AN APPARATUS FOR DISSOLVING A SOLID SUBSTANCE IN A SOLVENT

The present invention relates to a method for dissolving a solid substance in a solvent, and to an apparatus for performing said method. More specifically the invention relates to a method for performing an organic synthesis, such as a peptide synthesis, and to a peptide synthesis apparatus.

It is known i.a. from SE-B 7910353-7 to dissolve a solid substance in a solvent by passing a solvent through a flow-through container containing a solid substance which comprises a compound to be dis¬ solved in the solvent. This reference deals with the problem of gradually dissolving the compound, and the compound is placed in a by-pass conduit means.

DE-A1 35 08 915 and DE-A1 37 19 185 likewise disclose methods for gradually dissolving compounds contained in flow-through containers.

The trend of present developments with regard to the synthesis of peptides or proteins is mainly towards further development and modi¬ fication of the strategy developed by R.B. Merrifield for solid-phase synthesis, as solid-phase synthesis is generally far better than synthesis in a homogeneous solution in respect of product yield and the work effort required, and in respect of automation of the syn¬ thesis process.

The general method originally developed by Merrifield [vide for example J. Am. Chem. Soc. 85, 2149 (1963)] employs a functionalized crosslinked styrene/divinylbenzene copolymer as solid-phase carrier material. This copolymer is normally provided in the form of beads or particles which often have a dominant particle size of 20-80 μm. The functionalization originally preferred by Merrifield was a functiona- lization of the aromatic rings of the copolymer with chloromethyl groups, but a number of other functionalities, including aminomethyl, α-aminobenzyl and α-amino-4-methylbenzyl have later been used. Re¬ gardless of its kind, the purpose of the functionality is to provide an anchoring bond between the carrier material and the C terminal of the first amino acid which should be coupled to the carrier material. More recent refinements of the Merrifield method comprise the further incorporation between a functionality (for example one of the above functionalities) on the polystyrene chain of the carrier material and the C terminal of the first amino acid to be coupled, of a bifunc- tional linker group (also called a spacer or handle group) the reac¬ tivity of which is tailored among other things to satisfy certain requirements with regard to the coupling of the first amino acid to the carrier material and/or with regard to the ease with which the completed synthesized peptide chain is cleaved from the carrier material.

The present specification employs the following abbreviations:

DMF: N.N-dimethylformamide;

DMAP: 4-(N,N-dimethylamino)pyridine; Dhbt; 3,4-dihydro-4-oxo-l,2,3-benzotriazin-3-yl;

Dhbt-OH: 3,4-dihydro-3-hydroxy-4-oxo-l,2,3-benzotriazine; tBu: tert-butyl;

Fmoc: 9-fluorenylmethyloxycarbonyl;

Pfp: penta luorophenyl ; TFA: trifluoroacetic acid;

HPLC: high performance liquid chromatography

The abbreviations employed for amino acids are the abbreviations recommended by the IUPAC-IUB Commission of Biochemical Nomenclature [J. Biol. Che . , 247, 977-983 (1972)].

Owing to significant developments within the field of molecular biology from the beginning of the 1970's a need arose for a re-evalu¬ ation of solid-phase peptide synthesis methods, and it became clear that the reaction method developed by Merrifield was not necessarily the optimum one. These considerations led to the development of a new variant of a method for solid-phase peptide synthesis.

This new variant uses polar polydimethylacrylamide carrier material which incorporates acrylolylsarcosin methyl ester groups as functio¬ nalities to which may be coupled suitable linker groups. This polymer is freely permeated and solvated by a large number of solvents, including water, but especially by dipolar aprotic solvents of the dimethylformamide type. Dimethylformamide is also generally suitable as a solvent for protected peptides and is a preferred solvent for many peptide bond formation reactions.

An important aspect of this "polyamide" method for peptide synthesis is that the conventional acid-labile t-butoxycarbonyl protective groups are replaced by other base-labile N-protective groups such as Fmoc which are displaced within a few seconds upon treatment with secondary amines such as piperidine. At the same time the benzyl- based side-chain protective groups used in the conventional Merri¬ field method are replaced by for example t-butyl-based side chain protective groups. A combination of protective groups of the latter types permits solid-phase synthesis under considerably milder condi- tions than those used in the Merrifield strategy, which widens the field of use of the solid-phase method considerably.

Suitable solid-phase peptide synthesis methods using this polyamide carrier material are normally those in which the growing peptide chain is fastened to the carrier material during the synthesis by means of an acid- or base-cleavable linker, for example 4-hydroxyπ.et- hylphenoxyacetic acid, 3-methoxy-4-hydroxymethylphenoxyacetic acid which are both acid cleavable, or 4-hydroxymethylbenzoic acid which is base cleavable. Thus the methyl ester functions of the carrier material are first reacted with ethylenediamine. Then the free amino groups in the ethylenediamine units are acylated with the linker, whereupon the first amino acid in the form of Fmoc-amino acid an¬ hydride is coupled to the hydroxymethyl group of the linker; the coupling process is catalyzed by DMAP. Alternatively the first amino acid may be coupled to the carrier material, after packing the latter in suitable columns, wells or other reaction containers, by reaction of Fmoc-amino acid-0-Pfp ester under catalysis with DMAP.

After provision of the carrier material with the coupled, first, at least N-protected amino acid, the synthesis cycle is continued by cleavage of the Fmoc group of the coupled, first amino acid, followed by the coupling of the next Fmoc-amino acid with its carboxyl group activated in an appropriate manner, for example by introduction of the Fmoc-amino acid in the form of a symmetrical anhydride or in the form of an activated ester.

An apparatus for performing a peptide synthesis is described in "Continuous flow methods in organic synthesis", R. C. Sheppard,

Chemistry in Britain, May 1983. The reference discloses the conven¬ tional method for performing a continuous flow peptide synthesis according to the above "polyamide" technique.

In commercially available apparatuses for fully automated peptide synthesis, such as "Beck an System 990 Synthesizer" or "Biosearch Model 9500 Peptide Synthesizer", the solid substance is dissolved prior to dosing in a dosage unit.

In more advanced apparatuses the solid substances, which may be preactivated, are contained in containers covered by septa. The containers are lined up in a unit for positioning of said containers.

The containers may be lined up successively, as in "Applied Biosys- tems model 430A" or "LKB Biolynx 4170", or they may be placed in an

XY- able (MilliGen 9050 PepSynthesizer) . After penetration of septum with concentric needles a solvent is applied to the solid substance.

The dissolution of the solid substance is enhanced by agitation with a stream of inert gas.

Apparatuses according to the prior art involve a number of drawbacks. The penetration technique may lead to blocking of flow circuits in the apparatus resulting in a termination of the synthesis. The use of inert gas, such as argon or nitrogen, under pressure for dissolving the solid substance involves a risk of loosing active substance to the surroundings due to foaming. It is not possible to perform a peptide synthesis involving substances with a very low solubility, and the substance used having the lowest solubility is decisive for the amount of solvent to be used. Due to poor reproducability of the dosing stage it is necessary to use relatively large amounts of substances and solvents. Furthermore, the stages of dissolution and reaction take place sequentially. It is an object of the present invention to provide a method and an apparatus of the above kinds, which are especially applicable for dissolving a solid, preactivated amino acid in an apparatus for performing a peptide synthesis, which give a high yield, and which overcome the drawbacks of prior art apparatuses.

According to the present invention there is provided a method for dissolving a solid substance in a solvent, the method comprising the steps of:

- arranging the solid substance within a chamber defined in a substance-receiving body;

- establishing a closed fluid-flow circuit partly defined by said chamber; and

- circulating the solvent in said circuit so as to at least partly dissolve the solid substance in the solvent. The method gives a maximum content of dissolved solid substance in the circuit and effective mixing without the need for stirring means. Furthermore the method minimizes loss of active substances from the circuit.

When the circuit includes a reactor device, such as a reaction co¬ lumn, when the solid substance is a reagent for reacting with a reactant in the reactor device, and when at least part of the dis¬ solved reagent is recirculated through said chamber, the reaction in the reactor device will take place with maximum speed due to the fact that there will always be a maximum content of dissolved reagent in the solvent flowing in the circuit.

According to the invention it is an advantageous feature that the closed fluid circuit is established by moving flow circuit means into sealing engagement with flow connecting parts of the substance-re¬ ceiving body communicating with the chamber therein. In this way it is possible to accomplish a rapid change between different substance- receiving bodies, generally containing different active substances.

The method according to the present invention is especially appli¬ cable for performing a peptide synthesis in which the solid substance comprises a preferably preactivated amino acid and the reactant comprises a solid-phase peptide synthesis substrate which is substan¬ tially insoluble in said solvent.

It is preferred that the flow in the circuit is controlled so as to be substantially laminar. This feature provides for a low risk of mechanical collapse of the resin or reactive substance and compres¬ sion in the fluid circuit.

The method according to the invention for performing a continuous peptide synthesis in a circuit including a reaction column comprises the steps of: - arranging a variety of dosaged preactivated solid substances in substance-receiving bodies each defining flow paths,

- arranging the substance-receiving bodies in a predetermined pattern in a supporting device, said supporting device being dis- placeable, - displacing the supporting device to a position in which an inlet and an outlet of one of the bodies is in alignment with a pair of piston-like circuit members,

- moving said piston-like members into sealed engagement with the inlet and the outlet of the substance-receiving bodies, - passing a solvent through the substance-receiving body,

- bleeding air from the circuit,

- circulating the solvent in the circuit and gradually dissolv¬ ing the preactivated amino acid, and at the same time letting it react with a reactant in the reaction column, until a predetermined degree of reaction is achieved,

- displacing the reaction mixture with DMF,

- emptying the substance receiving body,

- washing the circuit,

- passing a deprotection liquid in the part of the circuit comprising the column for deprotecting the N-terminal of the last- coupled amino acid,

- displacing the reaction mixture with DMF,

- emptying the substance receiving body,

- cleaning the circuit, and - repeating the synthesis steps until a peptide with the desired length and composition is achieved. According to the invention an apparatus for dissolving a solid sub¬ stance in a solvent comprises:

- a body defining a chamber for receiving the solid substance therein,

- fluid-flow circuit means,

- connecting means for connecting said circuit means to said body so as to form a closed fluid-flow circuit partly defined by said chamber, and - means for circulating the solvent in the fluid-flow circuit.

In an apparatus according to the invention the flow circuit means preferably include a reactor device, such as a reaction column.

For selectively connecting the fluid-flow circuit to supplies of solvent, pressurized gas, washing liquid and/or deprotection fluid, the apparatus comprises valve means, and furthermore a bleeder valve for bleeding air from the system.

In the presently preferred embodiment of an apparatus according to the invention the substance-receiving bodies each define a through- going passage having an inlet and an outlet, said passage including the substance-receiving chamber. In this way, the substance-receiving bodies function as a combination of a container and a flow chamber.

It is preferred that the through-going passage runs in a generally vertical direction and that the inlet and outlet are placed around a common vertical axis.

To enable rapid change between different substance-receiving bodies, the apparatus according to the invention comprises first means for moving the connecting means into and out of sealing engagement with the inlet and the outlet, respectively.

In the presently preferred embodiment of the apparatus, the connect- ing means comprise piston-like members having sealing surfaces for engaging with corresponding, complementary shaped surface parts defined around said inlet and outlet, respectively, the piston-like members being reciprocatingly movable by the irst moving means.

When the apparatus according to the invention is to be used for a step-wise chemical synthesis, such as a peptide synthesis, it ad- vantageously includes:

- a plurality of substance-receiving bodies,

- a supporting device for supporting the plurality of bodies in a predetermined pattern,

- second moving means comprising means for relatively displacing the connecting means and the supporting device, and

- control means for controlling the displacing means, so as to successively position the connecting means in alignment with the inlet and the outlet of the respective bodies in accordance with a desired sequence.

To avoid the use of bottom closure means, such as a septum, to be penetrated by the lower piston-like member, it is preferred that the chamber in each of the substance-receiving bodies is partly defined by a bottom closure means which is permeable to fluid and impermeable to the substance in solid form.

In the presently preferred embodiment of the apparatus according to the invention the supporting device comprises a circular disk having apertures for receiving the substance-receiving bodies, the displac¬ ing means comprising a stepper motor for rotating the circular disc. These features provide for simple and effective controlling of the position of the subs ance-receiving bodies.

The invention will now be described in greater detail with reference to the accompanying drawings, in which

Figs, la, lb and lc are schematical views illustrating the steps of dissolving a solid substance in accordance with the present inven- tion,

Fig. 2 is an elevational view of a dosing or dosage apparatus of an apparatus according to the present invention,

Fig. 3 is a schematical view of a peptide synthesis apparatus accord¬ ing to the present invention, Fig. 4 is an HPLC-chromatogram of a peptide synthesized in accordance with the present invention, and

Fig. 5 is an HPLC-chromatogram of another peptide synthesized in accordance with the present invention.

In Fig. la, a through-flow container 10 is shown containing a solid substance 11 to be dissolved in a solvent. First and second pistons 12 and 13 containing flow paths 14 and 15 are located at lower and upper connecting parts 16 and 17, respectively, of the through-flow container 10. The pistons 12 and 13 are reciprocatingly movable into (Fig. lb) and out of (Fig. la) sealing engagement with connecting parts 16 and 17, respectively.

In Fig. lc a situation is illustrated in which the pistons 12 and 13 are brought into sealing engagement with the connecting parts 16 and 17, respectively, of the through-flow container 10. The container 10 consequently constitutes an integral part of a flow circuit 18. To dissolve the solid substance 11 in a solvent the solvent is let into the circuit via valve means 22 from a solvent container 19. The circuit 18 is gradually filled with the solvent put under pressure by means of a pump 20. During the filling of the circuit 18 the solvent passes, preferably from the first or lower piston 12, through the through-flow container 10, and the solid substance 11 is gradually dissolved in the solvent, and air in the system is gradually dis¬ placed by venting or bleeding the system through a bleeder valve 23. In Fig. lc, the circuit 18 includes a reaction column 21 in which the dissolved solid substance is to react. The solvent flowing from the reaction column 21 consequently contains a smaller amount of the dissolved solid substance than the solvent flowing into the reaction column 21, and the solvent is consequently able to dissolve more of the possibly slightly soluble solid substance. When the circuit is completely filled with solvent the admission of the solvent via the valve means 22 is stopped, and the circulation is continued in the closed circuit 18 until the desired reaction is completed. After this, the circuit can be drained through a waste conduit 24, and the circuit may for emptying and drying comprise a pressurized N -source (not shown) . In Fig. 2 a dosing apparatus is shown, generally designated 25, of a preferred embodiment of the apparatus according to the invention. In operation a part of the dosing apparatus 25 forms an integrated part of a flow circuit.

The dosing apparatus 25 comprises a frame 30 in which a circular rotatable disk 31 Is mounted made from PTFE (polytetrafluoroethylene) and having apertures 32 for receiving a plurality of substance-re¬ ceiving bodies 33 made from POM (polyoxymethacrylate) . Each of the bodies 33 defines a substantially vertical through-going flow passage 34 defining an inlet 35 and an outlet 36. In the bottom of each of the substance-receiving bodies a semi-permeable closure 37, such as a PTFE-filter, Is arranged permitting the passage of fluids, but block¬ ing any passage of the solid form substance. For fixing and maintain¬ ing the substance-receiving bodies 33 in the apertures 32 shrews 38 are preferably provided.

The circular rotatable disk 31 is movable by means of a stepper motor 39 via a gear 40.

Below and above the center of a circle having its center at the rotational axis of the disk 31 and defined by the center of the through-going flow passages 34 of the substance-receiving bodies 33 a pair of pistons 41 and 42 are located, each being reciprocatingly movable via moving means 43 and 44, respectively, such as pneumatic or hydraulic cylinders. The pistons 41 and 42 are provided with sealing rings 45, 46, respectively. Through the pair of pistons 41 and 42 flow passages 47 are provided (only the flow passage in the upper piston 42 is shown) , and the pistons 41 and 42 are each provi¬ ded with connection means, such as threaded bores (of which only the threaded bore designated 48 of the piston 42 Is shown) enabling fluid communication with corresponding flow circuit parts 50 and 51, re- spectively, so as to bring the through-going flow passage 34 of the substance-receiving bodies 33 Into fluid communication with a flow circuit when the pistons 41 and 42 are moved into sealing engagement with the inlet-35 and outlet 36, respectively of the substance-re¬ ceiving bodies 33. Furthermore, detection means 53 for detecting the angular position of the circular disk 31 are provided, such as optical detection means, proximity detection means, capacitive detection means etc., preferab¬ ly arranged below and above the disk. The stepper motor 39, the moving means 43 and 44 and the detection means 53 are preferably connected to and are controlled by or supplies information to an electronic control means 49, such as a microprocessor means compris¬ ing appropriate input/output interface means.

Fig. 3 scematically shows an automatic peptide synthesis apparatus generally denoted 60 comprising a dosing apparatus or sampler 61 similar to the dosing apparatus 25 shown in Fig. 2. The peptide synthesis apparatus comprises a first and a second or main circuit part 62 and 63, respectively, the first of which includes the dosing apparatus 61. The circuit parts 62 and 63 are seperable by means of a first valve means 64, and it is possible to control the flow of fluid through the dosing apparatus 61 in either direction by a valve means

65. The peptide synthesis apparatus 60 further comprises containers

66, 67, 68 and 69 for containing DMF, DMAP, 20% piperidine in DMF and 95% triflouroacetic acid, respectively. The containers 66, 67, 68 and 69 can all be put under pressure by means of a pressurized N2-source and via valve means 71, 72, 73 and 74. To control the flow of fluids from the containers to the main circuits 62 and 63 there are provided valve means 76, 77, 78 and 79. The latter valve means 79 is in com¬ munication with a waste conduit 80. For controlling the synthesis steps the apparatus 60 comprises a monitor 82, such as a solid phase spectrophotometer (L. Cameron, M. Meldal and R.C. Sheppard, J. Chem. Soc. Chem. Commun. , 270-272, (1987)) which can put be into communica¬ tion with the main circuit 63 via a valve means 83. The reaction column 52 may also be put into and out of communication with the second main circuit 63 via a valve means 84. When the linking steps of the peptide synthesis according to the present invention are performed, the circuit part 62 generally forms an integrated part of the main circuit 63, while the circuits 62 and 63 are generally separated during rinsing or washing, as described below. In the preferred embodiment, shown in Fig. 3, a second N2 inlet 86 in con¬ nection with a valve means 87 is provided _jor emptying and drying the circuit 62. A pump 85 provides for a sufficient flow rate in the circuits 62 and 63, and the apparatus further includes a UV-detection means 88.

When using the dosing apparatus 25, shown In Fig. 2, in connection with an automated peptide synthesizer, schematically shown i Fig. 3, for performing a solid-phase peptide synthesis according to the present invention, the first amino acid is coupled to the resin by either of the methods described above, and when the reaction is completed according to standard coupling time the reagent and the DMAP catalyst is removed by passing DMF through the reaction column 52. When the circuit 63 is rinsed, the part 62 of it comprising the substance-receiving body is emptied in the downwards direction by nitrogen gas pressure from the inlet 86. The circuits 62 and 63 are then filled with DMF in the upwards direction for rinsing or washing the conduits. The Nα-amino-group is deprotected by passing 20% plpe- ridine in DMF through the part of the circuit 63 comprising the column 52 only. The deprotection reagent is removed by passing DMF through the same part of the circuit 63. The substance receiving body 33 is then emptied and the pistons 41 and 42 are relaxed. The rotata¬ ble disk 31 is allowed to proceed to the next substance receiving body which is filled with the Fmoc amino acid Dhbt ester (or other activated derivatives) of the second amino acid in the required sequence. The pistons 41 and 42 are activated and DMF is passed through the substance receiving body in the upwards direction, allow¬ ing air to escape. When the circuits 62, 63 are filled the solution is recirculated through the column 52, while the dissolving process is continued in the substance receiving body. When the reaction is completed according to the monitor 82 the circuits 62, 63 are rinsed by passing through DMF. Rinsing, deprotection and rinsing is repeated as described above, and the next amino acid is added similarly from the succeeding substance receiving body. This process of deprotection and addition of an amino acid is repeated until the desired peptide is synthesized. After Nα-deprotection of the final peptide it is cleaved from the resin by means of a cleavage reagent such as a 95% aqueous trl luoroacetic acid.

It is obvious for a person skilled in the art that the dosing ap¬ paratus according to the present invention can be used for dosing and dissolving of solid substances for a great variety of applica¬ tions, such as dissolving prior to chromatography, dissolving highly activated substances for oligonucleotide preparation or for oligosac- charide synthesis, and in general for the introduction of highly insoluble substrates into solid matrices of various kinds.

The apparatus according to the present invention may further be subject to various modifications. For instance, the disk for support¬ ing the substance receiving bodies may be replaced with a linearly movable body, preferably a body movable in an orthogonal system of two coordinates (an X-Y table), and/or the pistons may be movable, preferably in an orthogonal system of two coordinates.

EXAMPLE:

The preferred embodiment, discussed in connection with Fig. 2, of a dosing apparatus according to the invention was used in conjunction with a fully automatic solid-phase peptide synthesizer for the syn¬ thesis of a large series of peptides containing from 11 to 25 amino acid moieties. The solid-phase peptide synthesis method employed was essentially that described by Sheppard (vide supra) . The peptide chains were built up on a solid support comprising poly-N,N-dimethyl- acrylamide polymerized in a hard kieselgtihr matrix. This solid sup¬ port is well suited to packing in a chromatography column. The amino acid side-chains were protected, where appropriate, using tert-butyl groups, and preactivated Dhbt esters of Fmoc-amino acids were used for peptide chain elongation. The first amino acids of the peptides were attached to the solid support via an acyl 4-oxymethylphenoxya- cetamide "linker" group, enabling the subsequent simultaneous clea¬ vage of the completed peptide chain from the solid support and remo¬ val of side-chain protection groups using trifluoroacetic acid.

The first amino acid was added to a packed column of the solid sup- port in form of its symmetrical anhydride, and after washing and reacting the support with 20% piperidine in DMF, a synthesis cycle was employed in which introduction of reagent (3 equiv. ) from the dosing apparatus and treatment with piperidine in DMF were carried out alternately. The resulting peptide was cleaved from the support, subjected to gel- iltration, and analysed by HPLC.

Using the above-outlined method, a 25 amino acid peptide which Is an analogue of rat and human E-caderln was synthesized. The amino acid sequence of this peptide Is as follows: H-Asp-Trp-Val-Ile-Pro-Pro- Ile-Val-Val-Pro-Glu-Asn-Glu-Lys-Gly-Pro-Phe-Pro-Lys-Asn-Leu-Val-Gln- Tyr-Cys-OH.

HPLC of the synthesized peptide gave the chromatogram shown in Fig. 4, which demonstrates the high purity of the product. Amino acid analysis gave the following results (in mol amino acid per mol pep¬ tide) : Asp, 2.6; Glu, 2.9; Gly, 1.1; Pro, 5.4; Tyr, 1.0; Val, 3,9; lie, 1.9; Leu, 1.2; Phe, 1.0; Lys, 2.0 (the amino acid analysis method could not analyse for Trp or Cys). Yield of peptide: 191 mg per g solid support.

Similarly prepared peptide resins were reacted automatically with biotin Dhbt ester or N-acridIn-9-yl-c-aminohexanoic acid Dhbt ester (Acr-Aha-Dhbt) dissolved by means of the described apparatus.

For example, the peptide Acr-Aha-Lys-Glu-Leu-Phe-Glu-Asp-Leu-Glu-Cys- Leu-Ala-Lys-Gln-Phe-Tyr-Gly-OH was prepared. Yield: 98 mg per g resin. Amino acid analysis: Ala 1.13. Gly 1.05, Phe 1.90, Asp 1.08,

Leu 2.91, Tyr 1.00, Glu 3.99, Lys 2.92. The HPLC of the crude product is shown In fig. 5.

Claims

1. A method for dissolving a solid substance in a solvent, the method comprising the steps of:

- establishing a closed fluid-flow circuit partly defined by said chamber; and

- circulating the solvent in said circuit so as to at least partly dissolve the solid substance in the solvent.

2. A method according to claim 1, wherein the flow circuit includes a reactor device.

3. A method according to claim 2, wherein the reactor device is a reaction column.

4. A method according to any of the claims 2 or 3, wherein the solid substance is a reagent for reacting with a reactant in the reactor device, at least part of the dissolved reagent being recirculated through said chamber.

5. A method according to any of the claims 1 to 4, wherein the closed fluid circuit is established by moving flow circuit means into seal- ing engagement with flow-connecting parts of the substance-receiving body communicating with the chamber therein.

6. A method according to claim 4 or 5 for performing a peptide syn¬ thesis, wherein the solid substance comprises a preactivated amino acid, and the reactant comprises a solid-state peptide synthesis substrate substantially insoluble in said solvent.

7. A method according to any of the claims 1 to 6, wherein the flow in the circuit is substantially laminar.

8. A method for performing a peptide synthesis in a circuit including a reaction column, the method comprising the steps of: - arranging a variety of dosaged preactivated solid substances in substance-receiving bodies each de ining low paths,

- arranging the substance-receiving bodies in a predetermined pattern in a supporting device, said supporting device being dis- placeable,

- displacing the supporting device to a position in which an inlet and an outlet of one of the bodies is in alignment with a pair of piston-like circuit members,

- moving said piston-like members into sealed engagement with the inlet and the outlet of the substance-receiving bodies,

- passing a solvent through the substance-receiving body,

- bleeding air from the circuit,

- displacing the reaction mixture with DMF,

- emptying the substance receiving body,

- washing the circuit, - passing a deprotection liquid in the part of the circuit comprising the column for deprotecting the N-terminal of the last- coupled amino acid,

- displacing the reaction mixture with DMF,

- emptying the substance receiving body, - cleaning the circuit, and

- repeating the synthesis steps until a peptide with the desired length and composition is achieved.

9. An apparatus for dissolving a solid substance In a solvent and comprising:

- a body defining a chamber for receiving the solid substance therein,

- fluid- low circuit means,

- connecting means for connecting said circuit means to said body, so as to form a closed fluid-flow circuit partly defined by said chamber, and

- means for circulating the solvent in the fluid-flow circuit.

10. An apparatus according to claim 9, wherein the fluid-flow circuit means include a reactor device.

11. An apparatus according to claim 10, wherein the reactor device is a reaction column.

12. An apparatus according to any of the claims 9 to 11, wherein the fluid-flow circuit means further comprise valve means for selectively connecting the fluid-flow circuit to supplies of solvent, pressurized gas, washing liquid and/or deprotection fluid.

13. An apparatus according to any of the claims 9 to 12, wherein the fluid-flow circuit means further comprise a bleeder valve for bleed¬ ing the system.

14. An apparatus according to any of the claims 9 to 13, wherein said substance-receiving bodies each define a through-going passage having an inlet and an outlet, said passage including the substance-receiv- ing chamber.

15. An apparatus according to claim 14 wherein the through-going passage runs in a generally vertical direction, and the inlet and outlet are placed around a common vertical axis.

16. An apparatus according to claim 14 or 15, further comprising first means for moving the connecting means into and out of sealing engagement with the inlet and the outlet, respectively.

17. An apparatus according to any of the claims 14 to 16, wherein the connecting means comprise piston-like members having sealing surfaces for engaging with corresponding complementary shaped surface parts defined around said inlet and outlet, respectively, the piston-like members being reciprocatingly movable by the first moving means.

18. An apparatus according to claim 17, comprising

- a plurality of substance-receiving bodies,

- a supporting device for supporting the plurality of bodies in a predetermined pattern, - second moving means comprising means for relatively displacing the connecting means and the supporting device, and

19. An apparatus according to any of the claims 9 to 18, wherein the chamber in each of the substance-receiving bodies is partly defined by a bottom closure means which is permeable to fluid and impermeable to the substance in solid form.

20. An apparatus according to claim 18 or 19, wherein the supporting device comprises a circular disk having apertures for receiving the substance-receiving bodies, the displacing means comprising a stepper motor for rotating the circular disk.