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WO1990011291A1 - Contrôle de reactions dans la synthese de peptides en phase solide par mesures de conductivite - Google Patents

Contrôle de reactions dans la synthese de peptides en phase solide par mesures de conductivite Download PDF

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Publication number
WO1990011291A1
WO1990011291A1 PCT/GB1990/000422 GB9000422W WO9011291A1 WO 1990011291 A1 WO1990011291 A1 WO 1990011291A1 GB 9000422 W GB9000422 W GB 9000422W WO 9011291 A1 WO9011291 A1 WO 9011291A1
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Prior art keywords
reaction
conductivity
synthesis
amino acid
reaction solution
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PCT/GB1990/000422
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English (en)
Inventor
Claus Schafer Nielsen
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Biotech Instruments Limited
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Publication date
Priority claimed from GB898906514A external-priority patent/GB8906514D0/en
Priority claimed from GB898919189A external-priority patent/GB8919189D0/en
Application filed by Biotech Instruments Limited filed Critical Biotech Instruments Limited
Publication of WO1990011291A1 publication Critical patent/WO1990011291A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/02—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance
    • G01N27/04—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance
    • G01N27/06—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating impedance by investigating resistance of a liquid
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/045—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers using devices to improve synthesis, e.g. reactors, special vessels
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00279—Features relating to reactor vessels
    • B01J2219/00281—Individual reactor vessels
    • B01J2219/00286—Reactor vessels with top and bottom openings
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00423—Means for dispensing and evacuation of reagents using filtration, e.g. through porous frits
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/0059—Sequential processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00596—Solid-phase processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/0068—Means for controlling the apparatus of the process
    • B01J2219/00698—Measurement and control of process parameters
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00718—Type of compounds synthesised
    • B01J2219/0072—Organic compounds
    • B01J2219/00725—Peptides
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures
    • C40B40/04—Libraries containing only organic compounds
    • C40B40/10—Libraries containing peptides or polypeptides, or derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • This invention concerns peptide synthesis.
  • Peptides consist of linear chains of amino acids (each comprising an amino group, a carboxyl group, a hydrogen atom, and a distinctive side chain, all bonded to. a carbon atom), linked by peptide bonds between the amino group and the carboxyl group of adjacent amino acids.
  • Techniques are known for the chemical synthesis of peptides by the sequential addition of desired amino acids, which form peptide bonds by a condensation reaction, resulting in a growing peptide chain.
  • Solid phase methods of peptide synthesis have been devised, in which the carboxyl terminus of the growing peptide chain is anchored to a solid support, and a desired sequence of amino acids added in stepwise manner to the amino terminus at the other end of the growing peptide chain.
  • carboxyl terminus of a first, fully protected amino acid is attached to a support, generally of polymeric material but typically a polystyrene or a polyamide-based resin.
  • the alpha-amino group is protected only temporarily, the protection being removed at each addition cycle.
  • the side chain protection, blocking the reactive groups of each amino acid is permanent and is only removed at the end of the synthesis. Side chain protection is usually by means of benzyl esters and ethers.
  • the stepwise synthesis cycle starts with the removal of the alpha-amino group protection (deprotection). After washing and neutralisation, the next amino acid, with a similarly protected alpha-amino group, is added in the presence of an activation agent. After this coupling reaction is complete, excess reagents are removed by washing. The procedure is repeated until the desired sequence of amino acids has been produced. At the end of a synthesis, all protecting groups are removed, and the peptide is cleaved from the solid phase support.
  • the pivotal reaction to monitor during a synthesis is the coupling reaction between the growing peptide chain and the next amino acid added to it. Near completeness of these acylation reactions during solid phase peptide synthesis by stepwise methods, such as that of Merrifield (8) and its later developments (6,9,10), is essential to ensure a high overall yield of the final peptides.
  • the time course of the coupling reaction in each step is essentially unpredictable, being dependent on the sequence of the peptide, the type of amino acid added and the activation method used.
  • the ideal monitoring technique will offer continuous, non interactive monitoring of coupling, deprotection and washing steps by means of a simple set-up. It should not introduce additional reagent cycles or other time consuming procedures and it should occur in real time in order to allow instant, preferably automatic, control of the synthesis. In addition, the ideal monitoring method should be based on measurements of unreacted groups during coupling, as this gives the most sensitive indication of the completeness of the coupling reaction.
  • Titration methods have also been used for monitoring peptide syntheses. These take place in a non-destructive way on the entire batch of support resin and require no sampling. Titration of amino groups by picric acid followed spectrofotometrically (16), by perchloric acid followed potentiometrically (17) or by other means (see discussion in (16)) determines the quantity of unreacted amino groups present during coupling reactions or of free amino groups present after deprotection. Titration methods can thus give an accurate picture of the reactions taking place during a synthesis and even allow for feedback operations. They do, however, share the drawback of introducing additional reagent cycles to the peptide synthesis, some of which may be harmful to the peptide chain or the protecting groups employed.
  • FMOC protecting groups (19) used to protect the alpha-amino groups of the growing peptide chain, can be measured in the liquid phase during deprotection steps and during couplings (20,21). This, however, gives only limited and inaccurate information about the time course of couplings (it measures small changes in a large amount of circulating surplus of FMOC-protected amino acid, yielding a not very easily interpreted set of data), while the deprotection step, in contrast, is monitored accurately.
  • the yellow colour produced when the carboxyl-activating group dihydroxybenzotriazole (Dhbt) (3) is deprotonized by unreacted amino groups on the resin can be measured and used in real time monitoring of coupling reactions (4,22).
  • This elegant method is applicable to coupling reactions employing Dhbt-esters and can be used with other ester types as well if free Dhbt is added to the coupling suspension.
  • the method requires technically complicated optical monitors for measurements directly on the resin and requires the use of resins with high transparency or with well defined light reflection properties (22).
  • the technique puts some restraints on the chemistry used, since reactions where tertiary amine is added as a catalyst (e.g. during esterification of the first amino acid to the linker (4) for the purposes of protection) cannot be monitored in this way.
  • the technique is further of little use in the monitoring of washing steps.
  • the present invention therefore aims to provide an alternative approach to monitoring reactions in solid phase peptide syntheses.
  • Acidic and basic species are involved in the reactions occuring in solid phase peptide synthesis, so by monitoring the conductivity of the reaction solution an indication can be obtained of the rate of reaction for both removal of protecting groups and coupling of incoming amino acid groups. From this kinetic data, predictions can be made as to the expected extent of reaction, and synthesis conditions can be modified as appropriate if required.
  • the invention also provides a method of solid phase peptide synthesis, characterised by monitoring the electrical conductivity of the reaction solution.
  • the invention is suitable for use during peptide synthesis in a batch mode, but is also applicable to continuous flow methods of solid phase peptide synthesis.
  • the invention is primarily applicable for use with the FMOC protection strategy of peptide synthesis, which is currently regarded as the most successful peptide synthesis method and an example of which is described below, but can also be used in other solid phase peptide syntheses involving acidic and basic species, such as the BOC method. Both of these techniques are described in the article by Newton and Fox referred to above. A number of variants and modifications of the FMOC and BOC methods have been devised, using different catalysts, linking groups etc., as are known to those skilled in the art, and the invention is not intended to be limited to any particular method of this type.
  • it is the change in conductivity of the reaction solution due to the production of ion pairs in the solution during a coupling step which is monitored during the synthesis.
  • the coupling step is acid catalysed so as to allow formation of ion pairs between the deprotected growing peptide chain and the acid catalyst.
  • the catalyst conveniently used is hydroxybenzotriazole (HObt). It is thought that this catalyst, acting as a proton donor, forms an ion pair with the deprotected alpha-amino group of the growing peptide chain (i.e. an NH + group is created at the free end of the chain). The formation of these ion pairs will cause an initial increase in conductivity of the reaction solution on addition of the HObt catalyst. As the incoming amino acid reacts with the growing peptide chain during the coupling step, the ion pairs are removed as the alph-amino groups become involved in coupling. Thus, conductivity will fall again during coupling, and these changes in conductivity can be used to monitor the progress of the coupling reaction.
  • HObt hydroxybenzotriazole
  • the conductivity is conveniently measured by use of two spaced apart electrodes located in a reaction vessel for synthesis reaction solutions, and by applying an AC voltage across the electrodes.
  • an AC voltage across the electrodes.
  • the present invention also provides apparatus for the solid phase synthesis of peptides, characterised in that it comprises a reaction vessel having two spaced apart electrodes located therein; means for applying an AC voltage across the electrodes; means for amplifying and rectifying the AC voltage resulting from current flowing through a reaction solution in the vessel; and means for displaying the resulting AC signal value.
  • the electrode geometry is not critical, and electrodes can be fitted to any suitable reaction vessel, possibly forming part of an automatic or semi-automatic solid phase peptide synthesiser.
  • the amplification and rectification circuitry may be of conventional construction, as will be well known to those skilled in the art.
  • the display means may comprise any convenient form of display such as a numerical display, a visual display unit or a graphical display, as will also be well known to those skilled in the art.
  • two platinum flashed, tungsten electrodes are located in a reaction vessel with a spacing of 10mm, and an electrode voltage of 0.2 volts AC is applied, operating at 1 KHz.
  • the ion pairs are formed when the incoming amino acid to be coupled with the peptide chain being sythesised is in the form of an amino acid ester, since the ester group will then be released in its free acid form during coupling.
  • a suitable proton acceptor an ion pair will be formed between the released acid group and the proton acceptor, resulting in an increase in the electrical conductivity of the reaction solution due to the presence of the ion pairs.
  • a suitable proton acceptor might conveniently be a tertiary amine such as diisopropylethyl amine (DIEA) .
  • DIEA diisopropylethyl amine
  • the proton acceptor should not itself be acylated in the presence of the incoming amino acid.
  • DIEA fulfils this criterion and also, usefully, reacts only slowly with the base labile FMOC group often used to protect the alpha-amino group of a growing peptide chain.
  • the incoming amino acid is preferably in the form of a pentafluorophenol ester, however, the method of the present invention is equally applicable for monitoring reactions involving dihydroxybenzotriazole esters or symmetric anhydrides.
  • the measurements made during the monitoring of a synthesis may be fed back to a computer or other data processor, and data obtained from the measurements may be used to control the duration of subsequent synthesis steps.
  • the method of the present invention is particularly useful in computer-controlled, or other automatic or semi-automatic, peptide syntheses, since measurements can be carried out on the system in real time and then used to provide control feed-back so as to maximise the efficiency of a synthesis.
  • conductivity is conveniently measured by means of two spaced apart electrodes located in a reaction vessel for synthesis reaction solutions, an AC voltage then being applied across the electrodes. Again, by amplifying and rectifying the AC voltage resulting from current flow through a reaction solution, a measure of the conductivity of the solution can be obtained, conductivity being proportional to the AC signal value.
  • reaction vessel preferably takes the form of a flow-through chromatographic column having an upper and a lower electrode, one at each of its ends.
  • the upper electrode is preferably a ⁇ -shaped rod inserted in the upper end of the reaction vessel, such that reactants injected into the reaction vessel are guided into the ⁇ -shaped cavity provided by the electrode and onto the surface of a solid phase support resin contained in the reaction vessel. In this way, reactants can be delivered onto the reaction surface without "splashing".
  • the upper electrode is conveniently made of electroplated stainless steel.
  • the lower electrode preferably comprises an end piece located in the lower end of the reaction vessel, which end piece is made of PTFE (polytetrafluoroethylene) containing 30% graphite.
  • PTFE polytetrafluoroethylene
  • a quantity of 3-hydroxy 4- oxodihydrobenzotriazol (Dhbt) is preferably included in the reaction solvent used during deprotection steps, so as to increase conductivity of the reaction solution and hence aid accurate measurement.
  • the invention can thus provide a novel, simple, non ⁇ destructive method of detecting the rate of reactions for both the removal of protecting groups and the subsequent coupling of the incoming amino acid.
  • the method is relatively insensitive to reactant concentration; capable of operating on any scale of synthesis; very cheap; and capable of providing accurate kinetic data, which enables a prediction to be made as to the expected extent of reaction.
  • adaptation of the reaction vessel to accommodate this new method is relatively simple.
  • Figure 1 illustrates the reactions involved in the solid phase synthesis of peptides using an example of FMOC chemistry
  • Figure 2 is a schematic illustration of automatic solid phase peptide synthesiser apparatus in accordance with the present invention, for use in a method of the first version of the present invention
  • Figure 3 illustrates the reaction vessel of the apparatus of Figure 2
  • Figure 4 illustrates the control circuitry of a conductivity sensor for monitoring conductivity of reaction solutions in the reaction vessel of the apparatus of Figure 2;
  • FIGs 5, 6 and 7 are graphs of typical outputs from the conductivity sensor of the apparatus of Figure 2;
  • Figure 8 illustrates a reaction vessel for use in a method of the second version of the present invention
  • Figure 9 is a graph illustrating the change in conductivity with concentration of solutions of FMOC- glycine, pentafluorophenol free acid and Dhbt in a DIEA/DMF mixture;
  • Figure 10 illustrates the correlation found between conductivity in a reaction solution during a peptide synthesis and the number of FMOC groups inserted on the support resin during coupling reactions
  • Figure 11 illustrates the baseline drift in conductivity of solutions of free pentafluorophenol acid
  • Figure 12 shows the results of a continuous conductivity recording made in accordance with the second version of the present invention.
  • FMOC (9-fluorenylmethoxycarbonyl-), the structure of which is illustrated in Figure 1 , is used to protect the alpha-amino group of the growing peptide chain, the caryboxyl terminus of which is attached to a polymeric support, e.g. functionalised polystyrene or polyamide beads, as is shown at (1).
  • the beads will typically be poured into a reaction vessel, and will undergo the following reactions.
  • the FMOC protecting group is removed by reaction with a base, e.g. 20% piperidine in DMF. This is the deprotection step, shown at (2).
  • the product of deprotection is compound (3), which is a weak base.
  • the next amino acid to be linked to the growing peptide chain (in this case AA_) is generally added as the activated o-pentafluorophenyl (OPFP) ester (4), in the presence of a suitable catalyst.
  • This catalyst may be a weak acid such as HObt, in which case ion pairs will be formed between the weak acid catalyst and the weak base (3). This causes an increase in the electrical conductivity of the reaction solution which will then fall off again as the coupling reaction proceeds, which changes in conductivity can be used to monitor the course of the coupling reaction in accordance with the first version of the present invention.
  • the coupling reaction involves acylation of the peptide chain by the incoming amino acid to form compound (5).
  • the ester group of the incoming acid is released in its free acid form.
  • This free acid in the presence of a proton acceptor, forms ion pairs, the presence of which affects conductivity of the reaction solution and can be used to monitor the coupling reaction, in accordance with the second version of the present invention (see below).
  • the FMOC technique may of course be modified or varied in a number of ways.
  • the incoming amino acid may be added in forms other than the OPFP ester.
  • FIG. 2 illustrates schematically the overall layout of automatic solid phase peptide synthesiser apparatus for use in carrying out a batch synthesis such as that described in Figure 1.
  • the apparatus comprises a reaction vessel 7 and sources 8 of reagents, including different amino acids (represented as AA. , AA_, etc.), linked by tubes and valves arranged in such a way that specified quantities of specified reagents can be delivered to the reaction vessel 7 in a specified sequence by a positive nitrogen gas pressure delivery system under control of control means (not shown) in conventional manner.
  • On-off valve 9 is positioned in the supply tubes between sources 8 and the reaction vessel.
  • the control means also controls delivery of nitrogen gas to the reaction vessel for agitating the contents and for removal of reagents from the reaction vessel at appropriate stages.
  • Reaction vessel 7 is illustrated in further detail in Figure 3.
  • the vessel is generally of conventional construction, and comprises a generally conical glass vessel with a sintered glass frit 10 positioned at its lower end and a ground glass Quickfit (Trade Mark) joint 11 at the top.
  • This construction allows the polymeric support material, usually polystyrene or polyamide based resin beads, to which the growing peptide is attached, to be poured into the reaction chamber.
  • Connector 12 includes an upper inlet tube 15 and vessel 7 includes a lower outlet tube 16, both of which tubes are modified to take standard Omnifit screw-on connectors, enabling the vessel to be connected into the apparatus of Figure 2.
  • vessel 7 has a pair of platinum flashed, tungsten electrodes 17 and 18 cemented into the sides of the vessel, the inner ends of the electrodes being spaced apart by a distance of 10mm.
  • Electrodes 17 and 18 are designed to measure the conductivity of reaction solutions contained in the vessel 7, and are linked to a conductivity sensor having circuitry as shown in Figure 4.
  • the variation of conductivity with time is conveniently obtained as a graphical representation such as is illustrated in Figures 5, 6 and 7, e.g. on a printer associated with the conductivity sensor, but other representations of conductivity can also be obtained, as will be apparent to those skilled in the art.
  • the illustrated apparatus is used in generally conventional manner for batch synthesis of peptides.
  • the base of the peptide chain (i.e. amino acid AA..), attached to its polymeric support material in reaction vessel 7, is washed with DMF and dried to remove solvent.
  • the FMOC protecting group is removed by reaction with 20% piperidine in DMF, to produce compound (3).
  • the rate of removal of the FMOC group varies according to the conformation of the peptide at the particular stage in the synthesis. Slow removal indicates probable folding or insolubility of the growing peptide chain. If the rate is excessively slow, then the reaction time can be extended or the reaction repeated to ensure completion. The peptide is then ready for the addition of the next amino
  • the normal method is to add the amino acid (e.g. AA_) as an active OPFP-ester with hydroxybenzotriazole (HObt) as catalyst.
  • the HObt is a weak acid and this protonates the exposed peptide amino group, causing an increase in conductivity of the reaction solution as the acid-amino group ion pairs are formed (during this reaction, the support resin behaves essentially as an ion exchange resin).
  • the protonated amino group reacts with the incoming amino acid the conductivity of the reaction solution falls again.
  • the rate of fall of signal from the conductivity sensor is directly proportional to the rate of formation of the peptide bond, this can be used to calculate the half life of the reaction. From the half life the time required for the reaction to be completed to the required specification (e.g. greater than 99.5%) can be calculated.
  • the peptide and resin are washed to remove excess reagents and are then ready for the next addition cycle.
  • the FMOC technique may be modified or varied in a number of ways.
  • catalysts such as dicyclohexyl- carbodiimide (DCC) may be used in place of HObt, and amino acids may be added in forms other than OPFP-esters.
  • DCC dicyclohexyl- carbodiimide
  • the method also offers an advantage over many other monitoring techniques in that no additional chemical ingredients are involved in the monitoring process; the HObt is present as a catalyst anyway.
  • a typical output from the conductivity sensor shows several distinct regions, as illustrated in Figure 5.
  • Region (a) indicates the start of the coupling cycle when there is little or no output from the sensor.
  • Region (b) is where piperidine has been added, and the sensor output rises as the FMOC group is removed.
  • the level of the response at (b) indicates the quantity of growing peptide present.
  • the unbound products of this reaction are then removed by solvent washing - region (c). Since the released FMOC group forms weak carbonates in solution, its release causes an increase in conductivity, whereas its removal by solvent washing causes conductivity to fall again.
  • a second quantity of piperidine is then added at (d): this ensures complete removal of the FMOC groups. All the unbound products of these reactions are removed at (e) with solvent washes: this results in a further decrease in monitor output until the next amino acid derivative to be coupled is added at (f). This results in a large increase in conductivity, which falls off during (g) as the coupling reaction proceeds until completion at (h). The slope of the region at (g) seems to be proportional to the rate of the coupling reaction.
  • Figures 6 and 7 help to illustrate the use of the conductivity sensor and also some of the typical problems encountered in solid phase peptide synthesis.
  • the reference lettes (a) to (g) refer to regions of the graphs corresponding to those shown in Figure 5.
  • Figure 6 shows the case where the coupling reaction is proceeding slowly and shows how the sensor can be used to follow the coupling reaction and extend the reaction time if necessary.
  • Figure 7 shows the case where removal of the FMOC group proceeds more slowly than normal and indicates how the deprotection step can be automatically extended following the output of the conductivity sensor. We have discovered that this seems to give an indication of steric hindrance occuring in the growing peptide chain and therefore potentially can be used to gain further information about possible mechanisms involved in the coupling reaction.
  • the reaction vessel shown in Figure 8 is for use in a the present invention.
  • the vessel is designed as a flow- through chromatographic column having a 12mm I.D. glass tube 19 equipped with end pieces 20 made of PTFE containing 30% graphite. This material is in itself electrically conducting and thus serves as an electrode in the lower end of the reactor.
  • the upper electrode 21 is a U-profiled rod made of electroplated stainless steel. This electrode is inserted in the centre of the upper end of the reactor so that reactants injected into the reactor are guided into the ⁇ -shaped cavity of the electrode to the surface of the support resin without "splashing" of reactants.
  • the reaction vessel is also equipped with a filter 22 and electrode jacks 23.
  • a typical peptide synthesis might be carried out in the reaction vessel shown in Figure 8 and monitored as follows.
  • the monitoring method is based on continuous measurement of electrical conductivity of reaction solutions in the reaction vessel. It is shown that there is a close correspondence between the degree of acylation of the growing peptide chain (as determined from the number of FMOC groups released during deprotection) and the conductivity profile obtained during coupling of incoming amino acids to the growing peptide chain. Measurements taken are fed back to a computer so as to provide data for software control of the duration of the acylation, deprotection and washing steps involved in the synthesis.
  • Pentafluorophenyl (Pfp) esters of FMOC amino acids and the acid labile resin (4-hydroxymethyl phenoxyacetic acid on polydimethylacrylamide in kieselguhr, Pepsyn KA) were obtained from Milligen, Bedford, MA. FMOC-Ser and FMOC-Thr were delivered as dihydroxybenzotriazole (Dhbt) esters from the same manufacturer. Dhbt (free acid, analytical grade) was purchased from Fluka, Switzerland. Dimethylformamide (DMF); pentafluorophenol (Pfp-OH) ester (free acid); piperidine and 4- ( dimethyl Jaminopyridine (DMAP), all of reagent grade, were purchased from Merck, Darrmstadt.
  • DMF dimethylformamide
  • Pfp-OH pentafluorophenol ester
  • DMAP dimethyl Jaminopyridine
  • N,N-diisopropylethylamine (DIEA) "peptide synthesis grade" was from Applied Biosystems, Foster City, CA. The syntheses was peroformed on an EASY-PEP computer controlled continuous flow peptide synthesizer from Kem- En-Tec, Copenhagen. A model 525 conduct imeter from CRISON, Barcelona, was used for recording electrical conductivity in the reaction vessels and the voltage output was interfaced to the computer by means of one of the 12-bit A/D converters in the synthesizer.
  • the reaction vessel of Figure 8 is incorporated into a standard continuous flow set-up for solid phase peptide synthesis. Between 4 and 5 ml of pre-swollen resin containing approximately 50 umoles of linker per ml is used as the solid phase support for the synthesis. Just prior to injection into the reaction vessel, a 3-fold molar excess of FMOC-amino acid ester (approx. 100 mM) is dissolved in 1.5 times the resin volume of 15 mM DIEA in DMF. The injection is carried out with a pump speed of 2.5 ml per min and no recirculation of the ester is performed after injection of the entire volume into the reaction vessel. Excess reactant solution is employed to ensure an even distribution of the amino acid ester throughout the resin.
  • DMAP 4,4 dimethylaminopyridine
  • the proton acceptor should not itself be acylated.
  • DIEA tertiary amines fulfil this criterion, and for our experiment we chose DIEA.
  • This amine is often used as a catalyst in acylation reactions with symmetrical anhydrides (11), and it has been reported to react only very slowly with the base labile FMOC group used for protection of the amino functionality (T ,.: 10.1 hours for FMOC-Val-OH in 50% DIEA in DMF, (23)).
  • DMAP has been reported to cause partial racemization of some BOC-amino acids (24), but to our knowledge no such effect has been reported with DIEA used with FMOC-amino acids.
  • the concentration of the base should be kept as low as possible.
  • Figure 9 shows the conductivity of 15 mM DIEA in DMF as a function of the concentration of Pentafluorophenol (Pfp- OH), Dhbt (free acids) and FMOC-protected Glycine.
  • Pfp-OH Pentafluorophenol
  • Dhbt free acids
  • FMOC-protected Glycine The conductivity recorded with 100 mM Pfp-OH, Dhbt or FMOC- Glycine in DMF without any DIEA present was less than 10 uS/cm.
  • 15 mM DIEA a continuous non linear increase in conductivity was seen up to a concentration of at least 100 mM of these acids, with Pfp-OH giving the highest values.
  • the deviation from linearity of the conductivity as a function of the concentration of free acid was moderate up to about 50 mM of the acid.
  • the deviation from linearity can be incorporated in computer software used to control the experiment, and good corrections can be made up to at least 100 mM of free acid, which is well above the levels encountered in most reactions.
  • concentration of DIEA was set to 15 mM in all subsequent experiments.
  • Figure 11 shows the baseline drift (expressed as uS/cm x min) calculated after subtraction of the initial measurements from those made after 90 min.
  • the drift decreases as a function of the concentration of free Pfp-OH in the system (curve 24).
  • DMAP rather than DIEA was used as a solvent
  • a significantly higher baseline drift was observed (curve 25).
  • the drift rate was low compared to the rate of the signal recorded during the initial phase of the acylation reactions (about 50 uS/cm.min).
  • the concentration of Pfp-OH released during the reaction was typically in the range of from 25 to 100 mM, which is more than adequate to quench the baseline drift in DIEA/DMF solutions.
  • FIG. 12 A monitoration of all steps in a typical peptide synthesis is shown in Figure 12.
  • the synthesis in this case was of the tetrapeptide Gly-Glu-Leu-Ile, and conductivity is shown in arbitrary units (A.U.).
  • the acylation steps were terminated by the computer program when the rate of change in the monitor signal was less than 2% per hour. The rate was calculated as the averaged rate over the 50 latest subsequent measuring points recorded at 4-second intervals. In a similar manner, the washing steps after the acylation and the deprotection reactions were terminated when the rate of change was less than.10% per hour.
  • the monitor profiles of Figure 12 show nonidentical reaction rates for individual acylation steps.
  • the recorded slow rate of the isoleucine coupling (region 26) is in accordance with the common experience that this amino acid couples relatively slowly.
  • the esterification of the first amino acid (Gly) to the linker (region 27) proceeded at a moderate rate, ais would be expected for this type of reaction.
  • the latter reaction (and that involving isoleucine) did not reach the rate threshold of 2% per hour and was terminated instead when reaching an arbitrary time limit of 3 hours defined for the duration of any reaction.
  • peaks 28 indicate signals recorded during deprotection in 20% v/v piperidine in DMF. Deprotection is seen to cause an increase in conductivity, which is probably as a result of the formation of ionizable species during deprotection. The fact that this occurs means that conductivity measurements can be used to monitor and control both the coupling and the deprotection steps.
  • the omission of a recirculation procedure has the advantage that the pump of the synthesizer is made available for other tasks, e.g. for injection of reagents into another reactor.
  • the above described technique allows for determination of both the reaction rate and the amount of ester consumed. Both recordings are generated by the released ester groups rather than by the unreacted amino groups on the resin. While this is less than ideal with respect to precision, it is believed that the simultaneous determination of both the rate and the amount provides a satisfactory basis for the decision of whether or not a reaction is completed. As an additional benefit, the technique is highly suited for monitoring deprotection and washing procedures. This defines precisely when to stop washing and, in our experience, may cut solvent consumption, compared to arbitrarily defined washings, by about 50%.

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Abstract

L'invention concerne un procédé de contrôle de réactions dans la synthèse de peptides en phase solide, consistant à contrôler la conductivité électrique de la solution de réaction. Les mesures de conductivité fournissent une indication du progrès de réactions jusqu'à leur terme, et peuvent ensuite être utilisées afin de permettre une régulation en retour dans des systèmes de synthèse automatique ou semi-automatique. On peut utiliser ledit procédé dans des synthèses par lots, ou dans des systèmes à écoulement continu. Ledit procédé est particulièrement adapté dans des synthèses basées sur la stratégie de protection FMOC (9 'fluorenylméthoxycarbonyle'). La conductivité contrôlée peut être due à la production de paires d'ions dans la solution de réaction, pendant l'étape d'accouplement. Ces paires d'ions peuvent être formées entre une chaîne peptidique croissante et un catalyseur d'acide utilisé pour catalyser l'étape d'accouplement, ou entre des groupes d'esters libérés par un acide aminé entrant, pendant l'accouplement, et un accepteur de protons présent dans ladite solution de réaction. Selon l'invention, on peut également contrôler les étapes de lavage et de déprotection dans une synthèse. L'invention concerne en outre un appareil adapté à une utilisation selon le procédé précité.
PCT/GB1990/000422 1989-03-21 1990-03-20 Contrôle de reactions dans la synthese de peptides en phase solide par mesures de conductivite WO1990011291A1 (fr)

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GB8906514.8 1989-03-21
GB898906514A GB8906514D0 (en) 1989-03-21 1989-03-21 Conductivity sensor for peptide synthesis
GB8916737.3 1989-07-21
GB898916737A GB8916737D0 (en) 1989-03-21 1989-07-21 Peptide synthesis
GB898919189A GB8919189D0 (en) 1989-08-23 1989-08-23 Peptide synthesis
GB8919189.4 1989-08-23

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994001214A1 (fr) * 1992-07-06 1994-01-20 Beckman Instruments, Inc. Procede de surveillance de la reaction et de l'ecoulement d'un processus en ligne
US5472880A (en) * 1988-05-24 1995-12-05 The Queen's University Of Belfast Conductance measurements in organic solvents
EP0648221A4 (fr) * 1992-06-30 1996-04-24 Applied Biosystems Surveillance du trityle dans la synthese automatique de polynucleotides.
WO1997011777A1 (fr) * 1995-09-29 1997-04-03 Pharmacopeia, Inc. Reacteur pour syntheses en phase solide et son mode d'utilisation
WO1997017310A1 (fr) * 1995-11-06 1997-05-15 Versicor, Inc. Sequestration reversible a base de charge sur un support solide
GB2379018A (en) * 2001-08-10 2003-02-26 Univ Hull Monitoring chemical reactions in a microreactor
CN105527320A (zh) * 2015-12-18 2016-04-27 盐城师范学院 四通道固相化学反应电导率分析方法
CN119375306A (zh) * 2024-12-27 2025-01-28 天津凯莱英医药科技发展有限公司 多肽固相合成在线监测方法及其在线监测系统

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JPS60115852A (ja) * 1983-11-29 1985-06-22 Olympus Optical Co Ltd 反応用カラムを含むフロ−ラインのモニタリング装置
WO1989011647A1 (fr) * 1988-05-24 1989-11-30 The Queen's University Of Belfast Mesures de conductance dans des solvants organiques

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US395642A (en) * 1889-01-01 Wire splicer and cutter
US4262253A (en) * 1978-04-26 1981-04-14 Phillips Petroleum Company Constant alternating current conductivity detector for a chromatograph
JPS60115852A (ja) * 1983-11-29 1985-06-22 Olympus Optical Co Ltd 反応用カラムを含むフロ−ラインのモニタリング装置
WO1989011647A1 (fr) * 1988-05-24 1989-11-30 The Queen's University Of Belfast Mesures de conductance dans des solvants organiques

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Acta Chem. Scand., Volume 23, No. 8, 1969, K. BRUNFELDT et al.: "Process Control in the Solid Phase Peptide Synthesis by Titration of Free Amino Groups", pages 2906-2907 *
CHEMICAL ABSTRACTS, Volume 77, No. 15, 9 October 1972, (Columbus, Ohio, US), K. BRUNFELDT et al.: "Automatic Monitoring of Solid Phase Synthesis of a Decapeptide", see page 484* Abstract 102222f, & FEBS (Fed. Eur. Biochem. Soc.) Lett. 1972, (2), 238-44* *
CHEMICAL ABSTRACTS, Volume 99, No. 3, 18 July 1983, (Columbus, Ohio, US), M.A. ZEWAIL: "Solidphase Synthesis of C-Terminal Nonapeptide Insulin B22-B30 Monitoring by Titration Method", see page 662* Abstract 22907x, 24(4-6), 347-56* *
PATENT ABSTRACTS OF JAPAN, Volume 9, No. 269 (P-400)(1992), 26 October 1985; & JP-A-60115852 (Olympus Kogaku Kogyo K.K.) 22 June 1985 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5472880A (en) * 1988-05-24 1995-12-05 The Queen's University Of Belfast Conductance measurements in organic solvents
EP0648221A4 (fr) * 1992-06-30 1996-04-24 Applied Biosystems Surveillance du trityle dans la synthese automatique de polynucleotides.
WO1994001214A1 (fr) * 1992-07-06 1994-01-20 Beckman Instruments, Inc. Procede de surveillance de la reaction et de l'ecoulement d'un processus en ligne
US5447692A (en) * 1992-07-06 1995-09-05 Beckman Instruments, Inc. On-line process flow and reaction monitor
WO1997011777A1 (fr) * 1995-09-29 1997-04-03 Pharmacopeia, Inc. Reacteur pour syntheses en phase solide et son mode d'utilisation
WO1997017310A1 (fr) * 1995-11-06 1997-05-15 Versicor, Inc. Sequestration reversible a base de charge sur un support solide
GB2379018A (en) * 2001-08-10 2003-02-26 Univ Hull Monitoring chemical reactions in a microreactor
EP1283071A3 (fr) * 2001-08-10 2004-06-16 Micro Chemical Systems Limited Surveillance des réactions chimiques dans des canaux d'un micro-réacteur
US6989090B2 (en) 2001-08-10 2006-01-24 Micro Chemical Systems Limited Method to monitor chemical reactions in a micro-reactor by measuring an electrical current
GB2379018B (en) * 2001-08-10 2006-02-22 Univ Hull Monitoring of chemical reactions
CN105527320A (zh) * 2015-12-18 2016-04-27 盐城师范学院 四通道固相化学反应电导率分析方法
CN119375306A (zh) * 2024-12-27 2025-01-28 天津凯莱英医药科技发展有限公司 多肽固相合成在线监测方法及其在线监测系统

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