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WO1994011388A1 - Support pour la synthese de polymeres modulaires - Google Patents

Support pour la synthese de polymeres modulaires Download PDF

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Publication number
WO1994011388A1
WO1994011388A1 PCT/AU1993/000546 AU9300546W WO9411388A1 WO 1994011388 A1 WO1994011388 A1 WO 1994011388A1 AU 9300546 W AU9300546 W AU 9300546W WO 9411388 A1 WO9411388 A1 WO 9411388A1
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WO
WIPO (PCT)
Prior art keywords
synthesis
support
modular
peptide
acid
Prior art date
Application number
PCT/AU1993/000546
Other languages
English (en)
Inventor
H. Mario Geysen
N. Joe Maeji
Andrew M. Bray
Robert M. Valerio
Original Assignee
Chiron Mimotopes Pty. Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chiron Mimotopes Pty. Ltd. filed Critical Chiron Mimotopes Pty. Ltd.
Priority to EP93923418A priority Critical patent/EP0672049A4/fr
Priority to JP6511520A priority patent/JPH08505604A/ja
Priority to AU53315/94A priority patent/AU680450B2/en
Publication of WO1994011388A1 publication Critical patent/WO1994011388A1/fr

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Classifications

    • 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
    • C07K1/042—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers characterised by the nature of the carrier
    • 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
    • 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
    • B01J2219/00306—Reactor vessels in a multiple arrangement
    • B01J2219/00322—Reactor vessels in a multiple arrangement the individual reactor vessels being arranged serially in stacks
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00504—Pins
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00504—Pins
    • B01J2219/00506—Pins with removable crowns
    • 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
    • B01J2219/0054—Means for coding or tagging the apparatus or the reagents
    • 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
    • B01J2219/0054—Means for coding or tagging the apparatus or the reagents
    • B01J2219/00545—Colours
    • 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
    • B01J2219/0054—Means for coding or tagging the apparatus or the reagents
    • B01J2219/00554—Physical means
    • 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
    • B01J2219/0054—Means for coding or tagging the apparatus or the reagents
    • B01J2219/00563—Magnetic means
    • 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
    • B01J2219/00718—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
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B70/00—Tags or labels specially adapted for combinatorial chemistry or libraries, e.g. fluorescent tags or bar codes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • This invention relates to a solid surface for the synthesis of peptides thereon, and in particular relates to a r od or pin having a plurality of surfaces on which peptides may be synthesized and assayed efficiently and economically.
  • Geysen. EP 198855. disclosed a method for the simultaneous synthesis of a large number of different peptides. Basically, this method involves the synthesis of peptides on a solid polymeric surface, such as polyethylene, which may be molded into the shape of a rod or pin. In a preferred embodiment of the method, these rods or pins are positioned in a holder so that they form a 12 by 8 matrix, with the rods or pins being positioned so that the spacing corresponds to that of the wells of microtiter plates which are widely used for ELISA (enzyme- linked immunosorbent assay) tests.
  • ELISA enzyme- linked immunosorbent assay
  • the quantity of peptide covalently bonded to the solid polymer surface by this method is sufficient to allow reaction of the peptide with specific binding entities such as antibodies to be readily detected.
  • specific binding entities such as antibodies
  • the quantity of peptide synthesized on each rod is relanvely small (typically less than 1 umole)
  • the ability to reuse the synthesized peptide after a test compensates for the small quantity of peptide on the rod.
  • the quantity of peptide needs to be greater. Examples of such applications include the removal of the peptide from the rod and recovery of specific oinding entities for further testing. Modifications to the process of synthesis and testing of the peptides were disclosed in PCT WO91/04266.
  • polyacrylic acid was grafted to the surface of a solid polyethylene support using gamma-irradiation.
  • the region on the rod on which peptide was grafted was completely defined by the volume of reagent used for the coupling of specific amino acids to the growing peptide. or more accurately, the depth into which the rod dipped into the amino acid solutions. As this depth inevitably varied slightly from cycle to cycle in the synthesis, the result was a small region on the rod where the peptide synthesized may have had appreciable amounts of deletion peptides (that is. peptides whose sequence varied from that intended by having one or more residues absent) present because slightly less of one of the activated amino acid solutions was dispensed in one or more cycles.
  • a further disadvantage of the earlier system is that the polymer layer grafted onto the rods during the radiation process is readily solvated by many sol vents and as a consequence, solvents will migrate upwards through this layer by capillary action. This results in depletion of the reservoir of reagent and consequently, as described above, a larger zone or uncertain .synthesis quality is created. In addition, unless extreme care is taken in extensively washing these rods, the polymer layer acts as a reservoir of the solvent used in synthesis leading to the contamination of subsequent solutions.
  • One aspect of the invention is an improved synthesis support, having a plurality of independent synthesis surfaces, which permits the simultaneous syntheses of peptides (or other modular poiymers) having different N- or C-groups, having different linkages to the support [e.g., permanent links, cleavable links, etc.), or otherwise differing in features other than sequence. Additionally, one can employ the new supports to make multiple copies of a collection of polymers.
  • Another aspect of the invention is a cleavable link which enables one to cleave modular polymers from the synthesis supports of the invention, leaving the modular polymer with an amide at the cleaved end.
  • Figure 1 depicts plan and cross-section views of an active surface com- ponent of the invention.
  • Figure 2 depicts plan views of support rod of the invention.
  • Figure 3 depicts ELISA results obtained using multiple epitope libraries prepared by the method of the invention.
  • Figure 4 depicts ELISA binding inhibition results obtained using mul- tiple epitope libraries prepared by the method of the invention Modes of Carrying Out The Invention
  • modular polymer refers to a polymer composed of non- identical subunits selected from a group of monomers. Modular polymers are generally .synthesized one monomer at a time.
  • monomer refers to a molecule which may be coupled or condensed to form an oligomer. To provide diversity, monomers are selected from sets whicn contain at least four distinct members. Suitable monomer sets include conventional D- and L-amino acids, nucleic acids/nucleotides. carbohydrates, nonconventionai D- and L-amino acids (e.g., cyclohexylalanine. benzhydrylglycine, chloroalanine. and the like), and "peptoids" as described in WO91/19735 (incorporated herein by reference).
  • conventional amino acid refers to the amino acids alanine (A), cysteine (C), aspartic acid (D).
  • nonconventionai amino acid refers to amino acids other than conventional amino acids. Presently preferred nonconventionai amino acids are
  • Nle L-norleucine
  • Aabu ⁇ -aminobutyric acid
  • Hphe L-homophenylalanine
  • Nva L-norvaline
  • Dpro D-proline
  • Dgln D-glutamine
  • Dthr D-threonine
  • Dval D-valine
  • Mleu L- ⁇ -methylleucine
  • Mmet L- ⁇ -methylmethionine
  • Mval L- ⁇ -methylvaline
  • Mtrp L- ⁇ -methyltryptophan
  • Dmpro D- ⁇ -methylproline
  • Dmgln D- ⁇ -methylglutamine
  • Dmtrp D- ⁇ -methyltryptophan
  • Dmtyr D- ⁇ -methyltyrosine
  • Nmala L-N-methylalanine
  • Nmcys L-N-methylcysteine
  • Nmile L-N-methylisoleucine
  • Nmlys L-N-methyllysine
  • Nmleu L-N-methylleucine
  • Nmmet L-N-methylmethionine
  • Nmval L-N-methylvaline
  • Nmt ⁇ L-N-methyltryptophan
  • Nmaib N-methylaminoisobuty ⁇ c acid:
  • Dnmglu D-N-methylglutamic acid
  • Dnmphe D-N-methylphenylalanine
  • Dnmpro D-N-methylproline
  • Dnmgln D-N-methylglutamine
  • Nala N-methylglycine ( sarcosine ):
  • Nasp N-(carboxymethyl)glycine:
  • Nmet N-(2-methylthioethyl)glycine
  • Nhser N-(hydroxyethyl)glycine
  • Ngln N-(2-carbamylethyl)glycine
  • Nthr N-(1-hydroxyethyl)glycine
  • Ncys N-(thiomethyl)glycine
  • Ncpro N-cyclopropylglycine
  • Ncbut N-cyclobutyglycine
  • Nchex N-cyclohexylglycine
  • Nchep N-cycloheptylglycine
  • Ncoct N-cyclooctylglycine
  • Ncdec N-cyclodecylglycine
  • Ncund N-cycloundecylglycine
  • Ncdod N-cyclododecylglycine
  • Nbhm N-(2,2-diphenylethyl)glycine
  • Nbhe N-(3,3-diphenylpropyl)glycine:
  • Nnbhm N-(N-(2,2-diphenylethyl)carbamylmethyl)glycine:
  • Nnbhe N-(N-(3,3-diphenylpropyl)carbamylmethyl)glycine
  • Nbmc 1-carboxy-1-(2,2-diphenylethylamino)cyclopropane
  • Naeg N-(2-aminoethyl)glycine.
  • active surface means a surface which is derivatized or otherwise rendered suitable for synthesis of modular polymers. It is "adapted” for use in the synthesis of modular polymers if modular polymers can be efficiently synthesized thereon.
  • the component which carries the active surface may be homogeneous or heterogeneous in composition.
  • the active surface may be bonded or grafted to a supporting structure or surface.
  • the active surface may be radiation grafted to any suppo ⁇ ing structure (which may be, for example, an "inert surface” as defined below).
  • inert surface refers to a surface which is stable to the modular polymer synthesis conditions, and does not react. Suitable inert surfaces include without limitation polyethylene, polyolefins. cellulose acetate, wool, cotton, chitin, and the like.
  • a plurality of supports for use in the synthesis of peptides or other polymeric compounds thereon which supports comprise an inert surface and a set of active surfaces each comprising an active region on which said synthesis may take place.
  • the inert surface is typically provided in the form of a rod or pin of generally cylindrical shape, having space for a plurality of active surface components.
  • the inert support is capable of retaining 2 to 20 active surface components, more preferably about 5 active surface components.
  • the support may have a cross-section that is circular, rectangular, or any other shape: circular and square are preferred.
  • the support may further be provided with projections or protrusions to assist in placing, locating and retaining the active surface components. Further, the support may be provided with indentations and/or incisions to impart sufficient flexibility to the support that the active surface components may be "snapped" on.
  • the active surface components may be positioned on a nail-shaped pin, followed by mounting the pin in a supporting array, where the "head" of the pin is shaped to retain all active surface components (the active surface components may be later removed by either removing the pin from its support, or by cleaving or removing the head of the pin).
  • the support rods may be grouped in an array to facilitate parallel processing, both during synthesis of the modular polymers and during their assay.
  • the supports (pins") are mounted in an 8 x 12 array on a block which matches the spacing of wells in a microwell assay plate.
  • the rods are mounted by press-fit or friction-fit into holes drilled or molded in the support block.
  • the rods may include retaining flanges or projections to insure that each rod projects the same distance from the block surface.
  • Alternative mounting means include threading the rods and holes, adhesives. one-piece molding, magnetic coupling, and the like. Other array formats are also considered within the scope of this invention.
  • the support rods may be positioned on a continuous belt, or may be gripped individually by robotic manipulators, to simplify automated handling.
  • the active surfaces are provided in a form which may easily be attached to (and removed from) the inert support by friction fitting or snapping into place.
  • other forms of attachment may be used, such as adhesive (which may or may not be permanent), heat fusing, threading (e.g., like a nut and bolt), Velcro®. slot and key, and magnetism.
  • adhesive which may or may not be permanent
  • heat fusing heat fusing
  • threading e.g., like a nut and bolt
  • Velcro® Velcro®. slot and key
  • magnetism magnetism
  • one may employ inert supports and active surface components having ferrous cores, and retain the active surface components by electromagnetism.
  • coupling reactions to the active surfaces may be accelerated by modulating the electric current thereby inducing vibration of the pins. Effective vibration frequencies range from about 40 Hz to 60 KHz or more, preferably about 50-60 Hz.
  • the supporting rods may be fashioned from, or affixed to, piezoelectric transducers which vibrate at the desired frequency when activated.
  • the active surface components are preferably annular in shape, but may be formed in any shape which can be retained on the inert support.
  • active surface components may be semiannular (e.g., completing about 270°) and still be retained by press-fit methods.
  • Active surface components which are retained by magnetism need not be completely annular.
  • the active surface components are preferably shaped to increase their surface area, so that they have a greater surface area than a cylinder of the same diameter. In one embodiment of the invention the active surface components are vaned or "gear" shaped, in order to maximize their surface area.
  • the inner surface (facing the inert support) may be provided with flanges, ledges and/or other surfaces which may engage corresponding surfaces on the inert support.
  • the terminal active surface component may have a different shape, because the inert support need not pass entirely through the component.
  • the terminal active surface component may be hemispherical.
  • Figure 1 illustrates one presently preferred embodiment of the active surface component (1 ).
  • Fig. 1A depicts a plan view of the component having a thickness t of 2.5 mm. a maximum outer diameter of 5.5 mm, a minimum internal diameter (between opposed internal projections (2)) of 2.0 mm. with a maximum internal diameter of 3.0 mm.
  • Figure 1 B depicts a cross section of the component The component is molded from polyethylene with 16 external "teeth" (3) and 4 internal teeth (2) arranged symmetrically.
  • Figure 2 illustrates one presently preferred embodiment of the support rod (10), designed for use with the components (1) of Fig. 1.
  • Rod 10 comprises a primary shaft having a diameter of about 3.8 mm. and a length of about 32.3 mm. End 12 is rounded to facilitate insertion into the support block, and is provided with flange 13 to insure uniform insertion.
  • Flange 13 is positioned about 9.8 mm from end 12, and measures 1.1 mm in thickness by 6.0 mm in outer diameter.
  • the secondary shaft (14) is coaxial with primary shaft 11, and extends about 21.0 mm therefrom.
  • Secondary shaft 14 has a diameter of about 2.0 mm, and is provided with a plurality of projections 15 of generally cylindrical shape, having a diameter of about 0.5 mm and extend -about 0.85 mm from the surface of the secondary shaft 14. These projections 15 serve to locate and position components 1 on secondary shaft 14. Secondary shaft 14 is further provided with a generally frustroconical end cap 16 having a minimum diameter of about 1.0 mm and a maximum diameter of about 2.4 mm, sloping at about 30°. End cap 16 facilitates addition of the components 1 to the support 10, and is responsible for retaining the bottom-most component.
  • the active surfaces may be of identical or different compositions, depending upon the chemistry to which they will be subjected.
  • the coating can be made of any of the porous resins which are used for conventional solid phase peptide and/or nucleic acid synthesis. Because these resins are porous, the surface area of the active region is increased dramatically and so allows a much greater yield of modular polymer.
  • the use of this embodiment of the invention makes it particularly convenient to change the chemistry used in the synthesis, and indeed, change the class of polymeric compounds to be synthesized by selecting the appropriate resin with which to coat the active surface component.
  • porous resins which may be used in such coatings include benzhydrylamine-polysty- rene resin and polyacrylamide gel inside kieselguhr.
  • Suitable surface materials include, without limitation, polyethylene glycol. cellulose and other natural polymers. Merrifield resin. Rink resin and polymers of acrylic acid, methylacrylate. methacrylic acid, methyl methacrylate. dimethylacrylamide. styrene, hydroxyethylacrylate, hydroxyethylmethacrylate. hydroxypropylmethacrylate. hydroxyethylmethacrylamide. methylmethacrylate. and polyethyleneglycol monomethacrylate. and the like, and combinations thereof.
  • the component bearing the active surface need not be homogeneous.
  • structural supports fashioned from not just polyethylene, polypropylene and its copolymers but also Teflon® (polytetra- fluoroethylene) or any other stable inert surface.
  • the active surface may then be attached to the supporting surface by any available means, including sintering, adhesive, heat fusing, and the like.
  • the presently preferred method is grafting, by placing the support surface in a solution of solvent and active surface material and irradiating the mixture with gamma radiation.
  • Preferred solvents are water, methanol (MeOH), H 2 O/MeOH mixtures, dimethylformamide (DMF), and dimethylsulfoxide
  • the surfaces are the n modified, if desired, for the selected coupling chemistry.
  • the modular polymers may be "permanent” (i.e., not easily removed from the surface) or "cleavable” (designed for facile cleavage and removal from the support).
  • Cleavable modular polymers will generally have a linkage to the active surface which facilitates cleavage from the surface under conditions not experienced during synthesis of the modular polymer.
  • Different linking chemistries may also provide for different N-terminal and C-terminal groups (in the case of peptides).
  • Cleavable mod ular polymers may be provided with labeling groups for detection, or binding ligands for purposes of separation and purificauon.
  • cleavable modular polymers may be biotinylated (or tagged with another similar ligand) to facilitate purification (e.g., using a streptavidin column), or labeled with fluorescein or radioactive atoms to simplify detection in a binding assay.
  • cleavable modular polymers may be biotinylated (or tagged with another similar ligand) to facilitate purification (e.g., using a streptavidin column), or labeled with fluorescein or radioactive atoms to simplify detection in a binding assay.
  • fluoro.sceinated. radioactive cleavable to acidic terminal groups
  • cleavable to amide terminal groups cleavable to neutral terminal groups.
  • lysine having a protected ⁇ -amino group (e.g., with BOC) is coupled to the support through the ⁇ -amino group.
  • Pro whose carboxy terminus is esterified with a suitably reactive spacer ("X") having an orthogonally protected (e.g., Fmoc) amino group is then coupled to the Lys carboxylate function.
  • Cleavage is effected by removing the BOC (or other group) protecting the Lys ⁇ - amino group, and neutralizing the resulting -NH 3 + .
  • the amine attacks the Pro carbonyl and displaces the Pro, and with it, the modular polymer.
  • a diketopiperazine moiety is left attached to the active surface.
  • This linkage may be used for any type of modular polymer.
  • the general formula for a bound modular polymer is (spacer)—Support
  • M is a monomer
  • n is an integer (preferably 2-30. inclusive)
  • Z is a protecting group.
  • the spacer is optional.
  • cleavable linkages may be employed.
  • photocleavable linkages which cleave upon exposure to light at a selected frequency.
  • linkages, and the linkages described above are generally most suitable for use with peptide- and peptoid-based modular polymers, but may also be used with nucleic acids.
  • the modular polymer is a nucleic acid, it may be conveniently removed by providing a cleavable linkage in the form of a restriction enzyme recognition site.
  • Nucleic acid modular polymers may also include poiymerase promoter/binding sites and amplification pnmer hybridization sites, to facilitate dupiex formation and amplification (e.g., by PCR).
  • the active surfaces may also carry identifying features to facilitate differentiation between different types of surface.
  • active surfaces of different types may have different colors and/or patterns, different sizes and/or shapes. different degrees of adherence to the pin or rod (e.g., requiring a different amount of force to remove a surface from a pin), different degrees of magnetization, etc.
  • Surfaces that differ in color and/or pattern may be separated by hand on inspection. Colors may be achieved by including dyes in the active surface component, either in the active surface layer or in the supporting structure underneath, or both.
  • Patterns should generally be simple, and may be obtained, e.g., by dying only half of each component
  • Surfaces that differ in degree of adherence may be separated from the pin or support by a measured shake or impact (removing first those surfaces that are loosely attached, followed by a more vigorous shake or impact to remove surfaces that are more tightly bound).
  • Surfaces that differ in size and/or shape may be separated by a simple screen that allows small. components to pass, while retaining larger components.
  • surfaces that are magnetized may be separated from non- magnetized surfaces using a magnet, electromagnet or ferrous metal. The last two systems are more suitable to automation.
  • one may prepare a collection or set of modular polymers as described above, in several different forms, in one operation. The different forms may then be easily separated for further research.
  • modular active surface components and inert components has several advantages. First, it permits one to prepare each component under optimal conditions, using the materials most suitable for the function of each component For example, the active surface components need not display the rigidity desired in the support rods or support blocik: by modular construction, the rods may be made of rigid materials, while the active surface components may be made of softer materials which are optimized for use as synthesis surfaces.
  • Another major advantage of manufacturing the portion providing the active region as a separate entity is the minimization of cross-contamination of solutions.
  • the polymer layer grafted onto the rods during the radiation process as described in EP 198855 is readily solvated by many solvents and as a consequence, solvents will migrate upwards through this layer by capillary action. This results in depletion of the reservoir of reagent and consequently, as described above, a larger zone of uncertain synthesis quality is created. Also, unless extreme care is taken in extensively washing these rods, the polymer layer acts as a reservoir of the solvents used in synthesis leading to the contamination of subsequent solutions. Where the portion providing the active region is manufactured separately as described herein, this migration of solvents and reagents cannot take place.
  • the portion providing the active region of the rod that is. the region of the rod on which the peptide or other polymeric molecule is to be synthesized, is a cylinder with a radius of 2 mm and a height of 4 mm, the surface area of which active region is 61.8 mm 2 (assuming that only one end of this cylinder is available for synthesis).
  • the surface area becomes 81.8 mm 2 ( 1.3 times the area of the solid cylindrical portion ). The surface area of the portion providing the active region can be increased even further by modification of the shape of this portion of the rod.
  • the surface area available for synthesis is increased to 124 mm 2 (twice the area of the unmodified cylindrical portion). It will be apparent that the surface area of the active region of the rod can be further increased by further modification of its geometric shape, and equally that such modifications can be made by molding the rod in its final desired shape or by machining a molded rod into its final desired shape.
  • the portion providing the active region of the rod is made by joining small particles of solid materials together, for instance, by sintering using pressure or heat or both.
  • This can be particularly useful where it is desired to use particular harsh chemistries or corrosive solvents.
  • glass is resistant to most solvents which would make most conventional plastic materials unstable.
  • an active region could be made by sintering together small spherical beads of glass. This could then be treated, for instance, by functionorfal-zing the surface with an amino-silane, to make it suitable as a base on which the peptide or other polymeric compound could be synthesized.
  • the inactive region would be made from a particularly resistant plastic such as polytetrafluoroethylene. In this way. a material such as glass, which would be an unsuitable material for the inactive region, can be used with advantage in the active region.
  • a further advantage of this .particular embodiment of the invention is the large increase in the surface area/volume ratio of the active region can be achieved.
  • 8885 spherical rigid particles each with a radius of 0.1 mm would occupy the volume of the portion providing the active region of the rod if close packed together.
  • the surface area of these spheres would be 1 1 16.4 mm 2 , 17.8 times the surrace area of a solid cylindrical portion. Decreasing the size of the particles to be sintered together, would provide a corresponding increase in the surface area available tor synthesis. For example, decreasing the radius of the rigid .spheres to be sintered to 0.055 mm increases the surface area to 2233 mm", about 35.5 times mat or the solid cylinder.
  • Polyethylene pins were molded as depicted in Fig. 2, and mounted in 8 x 12 blocks as described in WO91/04266.
  • cleavable crowns Removable synthesis surfaces for cleavable peptides ("cleavable crowns") were molded as depicted in Fig. 1. using polyethylene, and were then radiation-grafted as described in EP 198855 with methacrylic acid/dimethylacrylamide
  • HMB hydroxytmethylbenzoic acid
  • non-cleavable crowns Removable synthesis surfaces for non-cleavable peptides ("non-cleavable crowns") were molded as depicted in Fig. 1. using polyethylene, and were then radia- tion-grafted as described in EP 198855 with 30% hydroxypropylmethacrylate in
  • each pin was loaded with four non-cleavable crowns, followed by one cleavable crown.
  • the resulting synthesis structure was then used to synthesize a set of overlapping octapeptides derived from the Neisseria gonorrhea C30 strain pilin protein, amino acids 30-52 (RAQVSEAILLAEGQKSAVYEYYLNHGKWP).
  • any given pin carried five crowns having identical octapeptides, with each pin carrying an octapeptide different from the octapeptides carried on other pins.
  • the non-cleavable crowns were removed from the 5-position pins and were placed in corresponding positions on blocks holding 1-position pins (see WO91/ 04266) to provide four identical epitope libraries. Each block was tested for antibody binding by ELISA using a 1/20.000 dilution of a rabbit anti-piilin antiserum. The results (shown in Fig. 3) demonstrate mat all four sets of peptides are essentially identical.
  • Cleavable crowns were men removed from the pins, and the peptides removed. Each crown was treated with 800 uL of cleavage solution (0.1 M NH 4 HCO 3 , 40% CH 3 CN. pH 8.4). Aliquots were submitted to amino acid analysis. HPLC demonstrated high leveis of purity. The typical yield of peptide was about 600 nmole/crown.
  • Fig. 4 a concentration of 35 nmole/mL of the cleaved peptides

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Peptides Or Proteins (AREA)
  • Polyoxymethylene Polymers And Polymers With Carbon-To-Carbon Bonds (AREA)
  • Graft Or Block Polymers (AREA)
  • Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Un support pour la synthèse de multiples ensembles de plusieurs polymères modulaires, y compris des peptides, est constitué de composants généralement annulaires présentant des surfaces actives, maintenus coaxialement par des tiges de maintien. L'invention concerne également un polymère moléculaire lié, ayant la formule (I) dans laquelle chaque M représente indépendamment un monomère; n représente un nombre entier compris entre 2 et 100; et Z représente un groupe protecteur.
PCT/AU1993/000546 1992-11-06 1993-10-25 Support pour la synthese de polymeres modulaires WO1994011388A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP93923418A EP0672049A4 (fr) 1992-11-06 1993-10-25 Support pour la synthese de polymeres modulaires.
JP6511520A JPH08505604A (ja) 1992-11-06 1993-10-25 モジュラーポリマーを合成するための支持体
AU53315/94A AU680450B2 (en) 1992-11-06 1993-10-25 Support for the synthesis of modular polymers

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US97275592A 1992-11-06 1992-11-06
US07/972,755 1992-11-06

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JP (1) JPH08505604A (fr)
AU (2) AU680450B2 (fr)
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HU (1) HUT73150A (fr)
NZ (1) NZ257077A (fr)
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Cited By (20)

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US5609826A (en) * 1995-04-17 1997-03-11 Ontogen Corporation Methods and apparatus for the generation of chemical libraries
WO1998018550A1 (fr) * 1996-10-30 1998-05-07 Chiron Corporation Marquage par transpondeur de constituants utilises dans la synthese de composes
WO1998038158A1 (fr) * 1997-02-26 1998-09-03 Eli Lilly And Company Procede pour la preparation de composes pharmaceutiques
WO1999034214A1 (fr) * 1997-12-31 1999-07-08 Qiagen Genomics, Inc Embouts en phase solide et utilisations correspondantes
US5961923A (en) * 1995-04-25 1999-10-05 Irori Matrices with memories and uses thereof
US6054264A (en) * 1993-05-10 2000-04-25 Chiron Corporation Methods of typing hepatitis C virus and reagents for use therein
EP1018365A2 (fr) * 1995-10-26 2000-07-12 University Of Hertfordshire Particules codées destinées à la poursuite des séquences de procédés lors de la préparation de biblothèques de composés combinatoires
US6100026A (en) * 1995-04-25 2000-08-08 Irori Matrices with memories and uses thereof
US6171555B1 (en) 1995-04-17 2001-01-09 Ontogen Corporation Reaction block docking station
US6284459B1 (en) 1995-04-25 2001-09-04 Discovery Partners International Solid support matrices with memories and combinatorial libraries therefrom
US6362009B1 (en) 1997-11-21 2002-03-26 Merck & Co., Inc. Solid phase synthesis of heterocycles
US6365349B1 (en) 1997-07-22 2002-04-02 Qiagen Genomics, Inc. Apparatus and methods for arraying solution onto a solid support
EP1235766A1 (fr) * 1999-11-24 2002-09-04 Aventis Pharmaceuticals Inc. Appareil et procede permettant de synthetiser des bibliotheques combinatoires
WO2003039750A1 (fr) * 2001-11-07 2003-05-15 Genvault Corporation Appareil, systeme, et procede destines a archiver et a recuperer des echantillons
US6878557B1 (en) 1995-01-20 2005-04-12 Arqule, Inc. Logically ordered arrays of compounds and methods of making and using the same
US7584240B2 (en) 2001-11-07 2009-09-01 Genvault Corporation Automated biological sample archive for storage, retrieval and analysis of large numbers of samples for remote clients
US7718442B2 (en) 2002-11-22 2010-05-18 Genvault Corporation Sealed sample storage element system and method
US8273304B2 (en) 1996-01-16 2012-09-25 Affymetrix, Inc. Analytical biochemistry system with robotically carried bioarray
US8283165B2 (en) 2008-09-12 2012-10-09 Genvault Corporation Matrices and media for storage and stabilization of biomolecules
US8431384B2 (en) 2004-05-24 2013-04-30 Genvault Corporation Stable protein storage and stable nucleic acid storage in recoverable form

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Cited By (30)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6416946B1 (en) 1993-05-10 2002-07-09 Chiron Corporation Methods of typing hepatitis C virus and reagents for use therein
US6416944B1 (en) 1993-05-10 2002-07-09 Chiron Corporation Methods of typing hepatitis C virus and reagents for use therein
US6054264A (en) * 1993-05-10 2000-04-25 Chiron Corporation Methods of typing hepatitis C virus and reagents for use therein
US6878557B1 (en) 1995-01-20 2005-04-12 Arqule, Inc. Logically ordered arrays of compounds and methods of making and using the same
US5770157A (en) * 1995-04-17 1998-06-23 Ontogen Corporation Methods and apparatus for the generation of chemical libraries
US6171555B1 (en) 1995-04-17 2001-01-09 Ontogen Corporation Reaction block docking station
US5609826A (en) * 1995-04-17 1997-03-11 Ontogen Corporation Methods and apparatus for the generation of chemical libraries
US6284459B1 (en) 1995-04-25 2001-09-04 Discovery Partners International Solid support matrices with memories and combinatorial libraries therefrom
US5961923A (en) * 1995-04-25 1999-10-05 Irori Matrices with memories and uses thereof
US6100026A (en) * 1995-04-25 2000-08-08 Irori Matrices with memories and uses thereof
EP1018365A2 (fr) * 1995-10-26 2000-07-12 University Of Hertfordshire Particules codées destinées à la poursuite des séquences de procédés lors de la préparation de biblothèques de composés combinatoires
EP1018365A3 (fr) * 1995-10-26 2000-10-11 University Of Hertfordshire Particules codées destinées à la poursuite des séquences de procédés lors de la préparation de biblothèques de composés combinatoires
US8273304B2 (en) 1996-01-16 2012-09-25 Affymetrix, Inc. Analytical biochemistry system with robotically carried bioarray
US6265219B1 (en) 1996-10-30 2001-07-24 Mitokor Transponder tagging of constituents used in compound synthesis
AU732405B2 (en) * 1996-10-30 2001-04-26 Mimotopes Pty Ltd Transponder tagging of constituents used in compound synthesis
WO1998018550A1 (fr) * 1996-10-30 1998-05-07 Chiron Corporation Marquage par transpondeur de constituants utilises dans la synthese de composes
WO1998038158A1 (fr) * 1997-02-26 1998-09-03 Eli Lilly And Company Procede pour la preparation de composes pharmaceutiques
US6365349B1 (en) 1997-07-22 2002-04-02 Qiagen Genomics, Inc. Apparatus and methods for arraying solution onto a solid support
US6362009B1 (en) 1997-11-21 2002-03-26 Merck & Co., Inc. Solid phase synthesis of heterocycles
WO1999034214A1 (fr) * 1997-12-31 1999-07-08 Qiagen Genomics, Inc Embouts en phase solide et utilisations correspondantes
EP1235766A1 (fr) * 1999-11-24 2002-09-04 Aventis Pharmaceuticals Inc. Appareil et procede permettant de synthetiser des bibliotheques combinatoires
EP1235766A4 (fr) * 1999-11-24 2006-05-24 Aventis Pharma Inc Appareil et procede permettant de synthetiser des bibliotheques combinatoires
WO2003039750A1 (fr) * 2001-11-07 2003-05-15 Genvault Corporation Appareil, systeme, et procede destines a archiver et a recuperer des echantillons
US7584240B2 (en) 2001-11-07 2009-09-01 Genvault Corporation Automated biological sample archive for storage, retrieval and analysis of large numbers of samples for remote clients
US7718442B2 (en) 2002-11-22 2010-05-18 Genvault Corporation Sealed sample storage element system and method
US8431384B2 (en) 2004-05-24 2013-04-30 Genvault Corporation Stable protein storage and stable nucleic acid storage in recoverable form
US8283165B2 (en) 2008-09-12 2012-10-09 Genvault Corporation Matrices and media for storage and stabilization of biomolecules
US8951719B2 (en) 2008-09-12 2015-02-10 Gentegra, LLC. Matrices and media for storage and stabilization of biomolecules
US9637513B2 (en) 2008-09-12 2017-05-02 Gentegra Llc Matrices and media for storage and stabilization of biomolecules
US10160997B2 (en) 2008-09-12 2018-12-25 Gentegra Llc Matrices and media for storage and stabilization of biomolecules

Also Published As

Publication number Publication date
HUT73150A (en) 1996-06-28
AU2280697A (en) 1997-08-28
EP0672049A1 (fr) 1995-09-20
EP0672049A4 (fr) 1997-10-15
AU5331594A (en) 1994-06-08
NZ257077A (en) 1997-05-26
CA2148591A1 (fr) 1994-05-26
JPH08505604A (ja) 1996-06-18
HU9501322D0 (en) 1995-06-28
AU680450B2 (en) 1997-07-31

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