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 ..-. a i on or pin having a plurality of surfaces on which peptides may be synthesized and assayed efficiently and economically.
• EP 198X55. 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 S matrix, with the rods or pins being positioned so that the spacing corresponds to that of the wells of micro- titer plates which are widely used for ELISA (enzyme- linked immunosorbent assay) tests.
• ELISA enzyme- linked immunosorbent assay
• the appropriate amino acid is coupled to the peptide for each of the rods.
• 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.
• 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 WO 1/04266.
• 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.
• One aspect of the invention is an improved synthesis support, having a plurality of independent synthesis surfaces, which permits the simultaneous syn ⁇ theses of peptides (or other modular poiymers) having different N- or C-groups, hav ⁇ ing 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 lib ⁇ raries 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.
• modular polymer refers to a polymer composed of non- identical subunits selected from a group of monomers. Modular polymers are gen ⁇ erally .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. carbo ⁇ hydrates, nonconventionai D- and L-amino acids ( .#., cyclohexylalanine. benzhydryl- glycine, chloroalanine. and the like), and "peptoids" as described in WO91/1 735 (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
• Mnva L- ⁇ -methylnorvaline
• Mhphe L- ⁇ -methylhomophenylalanine
• 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-(i-hydroxyethyl)glycine
• Ncys N-(thiomethyl)glycine
• Ncpro N-cyclopropylglycine
• Ncbut N-cyclobutygiycine
• 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 l-carboxy-l-(2.2-diphenylethylamino)cyclopropane
• Naeg N-(2-aminoethyl)glycine.
• active surface means a surface which is derivatized or other ⁇ wise 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 mod ⁇ ular polymer synthesis conditions, and does not react. Suitable inert surfaces include without limitation polyethylene, polyolefins. cellulose acetate, wool, cotton, chitin , and the like.
• a plur ⁇ ality 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 ine ⁇ 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 compon ⁇ ents, 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 flex ⁇ ibility to the support that the active surface components may be "snapped" on.
• the active surface components may be positioned on a nail-shaped pin, fol ⁇ lowed 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 pro ⁇ cessing, 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
• 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 t his 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
• 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., complet ⁇ ing 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.
• the active surface components are vaned or "gear" shaped, in order to maxi ⁇ mize 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
• the terminal acnve surface component may be hemispherical.
• Figure 1 illustrates one presently preferred embodiment of the active surface component (/ ).
• Fig. IA depicts a plan view of the component having a 5 thickness r 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 I B depicts a cross section of the component The component is molded from polyethylene with 16 external "teeth" (3) and 4 inter ⁇ nal teeth (2) arranged symmetrically.
• Figure 2 illustrates one presently preferred embodiment of the support rod ( JO ), designed for use with the components (/) of Fig. 1.
• Rod 10 comprises a primary shaft having a diameter of about 3.8 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 15 end 12, and measures 1.1 mm in thickness by 6.0 mm in outer diameter.
• the sec ⁇ ondary shaft (14) is coaxial with primary shaft //, and extends about 21.0 mm there ⁇ from.
• 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. 20 These projections 15 serve to locate and position components / on secondary shaft 14.
• Secondary shaft 14 is further provided with a generally frustroconical end cap If ) 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 / to the support 10, and is responsible for retaining the bottom-most component.
• the a ⁇ tive 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
• SUBSTITUTE SHEET modular polymer 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.
• the component bearing the active surface need not be homo ⁇ geneous. It is presently preferred to employ 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 theii 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 sup ⁇ port).
• 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).
• SUBSTITUTE SHEET ular poiymers may be provided witn labeling groups for detection, or binding ligands for purposes of separation and pu ⁇ ficauon.
• cleavable modular poly ⁇ mers may be biotinylated (or tagged with another similar ligand) to facilitate purifi ⁇ cation (e.g., using a streptavidin column), or labeled with fluorescein or radioactive atoms to simplify detection in a Dinning assay.
• fluorescein or radioactive atoms to simplify detection in a Dinning assay.
• 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 Lys ⁇ -amino -NH 3 " is neutralized to -NH-,
• 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.
• the modular polymer is synthesized linked to the. active surface through a protected ⁇ -aminogly- cine. After the modular poiymer synthesis is completed, the protecting group is removed from the ⁇ -amino group. Immersing the active surface in an aqueous solu- tion at pH 7-10 results in cleavage, leaving an amide function at the site of cleavage on the modular polymer, as shown in the Scheme below:
• This linkage may be used for any type of modular polymer.
• the general formula for a bound modular polymer is
• 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 fre ⁇ quency.
• linkages, and the linkages described above are generally most suit ⁇ able for use with peptide- and peptoid-based modular polymers, but may also be used wi t h nucleic acids.
• the modular polymer is a nucleic acid, it may be conven ⁇ ien t ly removed by providing a cleavable linkage in the form of a restriction enzyme recognition site.
• Nucleic acid modular polymers may also include poiymerase pro ⁇ moter/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 dif ⁇ ferentiation between different types of surface.
• active surfaces of dif ⁇ ferent 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.
• Sur ⁇ faces 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 sep- arated by a simple screen that allows small . components to pass, while retaining larger components.
• surfaces that are magnetized may be separated from non- ma g netized surfaces using a magnet, electromagnet or ferrous metal. The last two sy.stems are more suitable to automation. Thus, one may prepare a collection or set
• 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 bloci : ">v modular construction, the rods may be made of rigid materials, while the active surrace 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 solu ⁇ tions. The polymer layer grafted onto the rods during the radiation process as des ⁇ cribed in EP 198855 is readily solvated by many solvents and as a consequence, sol ⁇ vents will .
• 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 active region as a sep ⁇ arate entity comes about because the active region will typically be much smaller than the complete unit. Therefore, more of the active region components can be treated simultaneously to create the active region with consequent savings in materials and time.
• the portion providing the active region of the rod. that is. the region of the rod on which the peptide or other poly ⁇ meric 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
• the portion pro ⁇ viding the active region of the rod is made by joining small particles of solid mater ⁇ ials 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 sin ⁇ tering together small spherical beads of glass. This could then be treated, for instance , by functiono ⁇ al-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.
• 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 fur her 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 clo.se packed together. The surface area of these spheres would be
• SUBSTITUTE SHEET be 1 1 16.4 mm-, 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 at or the solid cylinder. In practice, because of the process of joining the particles togetner. and the fact that the particles are neither uni ⁇ form in size nor rigid, the theors ⁇ .-u. increase in surface area would not be achieved. However, very significant gains in surface area available for synthesis can be achieved by making the solid support by sintering small particles of material together.
• 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 radia- tion-grafted as described in EP 198855 with methacrylic acid dimethylacrylamide
• 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 carry ⁇ ing 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/
• peptide was then used at three concentrations to illustrate solution- phase competition for binding of the antibody to the non-cleaved peptide set.
• serum preparations were tested: 1) 1/21,000 serum dilution, with no added peptide (Fig. 4A); 2) 1/21,000 serum dilution, peptide added to 35 nmole/mL (Fig. 4B);
• Fig. 4D 1/21,000 serum dilution, peptide added to 0.35 nmole/mL.
• the antiserum and peptides were mixed and incubated for 1 hr at room temperature before testing with the set of bound (noncleavable) peptides.
• the ELISA results are shown in Fig. 4: a concentration of 35 nmole/mL of the cleaved peptides

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• 1993
  • 1993-10-25 CA CA002148591A patent/CA2148591A1/fr not_active Abandoned
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  • 1993-10-25 WO PCT/AU1993/000546 patent/WO1994011388A1/fr not_active Application Discontinuation
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• 1997
  • 1997-05-16 AU AU22806/97A patent/AU2280697A/en not_active Abandoned

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