NZ257077A - Support for synthesis of modular polymers - Google Patents

Description

New Zealand No. 257077 International No. PCT/AU93/00546 Priority D*te(s): CompUrta Specification Filed: SSl.'.?.!?.;?..... Class: (6)C&l&.L/<?.H:i.£&I.&H.9$.....

Publlcefton Dato; ftALJ997- P.O. Journal No: l.kk.l.(ra NEW ZEALAND PATENTS ACT 1953 COMPLETE SPECIFICATION Title of Invention: Support for the synthesis of modular polymers Name, address and nationality of applicant(s) as in international application form: Cnc * CHIRON MIMOTOPES PTY LTD, of 44 Duerdin*Street Clayton, Victoria 3168, Australia ^ cvn * WO 94/11388 PCT/AU93/00546 77 - I Support For The Synthesis of Modular Polymers '5 Technical Field This invention reiates u a solid surface for the synthesis of peptides thereon, and in particular reiuto ..•! a ion or pin having a plurality of surfaces on which peptides may be synthesized and assayed efficiently and economically.

Background of the Invention Geysen. EP 19SX55. 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 15 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 micro-titer plates which are widely used for EL1SA (enzyme-linked immunosorbent assay) tests.

The method disclosed in this prior specification was based on the real-20 ization that for the solid-phase synthesis of any peptide, almost all of the steps of the synthesis are exactly the same for any peptide and are independent of the sequence of the peptide being synthesized. Thus, with the rods or pins arranged in the preferred format so that 96 are in a holder, all steps in the synthesis such as washing steps, neutralization steps and deprotection steps can be carried out simultaneously 25 in the synthesis of 9ft different peptides. .The only steps which must be carried out separately for each different peptide are the coupling of the appropriate amino acid residues. Each of these steps is convenientiy carried out by dispensing appropriate I SUBSTITUTE SHEET | - 2 activated amino acid solution into the corresponding wells of a microtiter plate or the like. Thus, 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 5 entities such as antibodies to be readily detected. Although the quantity of peptide synthesized on each rod is relatively 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. However, m some applications the quantity of peptide needs to be greater. Examples of such applications include the removal of the peptide from 10 the rod and recovery of specific binding entities for further testing. Modifications to the process of synthesis and testing of the peptides were disclosed in PCT W091/04266.

It is an object of the present invention to provide means by which the amount of peptide, or for that matter any other polymeric compound such as nucleic 15 acid which can be synthesized on a solid support can be increased while retaining the advantage of being able to synthesize many peptides with different sequences simultaneously.

In the earlier method polyacryiic acid was grafted to the surface of a solid polyethylene support using gamma-irradiation. In that earlier system, the region 20 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 25 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- SUBSTTTUTE SHEET i WO 94/11388 PCT/AU93/00546 - 3 • 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 of uncertain synthesis quality is created. In addition. unless extreme care is taken in extensively washing these rods, the polymer 5 layer acts as a reservoir of the solvent used in synthesis leading to the contamination of subsequent solutions.

Disclosure of the Invention One aspect of the invention is an improved synthesis support, having 10 a plurality of independent synthesis surfaces, which permits the simultaneous syntheses of peptides (or other modular polymers) having different N- or C-groups, having different linkages to the support it'.*'., 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. 15 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.

Summary of the Drawings Figure 1 depicts plan and cross-section views of an active surface com-20 ponent of the invention.

Figure 2 depicts plan views of support rod of the invention.

Figure 5 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-25 tiple epitope libraries prepared by the method of the invention.

SUBSTITUTE SHEET 4 - Modes of Carrying Out The Invention A. Definitions The term modular polymer" refers to a polymer composed of non-identical subunits selected from a group of monomers. Modular polymers are gen-5 erally synthesized one monomer at a time.

The term "monomer" as used herein refers to a molecule which may be coupled or condensed tc form an oligomer. To provide diversity, monomers are selected from sets which contain at least four distinct members. Suitable monomer sets include conventional D- and L-amino acids, nucleic acids/nucleotides. carbo-10 hydrates, nonconventionai D- and L-amino acids cyclohexylalanine. benzhydryl-glycine. chloroalanine. and the like ), and "peptoids" as described in W091/19735 (incorporated herein by reference).

The term "conventional amino acid" refers to the amino acids alanine (A), cysteine (C), aspartic acid (D). glutamic acid (E), phenylalanine (F), glycine (G), 15 histidine (H), isoleucine (I), lysine (K). leucine (L), methionine 4), asparagine (N), proline (P), glutamine (Q). arginine (R). serine (S), threonine (T). valine (V), tryptophan (W), and tyrosine (Y).

The term "nonconventionai amino acid" refers to amino acids other than conventional amino acids. Presently preferred nonconventionai amino acids are Nle = L-norleucine; Aabu = a-aminobutyric acid; Hphe = L-homophenylalanine: Nva = L-norvaline; Gabu = y-aminobutyric acid: Dala = D-ulanine; Dcys = D-cysteine: Dasp = D-aspartic acid: Dglu = D-glutamic acid: Dphe = D-phenylaianine: Dhis = D-histidine: Dile = D-isoleucine: Dlys = D-lysine; Dleu = D-leucine; Dmet = D-methionine: Dasn = D-asparagine; Dpro = D-proline: Dgln = D-glutamine: Darg = D-arginine: Dser = D-serine: Dthr = D-threonine: Dval = L)-valine; Dtrp = D-tryptophan: Dtyr = D-tyrosine; Dorn = D-ornithine: Aib = aminoisobutyric acid: Etg = L-ethylglycine: Tbug = L-r-butylglycine: SUBSTITUTE SHEET | Pen = penicillamine: Chexa = cyclohexylalanine: Cpro = aminocyclopropane earboxyiate: Mala = L-a-methylalanine: Masp = L-a-methylaspartic acid: Mphe = L-a-methylphenylalanine: Mile = i.-a-methylisoleucine: Mleu = L-a-methylleucine: Masn = L-a-methylasparagine: !() Mgln = L-a-methylglutamine: Mser = L-a-methylserine: Mval = L-a-methylvaline: Mtvr = L-a-methyltyrosine: Mnle = l-a-methylnorleucine: 15 Mnva = l-oc-mcthylnorvaline: Metg = l-a-methylethylglycine: Maib = a-methylaminoisobutync acid: Mpen = a-methylpenicillamine: Mchcxa = a-methylcyclohexylalanine: 20 Dmala = D-a-methylalanine: Dmcys = D-a-methylcysteine: Dmglu = d-a-methylglutamie acid: Dmhis = d-a-methylhisndine: Dmlys = D-a-methyllysine: 25 Dmmet = D-a-methylmethionine: Dmpro = D-a-methylproiine: Dmarg = d-a-methylarginine: Dmthr = D-a-methylthreonine: Dmtrp = D-a-methyitryptophan: 30 Nmala = l-N-methylalanine: Nmasp = L-N-methyla. lartic acid: Nmphe - L-N-methylphenylalanine: Nmile = L-N-methylisoleucine: Nmleu = l-N-methylleueine: 35 Nmasn = l-N-methylasparagine: Nmgln = L-N-methylglutamine: Nmser = L-N-methylserine: Nmval = L-N-methyivaline: Nmtyr = L-N-methyltyrosine: 40 Nmnle = L-N-methylnorleucine: Nmnva = L-N-methylnorvaline: Nmetg = L-N-meihylethvlalycine: Nmcpen = N-methylcvclopentylalanine: Anap = a-naphthylalanine; Cpen = eyciopentylalanine: Norb = aminonorbomylcarboxylate; Mcys = L-a-methylcysteine: Mglu = L-a-methyiglutamic acid: Mhis = L-a-mcthylhistidine: Mlys = L-a-methyllysine: Mmet = L-a-methylmethionine: Mpro = L-a-methylproline: Marc = L-a-methyiarginine: Mthr = L-a-methyithreonine: Mtrp = L-a-methyltryptophan: Mom = L-a-methylorni thine: Maabu = a-amino-a-methylbutyric acid: Mhphe = L-a-methylhomophenylalanine: Mgabu = a-mcthyl-7-aminobutyric acid; Mtbug = L-a-methyl-/-butylglycine; Manap = a-methyl-a-naphthylalanine; Mcpen = a-methylcyclopentylalanine; Dmorn = D-a-methylomithine: Dmasp = D-a-methylaspartic acid; Dmphe = D-a-methylphenylalanine; Dmile = D-a-methyiisoleucine; Dmleu = D-a-methylleucine: Dmasn = D-a-methylasparagine; Dmgln = D-a-methylglutamine; Dmser = D-a-methylserine; Dmval = D-a-methylvaline; Dmtyr = D-a-methyltyrosine; Nmcys = L-N-methylcysteine: Nmglu = L-N-methylglutamic acid; Nmhis = L-N-methylhistidine; Nmlys = L-N-methyllysine: Nmmet = L-N-methylmethionine; Nmchexa = N-methylcyclohexylalanine; Nmarg = L-N-methylarginine: Nmthr = L-N-methylthreonine: Nmtrp = L-N-methyltryptophan; Nmorn = L-N-methylornithine: Nmaabu = N-amino-a-methyl butyric acid; Nmhphe = L-N-methylhomophenylalanine: Nmgabu = N-methyl-7-aminobutyric acid; Nmtbug = L-N-methyl-f-butylglycine; SUBSTITUTE SHEET Nmpen = N-methylpenicillamine: Nmaib = N-methylaminoisobutyric acid: Dnmula = D-N-methylalanine: Dnmcys = D-N-methylcvsteine: Dnmglu = D-N-methylglutamic acid: Dnmhis = D-N-methylhistidine: Dnmiys = D-N-mcthyllvsine: Dnmmet = D-N-methylmethionine: Dnmpro = D-N-methyiproline: Dnmarg = D-N-methvlarginine: Dnmthr = D-N-methyithreonine: Dnmtrp = D-N-methyltryptophan: Nala = N-methylglycine (sarcosinei: Nglu = N-(2-carboxyethyl (glycine: Nhhis = N-(imidazolylethyl)glycine: Nlys = N-(4-aminobutyl)glycine: Nmet = N-(2-methylthioethyl)glycine: Nasn = N-(carbamylmethyl)glycine: Nval = N-( 1 -methylethyDglycine: Nhtrp = N-(3-indolylethyl)glycine: Nthr = N-(l-hydroxyethyl)glycine: Norn = N-(3-aminopropyl)glycine: Ncbut = N-cyclobutyglycine: Nchep = N-cycloheptylglycine: Ncdec = N-cyclodecylglycine: Ncdod = N-eyclododecylglyeine: 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- l-(2.2-diphenylethylamino)cyclopropane: Naeg as N-(2-aminoethyl)glyeine.

Nmanap = N-methyl-a-naphthyialanine: Dnmom = D-N-methylornithine: Dnmasp = D-N-methylaspanic acid: Dnmphe = D-N-methylphenylalanine: Dnmile = D-N-methylisoleucine: Dnmleu = D-N-methylleucine: Dnmasn = D-N-methylaspaiagine: Dnmgln = D-N-methylgiuramine: Dnmser = D-N-methylserine: Dnmval = D-N-mcthylvaiine: Dnmtyr = D-N-methyltyrosine: Nasp = N-(carboxymethyl)glycine: Nphe = N-benzyigiycme: Nile = N-(l-methyipropyl)glycine: Nleu = N-(2-methylpropyl)glycine; Nhser = N-(hydroxyethyl)glycine; Ngln = N-(2-carbaniylethyl)giycine: Narg = N-(3-guanidinopropyl)glycine: Nhtyr = N-(p-hydroxyphenethyl)glycine: Ncys = N-(thiomethyl)glycine; and Ncpro = N-cyclopropylglycine: Nchex = N-cyclohexylglycine: Ncoct = N-cyclooctylgiycine; Ncund = N-cycloundecylgiycine: Nbhm = N-(2.2-diphenylethyl)glycine; and The term "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. For example, the active surface may be bonded or grafted to a supporting structure or surface. In principle, the active surface may be radiation grafted to any supporting structure (which may be, for example, an "inert surface" as defined below).

SUBSTITUTE SHEET 7 - The term 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, polvolefins. cellulose acetate, wool, cotton, chitin, and the like.

B. General Method In a first embodiment it the present invention, there is provided 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 10 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. Preferably, the inert suppon is capable of retaining 2 to 20 active surface components. more preferably about 5 active surface components. The support may have a 15 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. Alter-20 natively, 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 pro cessing, both during synthesis of the modular polymers and during their assay. In a presently preferred embodiment 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. Preferably the rods are mounted by press-fit or friction-fit into holes drilled or molded in the SUBSTITUTE SHEET 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 ana holes, adhesives. one-piece molding, magnetic coupling, and the like. Other array formats are also considered within the scope of 5 this invention. For example, the support rods may be positioned on a continuous belt, or may be gripped individually by robotic manipulators, to simplify automated handling.

Preferably, 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 10 into place. However, 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. For example, one may employ inen supports and active surface components having ferrous cores, and retain the active surface components by electromagnetism. In this embodiment, coupling reactions to 15 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. Alternatively, 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 20 annular in shape, but may be formed in any shape which can be retained on the inert support For example, 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 25 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 inen support. The terminal active surface component may have a different SUBSTITUTE SHEET | - 9 shape, because the inen support need not pass entirely through the component. For example, the terminal active surface component may be hemispherical.

Figure 1 illustrates one presently preferred embodiment of the active surface component (/). Fig. 1A depicts a plan view of the component, having a 5 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 IB depicts a cross section of the component The component is molded from polyethylene with 16 external "teeth" (J) and 4 internal teeth (2) arranged symmetrically.

Figure 2 illustrates one presently preferred embodiment of the support rod {JO), designed for use with the components (J) of Fig. 1. Rod JO 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 15 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 J4. 20 These projections 15 serve to locate and position components J on secondary shaft 14. Secondary shaft 14 is further provided with a generally frustroconical end cap It) 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. 25 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 | SUBSTITUTE SHEET modular polymer. In addition, 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. Examples of 5 porous resins which may be used in such coatings include benzhydrylamine-polysty-rene resin and polyacrylamide gel inside kieselguhr. Other suitable surface materials include, without limitation, polyethylene glycol, cellulose and other natural polymers. Menifield resin. Rink resin and polymers of acrylic acid, methylacryiate. methacrylic acid, methyl methacrylate. dimethylacrylamide. styrene. hydroxyethylacrylate. 10 hvdroxyethylmethacrylate. hydroxypropylmethacrylate. hydroxyethylmethacrylamide. methylmethacrylate. and polyethyleneglycol monomethacrylate, and the like, and combinations thereof. The component bearing the active surface need not be homogeneous. It is presently preferred to employ structural supports fashioned from not just polyethylene, polypropylene and its copolymers but also Teflon® (polytetra-15 fiuoroethylene) or any other stable inen 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 r rthod is grafting, by placing the suppon surface in a solution of solvent and active surface material and irradiating the mixture with gamma radiation. Preferred solvents arc water, methanol 20 (MeOH). H20/Me0H mixtures, dimethylformamide (DMF). and dimethylsulfoxide (DMSO). The mixture is prepared and irradiated as described in EP 138855: see also D. Miiller-Schulte et al.. Polvmer Bull (1982-) 7:77-81.

The surfaces are then modified, if desired, for the selected coupling chemistry. The modular polymers may be "permanent" (/.«?., not easily removed from 25 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- SUBSTUUTE SHEET - II - ular polymers may be provided with labeling groups for detection, or binding iigands for purposes of separation and purification. For example, 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 pare a collection or library of moduiar polymers wherein one or more sets are permanent. and other sets are bionnviated. fluorosceinated. radioactive, cleavable to acidic terminal groups, cleavable to amide terminal groups, and cleavable to neutral terminal groups. (incorporated herein by reference), in which the link cyclizes to a diketopiperazine moiety with concomitant cleavage of the peptide chain from the support. In the preferred embodiment, lysine having a protected a-amino group (e.g., with BOC) is coupled to the support through the e-amino group. Pro, whose carboxy terminus is 15 esterified with a suitably reactive spacer ("X") having an orthogonally protected (e.g., Fmoc) amino group is then coupled to the Lys carboxy late function. Synthesis of the modular polymer then proceeds on the amino group on deprotection: atoms to simplify detection in a Dinciinu assay. Thus, one might simultaneously pre One form of cleavable linkage was described by Geysen. W090/09395 Pro—O—X—NH—Prot Pin Boc—Lys—Pro—O—X—NH—Prot Pin Boc—Lys—Pro—O—X—"NH2" Pin Alternatively, one may synthesize the group first and couple it to the pin: SUBSTITUTE SHEET 12 - Pin-0OOH - BQC—Lys-Pro—O—X—NH—Prot "NH2" Cleavage is effected by removing the BOC (or other group) protecting the Lys examine group, and neutralizing the resulting -NH?". When the Lys a-amino -NH^ is neutralized to -NH2. toe 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. Alternatively, one may couple the spacing group to the active surface, to form an ester link. Coupling of a suitably protected Lys to Pro will form the same basic linker. However, this configuration leaves a diketopiperazine moiety attached to the modular polymer after cleavage from the support.

We have also developed a new linker, which facilitates cleavage of a modular polymer to provide an amide function at the point of cleavage. The modular polymer is synthesized linked to the. active surface through a protected a-aminogly-cine. After the modular polymer synthesis is completed, the protecting group is removed from the a-amino group. Immersing the active surface in an aqueous solution at pH 7-10 results in cleavage, leaving an amide function at the site of cleavage on the modular polymer, as shown in the Schemc below: j SUBSTITUTE SHEET | 13 • BOC—NH Fmoc—NH—CH—CO—Support Synthesize peptide Boc—NH (protected peptide)—CO—NH—CH—CO—Support Side chain deprotection (H+) NH. peptide—CO—NH-ni H—CO—Support 1) wash(pH<5) 2) pH 7-10 NH2 peptide—CO—NH—CH—CO-Support peptide—CO—NH *NH—CH—CO—Support This linkage may be used for any type of modular polymer. The general formula for a bound modular polymer is NH—Z (M) (spacer)—Support H O 40 SUBSTITUTE SHEET | wherein M is a monomer, n is an integer (preferably 2-30. inclusive), and Z is a protecting group. The spacer is optional.

Other cleavable linkages may be employed. For example, there are known photocleavable linkages, which cleave upon exposure to light at a selected fre-5 quency. These 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. Where the moduiar 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 polymerase pro-10 moter/binding sites and amplification primer hybridization sites, to facilitate duplex formation and amplification (e.g.. by PCR).

The active surfaces may also carry identifying features to facilitate differentiation between different types of surface. For example, active surfaces of different types may have different colors and/or patterns, different sizes and/or shapes. 15 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 20 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-25 arated by a simple screen that allows small.components to pass, while retaining larger components. Obviously, 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. Thus, one may prepare a collection or set | SUBSTITUTE SHEET • of modular polymers as described above, in several different forms, in one operation. The different forms may then be easily separated for further research.

The use of modular active surface components and inert components has several advantages. First, it permits one to prepare each component under optimal 5 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 blocK: ">v .noauiur 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 solutions. The polymer layer grafted onto the rod", during the radiation process as described in EP 19X855 is readily solvated by many solvents and as a consequence, solvents will migrate upwards through this layer by capillary action. This results in 15 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, 20 this migration of solvents and reagents cannot take place.

Another major advantage of manufacturing xhe active region as a separate entity comes about because the active region will typica'ly be much smaller than the complete uniL Therefore, more of the active region components can be treated simultaneously to create the active region with consequent savings in materials 25 and time.

For the purpose of illustration only, 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 mm2 (assuming that only SUBSTITUTE SHEET one end of this cylinder is available for synthesis). However, if a slit 1 mm wide is made across the diameter of this portion of the rod. the surface area becomes Xl.X mm" (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 5 shape of this portion of the rod. For instance, if eight slits, each 0.4 mm wide and 1 mm deep are made into the surface of a cylindrical portion, the surface area available for synthesis is increased to 124 mm" (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 10 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.

However, in a preferred embodiment of the invention, 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 15 particularly useful where it is desired to use particular harsh chemistries or corrosive solvents. For instance, glass is resistant to most solvents which would make most conventional plastic materials unstable. Thus an active region could be made by sintering together small spherical beads of glass. This could then be treated, for instance, by fiinctionalizing the surface with an amino-silane, to make it suitable as 20 a base on which the peptide or other polymeric compound could be synthesized. In this example, the inactive region would be made from a Dirticulariy resistant plastic such as polytetrafluoroethyiene. In this way. a material *uch 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. Using the example given above. KHX5 spherical rigid particles each with a radius of 0.1 mm would occupy the volume of the portion providing the aciivt region of the rod if close packed together. The surface area of these spheres would [ SUBSTITUTE SHEET j be 1116.4 mm". 17.X times the surface 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 for synthesis. For example, decreasing the radius of the rigid spheres to be sintered to 0.055 mm increases the surface area to 2233 mm2, about 35.5 times mat or tne solid cylinder. In practice, because of the process of joining the particles togetner. and the fact that the particles are neither uniform in size nor rigid, the theoretical increase in surface area would not be achieved. However, very significant gams in surface area available for synthesis can be achieved by making the solid support by sintering small particles of material together.

Further features of the present invention are illustrated, by way of example only, in the following example.

C. Examples The examples presented below are provided as a further guide to the practitioner of ordinary skill in the an. and are not to be construed as limiting the invention in any way.

Example l (Synthesis of Peptides) Polyethylene pins were molded as depicted in Fig. 2, and mounted in X x 12 blocks as described in WOV1/04266.

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 19XX55 with methacrylic acid/dimethylacrylamide (10% MA, 20% DMA in methanol). Cleavable crowns were then derivatized with Boc-hexamethylenediamine according to W091/04266 by immersing in a 60 mM solution of r-butoxycarbonylhexamethylenediamine (Boc-HMD), triethylamine (TEA), dicyclohexylcarbodiimide (DCC) and hydroxybenzotriazole (HOBT) (1:1:1:1.2) in | SUBSTITUTE SHEET WO 94/11388 PCT/AU93/00546 IX - DMF. After Boc deprotection with trifluoroacetic acid, the active surface was neutralized by washing in MeOH (2 mini, 5% TEA in MeOH (2x5 min) and MeOH (5 min). The preformed diketopiperazine linker (Boc-Lys(Fmoc)-Pro-O-HMB) was coupled at 60 mM concentration with DCC/HOBT (1:1:1.2) for a set time to obtain 5 the desired loading (HMB = hydroxylmethylbenzoic acid). On a surface area of 130 mm2, approximately 1.5 umoles of linker can be coupled within 90 min. Unreacted amino groups on the surface are capped by acetvlation.

Removable synthesis surfaces for non-cleavable peptides ("non-cleavable crowns") were molded as depicted in Fig. 1. using polyethylene, and were then radia-10 tion-grafted as described in EP 19XX55 with 30% hydroxypropylmethacrylate in MeOH. The surface was then derivatized with Fmoc-Gly (30 mM). DCC and di-methylaminopyridine (DMAP) (1:1:0.2) in DMF/dichloromethane (1:3) for 15 min. The unreacted hydroxyl groups on the surface are capped by acetylation. After Fmoc deprotection in 20% piperidine/DMF and washing with DMF (2 min) and MeOH (3 15 x 2 min), a control Fmoc-fi-Ala coupling is performed to obtain a total loading of 50 to 100 nmoles. Fmoc-(3-Ala. DCC and HOBT (1:1:1.2. 30 mM) in DMF is coupled for approximately 10 min and the remaining amino groups of Gly capped by acetylation.

Each pin was loaded with four non-cleavable crowns, followed by one 20 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). Thus, any given pin carried five crowns having identical octapeptides, with each pin carrying an octapeptide different from the octapeptides carried on other pins. 25 Example 2 (Assay of Peptides) Assav of Non-Cleavable Peptides: The non-cleavable crowns were removed from the 5-position pins and were placed in corresponding positions on blocks holding 1-position pins (see W091/ SUBSTITUTE SHEET | 04266) to provide four identical epitope libraries. Each block was tested for antibody binding by ELISA using a 1/20.000 diluuon of a rabbit anti-pillin antiserum. The results (shown in Fig. 3) demonstrate tnat all four sets of peptides are essentially . identical.

D Assay of Cleavable Peptides: Cleavable crowns were men removed from the pins, and the peptides removed. Each crown was treated with X00 uL of cleavage solution (0.1 M NH4HC03, 40% CH-,CN. pH N.4i. Aliquots were submitted to amino acid analysis. HPLC demonstrated high levels of purity. The typical yield of peptide was about 10 600 nmole/crown.

Each peptide was then used at three concentrations to illustrate solution-phase competition for binding of the antibody to the non-cleaved peptide set. Four 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); 3) 1/21,000 serum dilution, peptide added to 3.5 nmole/mL (Fig. 4C); 4) 1/21,000 serum dilution, peptide added to 0.35 nmole/mL (Fig. 4D). The antiserum and peptides were mixed and incubated for 1 hr at room temperature before testing with the set of bound (noncieavable) peptides. The ELISA 20 results are shown in Fig. 4: a concentration of 35 nmole/mL of the cleaved peptides (Fig. 4B) was sufficient to inhibit antibody binding to the noncieavable peptides essentially completely.

SUBSTITUTE SHEET 257 07?

Claims (5)

WHAT WE CLAIM IS:

1. A synthesis support for use in the synthesis of modular polymers, said synthesis support comprising: a plurality of generally annular synthesis components, comprising an active surface adapted to support solid-phase synthesis of a modular polymer; and an elongate, inert support rod, positioned axially to said generally annular synthesis components, wherein said support rod retains said generally annular synthesis components in a collinear configuration.

2. The synthesis support of claim 1, wherein said inert support rod is designed to receive about 5 generally annular synthesis components.

3. The synthesis support of claim 1, wherein said inert support comprises polyethylene or polypropylene.

4. The synthesis support of claim 1, wherein a plurality of synthesis supports are fixed in an array.

5. The synthesis support of claim 4, wherein said array is an 8 x 12 array. w. f.C! . .Vii i i ifin "• ' By the authorised agents A J PARK & SON ^ /j pe, /-'A END OF CLAIMS N.Z. PATENT OFFICE \ APR 19# RECEIVED