WO2014058755A1

WO2014058755A1 - Synthesis support resin

Info

Publication number: WO2014058755A1
Application number: PCT/US2013/063636
Authority: WO
Inventors: John MAIKNER; Lester H. Mcintosh
Original assignee: Rohm and Haas Co
Current assignee: Rohm and Haas Co
Priority date: 2012-10-10
Filing date: 2013-10-07
Publication date: 2014-04-17
Anticipated expiration: 2015-04-10

Description

SYNTHESIS SUPPORT RESIN

[0001] A common method of making oligonucleotides or polypeptides is to start with a solid support resin that has a pendant linking group. Then, a nucleotide (or an amino acid) is bonded to that linking group. Further nucleotides (or amino acids) are then bonded to previous nucleotides (or amino acids) to form the oligonucleotide (or polypeptide).

[0002] It is desired to provide a method of making a solid support resin for

oligonucleotide synthesis or polypeptide synthesis, where that method involves the use of polymer having pendant epoxy groups. It is contemplated that the versatility and reactivity of the epoxy group will enable such solid supports to be made more efficiently and/or more cheaply. It is also desired to provide solid support resins made by such a method.

[0003] It is also desired to provide a method in which a solid support resin that contains polymer having a pendant epoxy group is reacted with one or more reagent; the product of the reaction is a polymer having a pendant intermediate functional group; in such a method, the pendant intermediate functional group is further reacted with one or more additional reagent to form a linking group that is useful in oligonucleotide synthesis. It is desired to provide solid support resins that are suitable for oligonucleotide synthesis and that are made by such a method.

[0004] In the case of oligonucleotide synthesis, it is also desired to provide a method in which a solid support resin that contains polymer having a pendant epoxy group is reacted with one or more reagent; the product of the reaction is a polymer having a pendant intermediate functional group; in such a method, the pendant intermediate functional group is further reacted with one or more additional reagent to form a linking group that is useful in oligonucleotide synthesis. It is desired to provide a resin that contains a polymer having such a pendant intermediate functional group. It is also desired to provide solid support resins that are suitable for oligonucleotide synthesis and that are made by such a method.

[0005] US 7,700,706 describes solid support media for use in oligomer synthesis. The solid support media described by US 7,700,706, contain a copolymer that contains a functionalizing monomer, which may be propanoyloxystyrene or acetoxystyrene.

[0006] The following is a statement of the invention.

[0007] The first aspect of the present invention is a functional resin comprising structure (I):

wherein said particle comprises a crosslinked styrenic polymer, wherein -INT is a functional group, wherein -R¹⁰¹ is hydrogen or alkyl, wherein -L¹⁰¹- and -L²⁰¹- are independent optional linking groups, wherein each of -R 201 , -R 202 , and -R 203 is independently hydrogen or a substituted or unsubstituted hydrocarbyl group, wherein said functionalized resin is suitable for one or more of use as a solid support for polypeptide synthesis or use in making a solid support for oligonucleotide synthesis.

[0008] The second aspect of the present invention is a method of making the functional resin of claim 1 comprising the steps of

(A) providing an ep

wherein said particle comprises a crosslinked styrenic polymer,

wherein -L 201 - is an independent optional linking group,

wherein each of -R 201 , -R 202 , and -R 203 is independently hydrogen or a substituted or unsubstituted hydrocarbyl group,

(B) reacting said epoxy-functional resin with one or more reagent so that said structure

III becomes said structure I.

[0009] The third aspect of the present invention is a functional resin comprising structure (XXXI)

wherein said particle comprises a crosslinked styrenic polymer, wherein -L¹⁰¹- and -L²⁰¹- are independent optional linking groups, wherein each of -R 201 , -R 202 , and -R 203 is independently hydrogen or a substituted or unsubstituted hydrocarbyl group, and wherein -R³⁰⁴ is a chemical group, wherein said functionalized resin is suitable for one or more of use as a solid support for polypeptide synthesis or use in making a solid support for

oligonucleotide synthesis. [0010] The fourth aspect of the present invention is a support resin suitable for oligonucleotide synthesis comprising structure (II)

wherein said particle comprises a crosslinked styrenic polymer, wherein -R is hydrogen or

102 201

alkyl, wherein -L - and -L - are independent optional linking groups, wherein each of

201 202 203

-R , -R , and -R^{^J} is independently hydro gen or a substituted or unsubstituted hydrocarbyl group, and wherein -L is a functional group that is capable of forming a phosphate linkage with a nucleotide.

[0011] The fifth aspect of the present invention is a method of making the support resin of claim 1 comprising the steps of

(A) providing a functional resin comprising structure (IV)

wherein said particle comprises a crosslinked styrenic polymer, wherein -INT is a functional group, wherein -R¹⁰¹ is hydrogen or alkyl, wherein -L¹⁰¹- and -L²⁰¹-

201 202 203 are independent optional linking groups, wherein each of -R , -R , and -R is independently hydrogen or a substituted or unsubstituted hydrocarbyl group, and (B) reacting said functional resin with one or more reagent so that said structure (IV) becomes said structure (II).

[00010] The sixth aspect of the present invention is a support resin suitable for oligonucleotide synthesis comprising structure (XXXII)

(XXXII)

wherein said particle comprises a crosslinked styrenic polymer, wherein -L¹⁰²- and -L²⁰¹-

201 202 203 are independent optional linking groups, wherein each of -R , -R , and -R is

305 independently hydrogen or a substituted or unsubstituted hydrocarbyl group, -R is a chemical group, and wherein -L is a functional group that is capable of forming a phosphate linkage with a nucleotide.

[0012] The following is a detailed description of the invention.

[0013] As used herein, the following terms have the designated definitions, unless the context clearly indicates otherwise.

[0014] Percentages are by weight unless otherwise stated. Ambient temperature is approximately 23 °C.

[0015] A particle is characterized by its diameter. If the particle is not spherical, its diameter is considered herein to be the diameter of a sphere that has the same volume as the particle. A collection of particles is characterized herein by the volume-average diameter.

[0016] A "polymer" is a relatively large molecule made up of the reaction products of smaller chemical repeat units. Polymers may have structures that are linear, branched, star shaped, looped, hyperbranched, crosslinked, or a combination thereof; polymers may have a single type of repeat unit ("homopolymers") or they may have more than one type of repeat unit ("copolymers"). Copolymers may have the various types of repeat units arranged randomly, in sequence, in blocks, in other arrangements, or in any mixture or combination thereof.

[0017] Some polymers are fully crosslinked. The molecular weight of fully crosslinked polymers cannot be measured. Fully crosslinked polymers are considered to have infinite weight-average molecular weight (Mw) and are considered to be insoluble in water and solvents. When a polymer is described herein as "crosslinked," it is meant that the polymer is fully crosslinked.

[0018] Molecules that can react with each other to form the repeat units of a polymer are known herein as "monomers." After polymerization, the residue of a monomer is known herein as a polymerized unit of that monomer.

[0019] A vinyl monomer is a compound that contains a vinyl group and that is capable of undergoing vinyl polymerization to form a polymer. Vinyl aromatic monomers are vinyl monomers that contain one or more aromatic ring, and a carbon atom within an aromatic ring is connected by a covalent single bond to one of the carbon atoms in the carbon-carbon double bond of the vinyl group. Vinyl aromatic monomers include, for example, styrene, alpha-substituted styrenes, ring-substituted styrenes, and mixtures thereof. Ring-substituted styrenes include, for example, styrenes in which one or more substituent is located the ortho, meta, or para position (or a combination thereof, or a mixture thereof) relative to the vinyl group. The substituent in a ring-substituted styrene may be, for example, an additional vinyl group, an epoxy group, a different substituent, combinations thereof, and mixtures thereof.

[0020] As used herein, a styrenic polymer is a polymer in which 50% or more by weight of the polymer is polymerized units of vinyl aromatic monomers.

[0021] As used herein, a crosslinking monomer is a monomer that contains two or more vinyl groups, both of which are capable of participating in a vinyl polymerization reaction.

[0022] As used herein, a functional group is capable of undergoing one or more useful chemical reaction. As used herein, a functional resin is a polymer that has a functional group pendant from the polymer chain.

[0023] As used herein, by vinyl benzene mono oxide monomer (VBMO) is meant a compound that contains an aromatic 6-carbon ring in which a vinyl group is attached to one of the carbon atoms of the aromatic ring and an epoxy group is attached to a different carbon atom of the same aromatic ring. The epoxy group has the structure (XI):

The epoxy group may be ortho, meta, or para in relation to the vinyl group.

[0024] As used herein, a nucleoside is a compound in which either ribose or deoxyribose is attached to a nucleobase via a beta-glycosidic link. Nuclear bases are adenine, guanine, cytosine, uracil, and thymine. Also considered herein to be nucleosides are derivatives of such compounds in which one or more hydrogen atom is replaced by a protecting group. Nucleosides are represented herein by schematic structure (XXI):

In schematic structure (XXI), the only atoms shown explicitly are the -OH groups attached to the 3' and 5' carbon atoms; the other features of the molecule, such as the 5-member sugar ring and the nucleobase, are not shown.

[0025] As used herein, a nucleotide is a compound in which a nucleoside is bonded to a phosphorous-containing group that is capable of reacting with an -OH group on a different nucleotide or nucleoside to form a covalent bond. As used herein, an oligonucleotide is a polymer in which each polymerized unit is a nucleotide. Oligonucleotides have from 2 to 1,000 polymerized units. [0026] As used herein, a polypeptide is a polymer in which each polymerized unit is an amino acid, and the polymerized units are linked to each other by peptide bonds.

Polypeptides have 2 or more polymerized units.

[0027] As used herein, a diacid is a compound having two carboxyl groups. A diacid has the structure HOOC-RA-COOH, where RA is an organic group. A monoester of a diacid has the structure HOOC-RA-COO-RB, where RB is an organic group. Regardless of how such a monoester is made, it is described as the monoester of the diacid and an alcohol having the structure HO-RB.

[0028] Preferred methods of the present invention involve the use of a functional resin, which is a polymer. The functional resin preferably comprises polymerized units of vinyl aromatic monomers. Preferably, the amount of polymerized units of vinyl aromatic monomers, by weight based on the weight of the functional resin is 50% or more; more preferably 75% or more; more preferably 90% or more; more preferably 95% or more; more preferably 99% or more.

[0029] Preferably the amount of polymerized units of VBMO is, by weight based on the total weight of the functional resin , 5% or more; more preferably 10% or more; more preferably 15% or more; more preferably 20% or more. Preferably the amount of polymerized units of VBMO is, by weight based on the total weight of the functional resin , 99% or less; more preferably 90% or less; more preferably 70% or less; more preferably 50% or less.

[0030] Preferably, the amount of polymerized units of crosslinking monomer is, by weight based on the weight of the functional resin , 0.5% or more; more preferably 2% or more; more preferably 5% or more; more preferably 10% or more. Preferably, the amount of polymerized units of crosslinking monomer is, by weight based on the weight of the functional resin , 40% or less; more preferably 20% or less; more preferably 20% or less.

[0031] Suitable crosslinking monomers include divinylbenzene, divinylpyridine, divinyltoluenes, divinylnaphthalenes, diallyl phthalate, ethylene glycol diacrylate, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, neopentyl glycol dimethacrylate, bis-phenol A dimethacrylate, pentaerythritol tetra- and trimethacrylates, divinylxylene, divinylethylbenzene, divinylsulfone, divinylketone, divinylsulfide, allyl acrylate, diallyl maleate diallyl fumarate, diallyl succinate, diallyl carbonate, diallyl malonate, diallyl oxalate, diallyl adipate, diallyl sebacate, divinyl sebacate, diallyl tartrate, diallyl silicate, triallyl tricarballylate, triallyl aconitate, triallyl citrate, triallyl phosphate, Ν,Ν'- methylenediacrylamide, Ν,Ν'-methylene dimethacrylamide, N,N'-ethylenediacrylamide, trivinylbenzene, trivinylnaphthalene, polyvinylanthracenes and the polyallyl and polyvinyl ethers of glycol, glycerol, pentaerythritol, resorcinol and the monothio or dithio derivatives of glycols.

[0032] Preferred crosslinking monomers are divinylbenzene, diethylene glycol divinyl ether, trimethylol propane trimethacrylate, and mixtures thereof. Most preferred is divinyl benzene.

[0033] Herein, reference is m

In structure (XX), "Particle" denotes a particle that contains crosslinked styrenic polymer. R is any chemical group. The line extending from the circle surrounding the word "Particle" denotes a covalent bond between a carbon atom in the styrenic polymer and a carbon atom of the aromatic ring displayed in structure (XX). The line extending from the center of the aromatic ring in structure (XX) denotes that R is attached by a covalent bond to a carbon atom of the aromatic ring; R may be attached in the ortho, meta, or para position relative to the bond that attaches to the Particle. There may be many of the structures shown in figure (XX) attached to a single particle.

[0034] A preferred method of making the support resin of the present invention involves the use of a functional resin having the structure (I):

The particle comprises a crosslinked styrenic polymer; -INT is a functional group; -R is hydrogen or alkyl; -L¹⁰¹- and -L²⁰¹- are independent optional linking groups; and each of -R 201 , -R 202 , and -IT 20"3 is independently hydro gen or a substituted or unsubstituted hydrocarbyl group.

[0035] Preferably, -L²⁰¹- is absent. Preferably, -R¹⁰¹ is H. Preferably, -R²⁰¹ is H.

Preferably, -R²⁰² is H. Preferably, -R²⁰³ is H. Preferably, -L¹⁰¹- is absent or is selected from the following: ii— R³⁰⁵ or o— R³⁰⁵ or s— R³⁰⁵

where -R - is any divalent group and -R^JUD is hydro gen or a substituted or unsubstituted hydrocarbyl group. Preferably, -R is hydrogen. Preferably, when -R - is present, -R - is a substituted or unsubstituted hydrocarbyl group; more preferably; -R - is a substituted or unsubstituted alkyl group; more preferably -R - is an unsubstituted alkyl group; more preferably -R - is an unsubstituted alkyl group having 1 to 6 carbon atoms.

305 101 305

More preferably, -L - is absent or is -NH-R^JUJ- ; more preferably IS -NH-R

305

and -R - is an unsubstituted alkyl group having 1 to 6 carbon atoms. Preferably, each of ,201 ,202 . ,203 · . .

-R , -R , and -R is hydrogen.

[0036] Preferred -INT groups are halogen, hydroxyl, thio, amino, N-alkylamino, halo- substituted alkyl, amino-substituted alkyl, amino- and aryl-substituted alkyl, amino- and alkylaryl-substituted alkyl, hydroxy- substituted alkyl, and compounds of any of structures XIII through XIX.

[0037] Structures XIII to XVII are defined as follows:

derivatives thereof in which one of the H atoms attached to a nitrogen atom is replaced by a protecting group. Preferred protecting groups are Fmoc (9-fluorenylmethyl carbamate) and t- Boc (Di-tert-butyl dicarbonate). The unattached line segment in each of structures XIII to XIX denotes a covalent bond that attaches to some other atom; it does not denote a terminal methyl group.

[0039] Among embodiments in which -INT is halo-substituted alkyl, preferred are chloro-, bromo-, and iodo-substituted alkyl; more preferred is chloromethyl. Among embodiments in which -INT is amino-substituted alkyl, preferred is aminomethyl. Among embodiments in which -INT is amino- and alkyl-substituted aryl, preferred is alpha- aminobenzyl. Among embodiments in which -INT is amino- and alkylaryl-substituted alkyl, preferred are alpha-amino-3-methylbenzyl and alpha-amino-4-methylbenzyl. Among embodiments in which -INT is hydroxy-substituted alkyl, preferred is hydroxymethyl.

[0040] In some embodiments, the functional resin of the present invention is suitable for use as a solid support for polypeptide synthesis; such a resin is known herein as a

"polypeptide support resin." In preferred embodiments, the functional resin of the present invention is suitable for use in making a solid support for oligonucleotide synthesis. A resin suitable as a solid support for oligonucleotide synthesis is known herein as an

"oligonucleotide support resin."

[0041] Among polypeptide support resins, preferred are those in which -L¹⁰²- is not present. Among polypeptide support resins, preferred are those with -INT groups described herein above as preferred for the functional resin of the present invention.

[0042] Among oligonucleotide synthesis support resins, the more preferred -INT are -OH, -SH, and -NH2; even more preferred is -NH2.

[0043] The functional resin is in the form of particles. Preferably, the amount of the functional resin of the present invention that is contained in the particles is 50% or more by weight, based on the weight of the particles; more preferably 90% or more; more preferably 99% or more.

[0044] Preferably, the volume average diameter of the particles that contain the functional resin is 10 micrometers or larger; more preferably 20 micrometers or larger; more preferably 40 micrometers or larger. Preferably the volume average diameter of the particles that contain the functional resin of the present invention is 1000 micrometers or smaller; more preferably 500 micrometers or smaller; more preferably 200 micrometers or smaller.

[0045] Preferably, the functional resin is made by starting with a precursor resin that contains structure (III):

In Structure (III), the particle contains a crosslinked styrenic polymer. -L - , -R , -R ,

203 201 201 202 and -R are defined as in structure (I). Preferred embodiments of -L - , -R , -R ,

203

and -R are the same as those described herein above as preferred for structure (I).

The particle contains a crosslinked styrenic polymer. The precursor resin is either a gel resin or a macroreticular resin. Preferably, the precursor resin is a macroreticular resin.

[0046] In making a gel precursor resin, a wide variety of polymerization conditions and processes can be used. Suspension polymerization is the preferred method of polymerization. An exemplary method is the following: a first polymerization step is carried out in an aqueous solution. The aqueous solution in addition to water may optionally contain buffer agents, such as for example, boric acid and/or sodium borate. The aqueous solution preferably is water containing suspending agents such as for example: polyvinyl alcohol, gelatin, and/or carboxymethylmethylcellulose. To the aqueous solution are added VBMO, at least one crosslinking monomer, at least one initiator, and optionally one or more of styrene and one or more alpha-alkyl styrene, to form a polymer resin. Subsequently, in a

functionalization step, the polymer resin is mixed with an optional swelling solvent and a functional agent. The swelling solvent may be mixed with the polymer resin before, during, or after the functional agent or not at all. The residual functional agent and swelling solvents are then removed by conventional means to provide the final resin product.

[0047] A suitable class of free-radical generating compounds which can be used as initiators for polymerization are the azo initiators, which contain an N=N group attached to aliphatic carbon atoms, at least one of which is tertiary. Preferred azo initiators are azodiisobutyronitrile, azodiisobutyramide, azobis(. alpha.,. alpha. -dimethylvaleronitrile), azobis(. alpha. -methyl-butyronitrile) dimethyl, diethyl, and dibutyl azobis(methyl-valerate).

[0048] When an azo initiator is used, the preferred amount of initiator is, by weight based on the total weight of monomer is 0.01 or larger. Preferably, the amount of initiator is, by weight based on the total weight of monomer is 2% or less; more preferably 1.5% or less; most preferably 1.0 percent or less. [0049] Non-azo initiators may also be used. Preferred non-azo initiators include common peroxide initiators known in this art, such as benzoyl peroxide, cumene hydroperoxide, acetyl peroxide, caproyl peroxide, and the like. When a non-azo initiator is used, the preferred amount is, by weight based on the total weight of monomer, 0.01% or more. When a non-azo initiator is used, the preferred amount is, by weight based on the total weight of monomer, 5% or less; more preferably 2.5% or less; more preferably 1% or less. Any of the initiators described herein may be used alone or in combination with one or more additional initiators.

[0050] Swelling solvents useful in the present invention include but are not limited to methylal, dimethoxymethane, N-methylpyrrolidone, dimethylformamide, dimethylacetamide, and toluene.

[0051] Preferred swelling solvents of the present invention are methylal and N- methylpyrrolidone.

[0052] Preferably, the amount of swelling solvent present during functionalization is, by weight based on the total weight of monomers, 40% or more; more preferably 50% or more; more preferably 55% or more. Preferably, the amount of swelling solvent present during functionalization is, by weight based on the total weight of monomers, 99% or less; more preferably 80% or less; more preferably 65% or less.

[0053] Macroreticular precursor resins are prepared by a method similar to the gel precursor resin preparation. As in the case of making gel resins, suspension polymerization is the preferred method. An exemplary method is the following: a first polymerization step is carried out in an aqueous solution. The aqueous solution preferably is based on water as the continuous phase. The polymerization composition contains a substance which is a solvent for the monomer or monomer mixture and a non-solvent for the polymer, said solvent being referred to hereinafter as a "phase extender." To the aqueous solution are added VBMO, at least one crosslinking monomer, a phase extender, at least one initiator, and optionally one or more of styrene and one or more alpha-alkyl styrene, to form a polymer resin. Subsequent to polymerization, the phase extender must be removed, e.g., by washing the bead with another solvent or, more commonly, by azeotropic distillation of the aqueous suspension slurry.

[0054] In making a macroreticular resin, the preferred types and amounts of monomers are the same as those in making gel resins.

[0055] The phase extender, when present, is present in an amount of between 20% and 80%, preferably 25 to 60, more preferably 30 to 35 %, i.e., the mixture of monomer, crosslinker and phase extender. Preferably, the phase extender comprises 30 - 50% by weight of the organic phase and the ratio of aqueous to organic phase is 1.5: 1. Polymerization temperatures normally are held in the range of 60-80 °C. Preferred phase extenders are o- xylene, methyl isobutyl carbinol (MIBC), 2,4,4-trimethylpentane, and mixtures thereof.

[0056] Subsequently, the precursor resin is optionally mixed with a swelling solvent. The phase extender and any swelling solvents are then removed to provide the functional resin.

[0057] Preferably, the precursor resin is reacted with one or more reagent to form a functional resin. When the precursor resin is a gel resin, the functional resin will also be a gel resin. When the precursor resin is a macroreticular resin, the functional resin will also be a macroreticular resin.

[0058] Also contemplated are embodiments in which a precursor resin that contains structure (III) is reacted with one or more reagent to form a functional resin that contains structure (XXXI).

[0059] In some embodiments, the present invention involves an oligonucleotide synthesis support resin. In such embodiments, the oligonucleotide synthesis support resin contains structure (II):

The particle comprises a crosslinked styrenic polymer; -R is hydrogen or alkyl; -L - and -L 201 - are independent optional linking groups; each of -R 201 , -R 202 , and -R 203 is independently hydrogen or a substituted or unsubstituted hydrocarbyl group; and

-L is an functional group that is capable of forming a phosphate linkage with a nucleotide.

[0060] For oligonucleotide synthesis support resins, preferred embodiments of -L²⁰¹- , -R²⁰¹, -R²⁰², and -R²⁰³ are the same as those described herein above as preferred for structure (I).

[0061] In embodiments in which the support resins of the present invention are synthesis support resins suitable for use as a solid support for oligonucleotide synthesis (herein "oligonucleotide support resins"), preferably, -L¹⁰²- is absent or is selected from the following: ii— R³⁰⁵ or o— R³⁰⁵ or s— R³⁰⁵

where -R 305 - is any divalent group and -R 306 is hydrogen or a substituted or unsubstituted hydrocarbyl group. Preferably, -R 306 is hydrogen. Preferably, when -R 305 - is present, -R 305 - is a substituted or unsubstituted hydrocarbyl group; more preferably; -R 305 - is a substituted or unsubstituted alkyl group; more preferably -R - is an unsubstituted alkyl

305

group; more preferably -R - is an unsubstituted alkyl group having 1 to 6 carbon atoms. More preferably, -L¹⁰²- is absent or is -NH-R³⁰⁵- ; more preferably -L¹⁰²- is -NH-R³⁰⁵-

305

and -R - is an unsubstituted alkyl group having 1 to 6 carbon atoms. Preferred -L groups are residues of diacid monoesters of nucleosides.

[0062] A residue of a diacid monoester of a nucleoside is defined herein is the structure that would result if one carboxyl group of a diacid were first reacted with an -OH group on a nucleoside to form a monoester and then the other carboxyl group of the diacid were reacted with -L102- to form a covalent bond.

[0063] More preferred -L groups have structure (XXIII):

Prot— o

(XXIII)

Prot is a protecting group or hydrogen. Preferably, Prot is a protecting group. Preferred protecting group is DMT (4,4'-dimethoxytrityl). Preferably, RA is a hydrocarbon aliphatic group or a hydrocarbon aromatic group; more preferably RA is an alkyl group; more preferably -RA- is -CH₂CH₂-.

[0064] Preferably, the synthesis support resin of the present invention is in the form of particles. Preferably, the amount of the synthesis resin of the present invention that is contained in the particles is 50% or more by weight, based on the weight of the particles; more preferably 90% or more; more preferably 99% or more.

[0065] Preferably, the volume average diameter of the particles that contain the synthesis support resin of the present invention is 10 micrometers or larger; more preferably 20 micrometers or larger; more preferably 40 micrometers or larger. Preferably the volume average diameter of the particles that contain the synthesis support resin of the present invention is 1000 micrometers or smaller; more preferably 500 micrometers or smaller; more preferably 200 micrometers or smaller.

[0066] In the synthesis support resin, the preferred amounts of crosslinking monomer and the preferred types of crosslinking monomer are the same as those described herein above for the functional resin.

[0067] The preferred method of producing a synthesis support resin that is suitable for oligonucleotide synthesis (i.e., a oligonucleotide support resin) is as follows. Preferably, a precursor resin is reacted with reagents that include an alkyl multi-amine (i.e., an alkyl compound substituted with two or more amine groups) to produce a functional resin that contains structure (I) in which -INT is -NH2. Preferably, the functional resin is reacted with one or more reagent to produce an oligonucleotide support resin that contains structure (II). More preferably, the functional resin is reacted with reagents that include one or more carbodiimide and one or more diacid monoester of a nucleoside.

[0068] A carbodiimide has the structure R1-N=C=N-R2, where Rl and R2 are organic groups. Preferably, Rl and R2 are identical to each other. Preferably Rl and R2 are alkyl; more preferably Rl and R2 are secondary alkyl. Preferably Rl and R2 have 8 or fewer carbon atoms.

[0069] Preferred diacid monoester of a nucleoside has the structure (XXIV):

Prot— o.

The features in structure XXIV are defined as in structure (XXIII). Preferably Prot- is DMT-. -RA- is preferably alkyl, more preferably -CH2CH2-.

[0070] When the synthesis support resin of the present invention is an oligonucleotide support resin, it is preferred to make the oligonucleotide support resin by starting with a functional resin of the present invention and then reacting that functional resin with one or more reagent to convert it to an oligonucleotide support resin.

[0071] Preferably, the reagent or reagents that are reacted with the functional resin of the

102

present invention include hexamethylene tetramine. Preferably, -L - has the structure -L¹⁰²-L¹⁰⁴-, where -L¹⁰⁴- has the structure of -L with one hydrogen atom removed.

[0072] The synthesis support resin of the present invention is in the form of particles. Preferably, the amount of the synthesis support resin of the present invention that is contained in the particles is 50% or more by weight, based on the weight of the particles; more preferably 90% or more; more preferably 99% or more.

[0073] When the functional resin is a gel resin, a synthesis support resin made from that functional resin will also be a gel resin. Similarly, when the functional resin is a

macroreticular resin, a synthesis support resin made from that functional resin will also be a macroreticular resin.

[0074] When a synthesis support resin of the present invention is put to use, it is contemplated that methods appropriate for oligonucleotide synthesis would be used, including the use of appropriate reagents in addition to or instead of the reagents described herein, and also including the use of appropriate protecting groups in addition to or instead of the protecting groups described herein.

[0075] When a synthesis support resin is put to use, it is contemplated that methods appropriate for polypeptide synthesis and/or oligonucleotide synthesis would be used, including the use of appropriate reagents in addition to or instead of the reagents described herein, and also including the use of appropriate protecting groups in addition to or instead of the protecting groups described herein.

[0076] When a polypeptide support resin of the present invention is put to use, a preferred method includes the following steps. Preferably, one step is to react the resin with a protected amino acid in the presence of a carbodiimide. Preferably, the protected amino acid has a protecting group attached to the amine nitrogen atom of the the amino acid. Preferably the carboxylic acid group of the protected amino acid reacts with a functional group on the polypeptide support resin to form a covalent bond. Preferably, the protecting group is then removed from the amine group, and the amine group subsequently reacts with the carboxylic acid group of a fresh protected amino acid. Preferably the process is repeated until the desired polypeptide is formed. Preferably, additional steps will be performed, including steps to remove any remaining protecting groups and to sever the polypeptide from the polypeptide support resin.

[0077] When an oligonucleotide synthesis support resin of the present invention is put to use, a preferred method includes the following steps. Preferably, the oligonucleotide support resin is reacted with one or more reagent to remove the protecting group from the oxygen attached to the 5' carbon, and to replace the protecting group with a hydrogen. Preferably, the de-protected oligonucleotide support resin is then reacted with one or more reagents that include a reagent having structure (XXV):

Prot— o

Phos is a phosporous-containing chemical group suitable for forming a covalent bond with the oxygen attached to the 5' carbon on a different nucleoside. The other features of structure (XXV) are defined in structure (XXIII). Preferably Phos is N,N-diisopropyl

phosphoramidite. Preferably, Phos reacts with the -OH group attached to the 5' carbon of the nucleoside that is attached to the oligonucleoside support resin. Preferably, the process is repeated until the desired oligonucleotide is formed. Preferably, additional steps will be performed, including steps to remove any remaining protecting groups and to sever the oligonucleotide from the oligonucleotide support resin.

[0078] The following are examples of the present invention.

[0079] Preparation A: Precursor Resin

[0080] Aqueous phase was prepared for suspension polymerization as follows. Culminal MHEC 8000 (protective colloid, 2.4 g) was dissolved in lOOOmL of deionized water at 80°C. The solution was held at 80°C for 1 hour and then coolled to ambient temperature

(approximately 23 °C).

[0081] Macro porous (i.e., macroreticular) copolymer synthesis with 2,2,4- Trimethylpentane was performed as follows. 1000 g aqueous phase was placed in a 2 liter, 3 neck glass reactor. To the aqueous phase were added the following ingredients in the order shown: 41.5 g of divinyl benzene (DVB, 80 wt divinylbenzene, 20 wt

ethylvinylbenzene), 61.1 g of VBMO (78.23 wt vinyl benzene mono oxide monomer, 11.7 wt% Ethyl Vinyl Benzene Oxide, 10.07 wt% impurity), 156 g of styrene, 425 g of 2,2,4- Trimethylpentane (425), and 3.0 g of tert-Butyl peroxy-2-ethylhexanoate. While stirring at 350 rpm, under nitrogen atmosphere, the following temperature ramp was applied: 25°C for 30 min, ramp to 80°C over 60 min, held at 80°C for 18 hour, then cooled to ambient temperature. The resulting beads were isolated by filtration by using a 2 liter sintered glass funnel, 2 liter filter flask, and vacuum. The isolated beads were then rinsed with 8 liters of methanol and followed by 8 liters of deionized water. Beads were placed into an oven, and then 40 mm of vacuum was applied. The beads were heated in the oven at 40°C for 18 hours, then cooled to ambient temperature. The beads were screened between 53 um to 90 um using stainless steel sieve. Volume average particle size was 75 micrometers.

[0082] Example 1 : Functional Resin

[0083] Macro porous poly(65 styrene/25 % VBMO/10 % divinylbenzene) beads (50.0 g) made by the process of Preparation A were charged to a 1-liter 3-neck round-bottom flask equipped with a overhead stirrer. Hexamethylenetetramine (159 g), deionized water (112.5 g), and Dimethoxymethane (221.3 g) were added to the reactor flask. Using mechanical stirring, the resulting mixture was gently agitated for 4 hours at 44°C under nitrogen atmosphere, then cooled to ambient temperature.

[0084] The reaction mixture was filtered using a 1 liter sintered glass funnel, 2 liter filter flask, and vacuum. The resin was washed with deionized water (300 mL) and filtered.

[0085] The resulting resin was charged to a 1 -liter 3-neck round-bottom flask equipped with an overhead stirrer. To the reactor was sequentially added hydrochloric acid (35%) (156.3g) and deionized water (156.3 g). Using mechanical stirring, the resulting mixture was gently agitated for 3 hours at 45 °C under nitrogen atmosphere, then cooled to ambient temperature. It is contemplated that the functional resin that was produced had structure (IV) in which -L was -OH.

[0086] The reaction mixture was filtered using a 1 -liter sintered glass funnel, 2-liter filter flask, and vacuum. The resin was washed with deionized water (500 mL) and filtered.

[0087] Example 2: Synthesis support resin

[0088] To a 1-liter 3-neck round-bottom flask equipped with a overhead stirrer and under nitrogen atmosphere, the following were successively added: the functional resin beads of Example 1 (37.5 g), anhydrous N,N-dimethylformaide (375 mL), anhydrous pyridine (110 mL), N, N dimethylamniopyridinne (137 mg), 1,3-dissopropylcarbodiimide (4.26g),and 5'- dimethoxytrityl-N2-isobutyryl-2-deoxyguanosine-3' -succinate ester (9.25 g). Using mechanical stirring, the resulting mixture was gently agitated for 6 hours at 25 °C. The reaction mixture was filtered using a 1 -liter sintered glass funnel, 2-liter filter flask, and a vacuum. The loaded resin was washed with acetonitrile (300 mL) and filtered. The degree of substitution was determined by dimethoxytriyl cation assay outlined in the procedure given below

[0089] To 10.0 mg of the functionalized resins was added 200 ml of 10 % v/v

dichloroacetic acid in dichloromethane. The resulting solution was diluted to 800 (mL) with 0.1 M p-toluenesulfonic acid in acetonitrile. The absorption of this solution was measured using standard 1-cm quartz cuvette and a HP UV/VIS spectrophotometer at 498 nm, and the degree of substitution was calculated using equation

[0090] Substitution (micromol/g) = A498 X 14.3 X 800

[0091] After the desired loading was achieved, the unfuctionalized hydroxyl groups remaining in the resin were "capped" with acetyl group by the sequential addition of anhydrous pyridine (150 mL) to the reaction mixture, followed by acetic anhydride. This mixture was transferred back into 1 -liter 3-neck round-bottom flask equipped with an overhead stirrer and sealed under a nitrogen atmosphere. The mixture was stirred for 1 hour at ambient temperature.

[0092] The functionalized and capped solid support was transferred a 1 liter sintered glass funnel, 2 liter filter flask, and washed sequentially with fresh dichloromethane (3 x 500 mL), methanol (3 x 300 mL) and diethyl ether (3 x 300 mL) and then dried under vacuum at ambient temperature. With the procedure described above, functionalized solid phase support having nucleoside substitution value of 220 micromol/g was obtained. [0093] Example 3 : Functional Resin

[0094] Macro porous beads (50.0 g) made by preparation A were charged to a 1-liter 3-neck round-bottom flask equipped with a overhead stirrer. 1 ,2-Diaminopropane (260 g) and Dimethoxymethane (221.3 g) were added to the reactor flask. Using mechanical stirring, the resulting mixture was gently agitated for 8 hours at 45 °C under nitrogen atmosphere, then cooled to ambient temperature (approximately 23 °C).

[0095] The reaction mixture was filtered using a 1 liter sintered glass funnel, 2 liter filter flask, and vacuum. The resin was washed with deionized water (300 mL) and filtered.

[0096] The resulting resin was charged to a 1 liter 3 neck round-bottom flask equipped with an overhead stirrer. To the reactor was sequentially added, hydrochloric acid (35%) (156.3g) and deionized water (156.3 g). Using mechanical stirring, the resulting mixture was gently agitated for 3 hours at 45 °C under nitrogen atmosphere, then cooled to ambient temperature. The reaction mixture was filtered using a 1 liter sintered glass funnel, 2 liter filter flask, and vacuum. The resin was washed with deionized water (500 mL) and filtered.

[0097] Example 4: Synthesis support resin

[0098] To a 1 liter 3 neck round-bottom flask equipped with a overhead stirrer and under nitrogen atmosphere, successively added were beads produced in Example 3 (37.5 g), anhydrous N,N-dimethylformaide (375 mL), anhydrous pyridine (110 mL), N, N

dimethylamniopyridinne (137 mg), 1,3-dissopropylcarbodiimide (4.26g),and 5'- dimethoxytrityl-N2-isobutyryl-2-deoxyguanosine-3' -succinate ester (9.25 g), Using mechanical stirring, the resulting mixture was gently agitated for 6 hours at 25 °C. The reaction mixture was filtered using a 1 liter sintered glass funnel, 2 liter filter flask, and a vacuum. The loaded resin was washed with acetonitrile (300 mL) and filtered. The degree of substitution was determined as in Example 2.

[0099] After the desired substitution was achieved, the unfuctionalized amino groups remaining in the resin were "capped" with acetyl group by the sequential addition of anhydrous pyridine (150 mL) to the reaction mixture, followed by acetic anhydride. This mixture was transferred back into 1 liter 3 neck round-bottom flask equipped with a overhead stirrer and sealed under a nitrogen atmosphere. The mixture was stirred for 1 hour at room temperature.

[00100] The functionalized and capped solid support was transferred to a 1 liter sintered glass funnel, 2 liter filter flask, and washed sequentially with fresh dichloromethane (3 x 500 mL), methanol (3 x 300 mL) and diethyl ether (3 x 300 mL) and then dried under vacuum at room temperature (approximately 23 °C). With the procedure described above, functionalized solid phase support having nucleoside substitution value of 270 micromol/g was obtained.

Claims

1. A functional resin comprising structure (I)

wherein said particle comprises a crosslinked styrenic polymer,

wherein -INT is a functional group,

wherein -R¹⁰¹ is hydrogen or alkyl,

wherein -L¹⁰¹- and -L²⁰¹- are independent optional linking groups,

wherein said functionalized resin is suitable for one or more of use as a solid support for polypeptide synthesis or use in making a solid support for oligonucleotide synthesis.

2. The functional resin of claim 1 , wherein said -INT is -NH₂.

3. The functional resin of claim 1, wherein each of -R²⁰¹, -R²⁰², and -R²⁰³ is hydrogen.

4. A method of making the functional resin of claim 1 comprising the steps of

(A) providing an epo -functional resin comprising structure III

wherein said particle comprises a crosslinked styrenic polymer,

wherein -L 201 - is an independent optional linking group,

(B) reacting said epoxy-functional resin with one or more reagent so that said structure III becomes said structure I.

5. The method of claim 7, wherein -L 201 - is absent, and wherein each of -R 201 , -R 202 , and -R²⁰³ is hydrogen.

6. A support resin suitable for oligonucleotide synthesis comprising structure (II)

wherein said particle comprises a crosslinked styrenic polymer,

wherein -R¹⁰¹ is hydrogen or alkyl,

wherein -L 102 - and -L 201 - are independent optional linking groups,

wherein each of -R 201 , -R 202 , and -R 203 is independently hydrogen or a substituted or unsubstituted hydrocarbyl group, and

wherein -L is an functional group that is capable of forming a phosphate linkage with a nucleotide.

7. The support resin of claim 1, wherein -L 201 - is absent.

8. The support resin of claim 1, wherein said -L is a succinate ester of a nucleoside.

9. A method of making the support resin of claim 6 comprising the steps of

(A) providing a functional resin comprising structure (IV)

wherein said particle comprises a crosslinked styrenic polymer,

wherein -INT is a functional group,

wherein -R¹⁰¹ is hydrogen or alkyl,

wherein -L¹⁰¹- and -L²⁰¹- are independent optional linking groups, wherein each of -R 201 , -R 202 , and -R 203 is independently hydrogen or a substituted or unsubstituted hydrocarbyl group,

(B) reacting said functional resin with one or more reagent so that said structure (IV) becomes said structure (II).

10. The method of claim 9, wherein said -L is a residue of a succinate ester of a nucleoside.