Functionalized silicon compounds and methods for their synthesis and use Number:7,129,307 from the United States Patent and Trademark Office (PTO) owispatent

Title: Functionalized silicon compounds and methods for their synthesis and use

Abstract: Provided are functionalized silicon compounds and methods for their synthesis and use. The functionalized silicon compounds include at least one activated silicon group and at least one derivatizable functional group. Exemplary derivatizable functional groups include hydroxyl, amino, carboxyl and thiol, as well as modified forms thereof, such as activated or protected forms. The functionalized silicon compounds may be covalently attached to surfaces to form functionalized surfaces which may be used in a wide range of different applications. In one embodiment, the silicon compounds are attached to the surface of a substrate comprising silica, such as a glass substrate, to provide a functionalized surface on the substrate, to which molecules, including polypeptides and nucleic acids, may be attached. In one embodiment, after covalent attachment of a functionalized silicon compound to the surface of a solid silica substrate to form a functionalized coating on the substrate, an array of nucleic acids may be covalently attached to the substrate. Thus, the method permits the formation of high density arrays of nucleic acids immobilized on a substrate, which may be used, for example, in conducting high volume nucleic acid hybridization assays.

Patent Number: 7,129,307 Issued on 10/31/2006 to McGall,   et al.

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Inventors:	McGall; Glenn (Mountain View, CA), Forman; Jonathan Eric (San Jose, CA)
Assignee:	Affymetrix, Inc. (Santa Clara, CA)
Appl. No.:	10/853,378
Filed:	May 25, 2004

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Related U.S. Patent Documents

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	Application Number	Filing Date	Patent Number	Issue Date
	10282979	Oct., 2002	6743882
	09418044	Nov., 2002	6486286
	09172190	Jul., 2001	6262216

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Current U.S. Class:	528/10 ; 528/25; 528/28; 528/33
Current International Class:	C08G 77/00 (20060101); C08G 77/04 (20060101)
Field of Search:	528/10,25,28,33

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Primary Examiner: Riley; Jezia
Attorney, Agent or Firm: Cooley Godward LLP

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Parent Case Text

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CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 10/282,979 filed Oct. 29, 2002, now U.S. Pat. No. 6,743,882 issued on Jun. 1, 2004, which is a divisional of U.S. application Ser. No. 09/418,044 filed on Oct. 13, 1999, now U.S. Pat. No. 6,486,286 issued on Nov. 26, 2002, which is a continuation-in-part of U.S. application Ser. No. 09/172,190 filed Oct. 13, 1998, now U.S. Pat. No. 6,262,216 issued Jul. 17, 2001; the disclosures of which are incorporated herein by reference.

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Claims

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What is claimed is:

1. A functionalized silicon compound of Formulas 17, 18, 19, or 20: ##STR00013## wherein: R.sub.1 and R.sub.2 are independently alkoxy or halide, and R.sub.3 is alkoxy, halide or alkyl; L.sub.1, L.sub.2 and L.sub.3 are independently linear or branched alkyl, or linear or branched heteroalkyl, optionally comprising one or more derivatizable groups; A.sub.1 and A.sub.2 independently are H or a moiety comprising one or more derivatizable functional groups; B.sub.1 and B.sub.2 are independently alkyl, a heteroatom, or heteroalkyl; L.sub.4 is a direct bond, alkyl or heteroalkyl optionally comprising one or more derivatizable groups; and R is H, alkyl, heteroalkyl, or acyl.

2. The silicon compound of claim 1, wherein the derivatizable functional groups are independently selected from the group consisting of hydroxyl, amino, carboxyl, thio, halo, amido and sulfonate.

3. The silicon compound of claim 1, wherein the compound is selected from the group consisting of ##STR00014## ##STR00015##

4. The compound of claim 1, wherein R.sub.3 is an alkoxy group.

5. The compound of claim 4, wherein R.sub.1, R.sub.2, and R.sub.3, are independently, --OCH.sub.3, or --OCH.sub.2CH.sub.3.

6. The compound of claim 5, wherein R.sub.1, R.sub.2 and R.sub.3 are each --OCH.sub.3.

7. The compound of claim 4, wherein R.sub.1, and R.sub.2, are independently, --OCH.sub.3 or --OCH.sub.2CH.sub.3, and R.sub.3 is --CH.sub.3, or a substituted alkyl group.

8. The compound of claim 2, wherein L.sub.1, L.sub.2, and L.sub.3 are independently, --C.sub.1-25 alkyl; or a heteroalkyl; and the derivatizable groups are hydroxyl or amino.

9. The compound of claim 8, wherein L.sub.1, L.sub.2, and L.sub.3 are independently, --(CH.sub.2).sub.n--, wherein n is an integer from 2 to 10.

10. The compound of claim 8, wherein L.sub.1, L.sub.2, and L.sub.3 are independently, --(CH.sub.2).sub.n--, wherein n is 2, 3, or 4.

11. The compound of claim 1, wherein A.sub.1 and A.sub.2 independently comprise, a linear or branched alkyl or linear or branched heteroalkyl, having 1, 2, or 3 derivatizable groups.

12. The compound of claim 11, wherein the derivatizable groups are amino, or hydroxyl.

13. The compound of claim 12, wherein A.sub.1 and A.sub.2 are independently --CH.sub.2CH.sub.2OH, or an amide.

14. The compound of claim 1, wherein B.sub.1 and B.sub.2 are independently C.sub.1-12 alkyl, branched C.sub.1-12 alkyl, a heteroatom, or branched C.sub.1-12 heteroalkyl.

15. The compound of claim 1, wherein L.sub.4 is C.sub.1-12 alkyl or C.sub.1-12 heteroalkyl optionally comprising one more derivatizable groups.

16. The compound of claim 1, wherein acyl is HC(.dbd.CH.sub.2)CO-- or MeC(.dbd.CH.sub.2)CO--.

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Description

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TECHNICAL FIELD

This application relates to silicon compounds, methods of making silicon compounds, and methods for use of silicon compounds as silylating agents in the treatment of surfaces, such as glass.

BACKGROUND ART

Silylating agents have been developed in the art which react with and coat surfaces, such as silica surfaces. For example, silylating agents for use in modifying silica used in high performance chromatography packings have been developed. Monofunctional silylating agents have been used to form monolayer surface coatings, while di- and tri-functional silylating agents have been used to form polymerized coatings on silica surfaces. Many silylating agents, however, produce coatings with undesirable properties including instability to hydrolysis and the inadequate ability to mask the silica surface which may contain residual acidic silanols.

Silylating agents have been developed for the silylation of solid substrates, such as glass substrates, that include functional groups that may be derivatized by further covalent reaction. The silylating agents have been immobilized on the surface of covalent reaction. The silylating agents have been immobilized on the surface of substrates, such as glass, and used to prepare high density immobilized oligonucleotide probe arrays. For example, N-(3-(triethoxysilyl)-propyl)-4-hydroxybutyramide (PCR Inc., Gainesville, Fla. and Gelest, Tullytown, Pa.) has been used to silylate a glass substrate prior to photochemical synthesis of arrays of oligonucleotides on the substrate, as described in McGall et al., J. Am. Chem. Soc., 119:5081 5090 (1997), the disclosure of which is incorporated herein by reference.

Hydroxyalkylsilyl compounds that have been used to prepare hydroxyalkylated substances, such as glass substrates. N,N-bis(hydroxyethyl) aminopropyl-triethoxysilane has been used to treat glass substrates to permit the synthesis of high-density oligonucleotide arrays. McGall et al., Proc. Natl. Acad. Sci., 93:13555 13560 (1996); and Pease et al., Proc. Natl. Acad. Sci., 91:5022 5026 (1994), the disclosures of which are incorporated herein. Acetoxypropyl-triethoxysilane has been used to treat glass substrates to prepare them for oligonucleotide array synthesis, as described in PCT WO 97/39151, the disclosure of which is incorporated herein. 3-Glycidoxy propyltrimethoxysilane has been used to treat a glass support to provide a linker for the synthesis of oligonucleotides. EP Patent Application No. 89 120696.3.

Methods have been developed in the art for stabilizing surface bonded silicon compounds. The use of sterically hindered silylating agents is described in Kirkland et al., Anal. Chem. 61:2 11 (1989); and Schneider et al., Synthesis, 1027 1031 (1990). However, the use of these surface bonded silylating agents is disadvantageous, because they typically require very forcing conditions to achieve bonding to the glass, since their hindered nature makes them less reactive with the substrate.

It is an object of the invention to provide functionalized silicon compounds that are provided with derivatizable functional groups, that can be used to form functionalized coatings on materials, such as glass. It is a further object of the invention to provide functionalized silicon compounds that can be used to form coatings on materials that are stable under the conditions of use.

DISCLOSURE OF THE INVENTION

Provided are functionalized silicon compounds and methods for their use. The functionalized silicon compounds include an activated silicon group and a derivatizable functional group. Exemplary derivatizable functional groups include hydroxyl, amino, carboxyl and thiol, as well as modified forms thereof, such as activated or protected forms. The functionalized silicon compounds may be covalently attached to surfaces to form functionalized surfaces which may be used in a wide range of different applications. In one embodiment, the silicon compounds are attached to the surface of a substrate comprising silica, such as a glass substrate, to provide a functionalized surface on the silica containing substrate, to which molecules, including polypeptides and nucleic acids, may be attached. In one preferred embodiment, after covalent attachment of a functionalized silicon compound to the surface of a solid silica substrate to form a functionalized coating on the substrate, an array of nucleic acids may be covalently attached to the substrate. Thus, the method permits the formation of high density arrays of nucleic acids immobilized on a substrate, which may be used in conducting high volume nucleic acid hybridization assays.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of the functionalized silicon compounds VI and VII and compounds of Formula 6a.

FIG. 2 shows the structure of the functionalized silicon compound VIII.

FIG. 3 shows schemes for the synthesis of compounds IX and X.

FIG. 4 is a scheme showing the synthesis of compounds of Formula 5 or 6.

FIG. 5 show schemes for the synthesis of compounds XII, XIII and compounds of Formula 8.

FIG. 6 shows schemes for the synthesis of compounds XV, VI and compounds of Formula 9.

FIG. 7 shows schemes showing the synthesis of compounds of Formula 10 or 11.

FIG. 8 shows schemes showing the synthesis of compounds of Formula 12 or 13.

FIG. 9 is a scheme of the synthesis of compounds of Formula 16b.

FIG. 10 illustrates the structure of compounds of Formula 14 and 15.

FIG. 11 is a graph of stability of silicon compound bonded phases vs. time.

FIG. 12 is a graph of hybridization fluorescence intensity vs. silane.

FIG. 13 is a scheme showing the synthesis of silicon compounds XVIa e.

FIG. 14 is a scheme showing the synthesis of silicon compounds XVIIa f.

FIG. 15 shows the structure of compounds of the general Formulas 17 21.

FIG. 16 shows the structure of some exemplary silicon compounds.

FIG. 17 shows another embodiment of exemplary silicon compounds XXI and XXII.

FIG. 18 shows the structure of exemplary silicon compounds XXIII, XXV and XXVI.

FIG. 19 shows the structure of exemplary silicon compounds XXIX and XXX.

FIG. 20 shows the structure of exemplary silicon compounds XXIa b, XXIIa b and XXIIIa b.

FIG. 21 is a scheme showing the synthesis of silicon compounds XIX and XX.

FIG. 22 is a scheme showing the synthesis of silicon compounds XXI and XXII.

FIG. 23 is a scheme showing the synthesis of silicon compound XXIII.

FIG. 24 is a scheme showing the synthesis of silicon compound XXIV.

FIG. 25 is a scheme showing the synthesis of silicon compound XXVIII.

FIG. 26 is a scheme showing the synthesis of silicon compounds XXVI and XXV.

FIG. 27 is a scheme showing the synthesis of silicon compounds XXIX and XXX.

FIG. 28 is a scheme showing the synthesis of silicon compounds XXXIa b and XXXIIIa b.

FIG. 29 is a scheme showing the synthesis of silicon compounds XXXIIa b.

FIG. 30 is a graph of normalized intensity vs. silane for silicon compounds bound to a solid substrate.

FIG. 31 is a graph of normalized hybridization fluorescence intensity vs. silane.

MODES FOR CARRYING OUT THE INVENTION

Functionalized silicon compounds are provided, as well as methods for their synthesis and use. The functionalized silicon compounds may be used to form functionalized coatings on a variety of surfaces such as the surfaces of glass substrates.

Functionalized Silicon Compounds

A variety of functionalized silicon compounds, which are available commercially, or which may be synthesized as disclosed herein, may be used in the methods disclosed herein to react with surfaces to form functionalized surfaces which may be used in a wide range of different applications. In one embodiment, the functionalized silicon compounds are covalently attached to surfaces to produce functionalized surfaces on substrates. For example, the silicon compounds may be attached to the surfaces of glass substrates, to provide a functionalized surface to which molecules, including polypeptides and nucleic acids, may be attached.

As used herein, the term "silicon compound" refers to a compound comprising a silicon atom. In a preferred embodiment, the silicon compound is a silylating agent comprising an activated silicon group, wherein the activated silicon group comprises a silicon atom covalently linked to at least one reactive group, such as an alkoxy or halide, such that the silicon group is capable of reacting with a functional group, for example on a surface of a substrate, to form a covalent bond with the surface. For example, the activated silicon group on the silicon compound can react with the surface of a silica substrate comprising surface Si--OH groups to create siloxane bonds between the silicon compound and the silica substrate. Exemplary activated silicon groups include --Si(OMe).sub.3; --SiMe(OMe).sub.2; --SiMeCl.sub.2; SiMe(OEt).sub.2; SiCl.sub.3 and --Si(OEt).sub.3.

As used herein, the term "functionalized silicon compound" refers to a silicon compound comprising a silicon atom and a derivatizable functional group. In a preferred embodiment, the functionalized silicon compound is a functionalized silylating agent and includes an activated silicon group and a derivatizable functional group. As used herein, the term "derivatizable functional group" refers to a functional group that is capable of reacting to permit the formation of a covalent bond between the silicon compound and another substance, such as a polymer. Exemplary derivatizable functional groups include hydroxyl, amino, carboxy, thiol, and amide, as well as modified forms thereof, such as activated or protected forms. Derivatizable functional groups also include substitutable leaving groups such as halo or sulfonate. In one preferred embodiment, the derivatizable functional group is a group, such as a hydroxyl group, that is capable of reacting with activated nucleotides to permit nucleic acid synthesis. For example, the functionalized silicon compound may be covalently attached to the surface of a substrate, such as glass, and then derivatizable hydroxyl groups on the silicon compound may be reacted with an activated phosphate group on a protected nucleotide phosphoramidite or H-phosphonate, and then stepwise addition of further protected nucleotide phosphoramidites or H-phosphonates can result in the formation of a nucleic acid covalently attached to the support. The nucleic acids also may be attached to the derivatizable group via a linker. In a further embodiment, arrays of nucleic acids may be formed covalently attached to the substrate which are useful in conducting nucleic acid hybridization assays.

The term "polynucleotide" or "nucleic acid" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, that comprise purine and pyrimidine bases, or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The backbone of the polynucleotide can comprise sugars and phosphate groups, as may typically be found in RNA or DNA, or modified or substituted sugar or phosphate groups. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. The sequence of nucleotides may be interrupted by non-nucleotide components.

The functionalized silicon compounds used to form coatings on a surface may be selected, and obtained commercially, or made synthetically, depending on their properties under the conditions of intended use. For example, functionalized silicon compounds may be selected for silanization of a substrate that are stable after the silylation reaction to hydrolysis.

For example, in one embodiment, the functionalized silicon compounds are used to form a coating on a solid substrate, and include functional groups that permit the covalent attachment or synthesis of nucleic acid arrays to the solid substrate, such as glass. The resulting substrates are useful in nucleic acid hybridization assays, which are conducted, for example in aqueous buffers. In one embodiment, preferred are silicon compounds that produce coatings that are substantially stable to hybridization assay conditions, such as phosphate or TRIS buffer at about pH 6 9, and at elevated temperatures, for example, about 25 65.degree. C., for about 1 to 72 hours, such that hydrolysis is less than about 90%, e.g., less than about 50%, or e.g, less than about 20%, or about 10%. The functionalized surfaces on the substrate, formed by covalent attachment of functionalized silicon compounds, advantageously are substantially stable to provide a support for biomolecule array synthesis and to be used under rigorous assay conditions, such as nucleic acid hybridization assay conditions.

The functionalized silicon compound in one embodiment includes at least one activated silicon group and at least one derivatizable functional group. In one embodiment, the functionalized silicon compound includes at least one activated silicon group and a plurality of derivatizable functional groups, for example, 2, 3, 4 or more derivatizable functional groups. In another embodiment, the functionalized silicon compound includes at least one derivatizable functional group and a plurality of activated silicon groups, for example, 2, 3, 4 or more activated silicon groups. Methods of making the functionalized silicon compounds are provided as disclosed herein, as well as methods of use of the functionalized silicon compounds, including covalent attachment of the silicon compounds to surfaces of substrates to form functionalized surfaces, and further derivation of the surfaces to provide arrays of nucleic acids for use in assays on the surfaces.

In one embodiment, there is provided a method of functionalizing a surface, the method comprising covalently attaching to the surface a functionalized silicon compound, wherein the functionalized silicon compound comprises at least one derivatizable functional group and a plurality of activated silicon groups, for example, 2, 3, 4 or more activated silicon groups. The method may further comprise covalently attaching a plurality of functionalized silicon compounds to the surface, and forming an array of nucleic acids covalently attached to the functionalized silicon compounds on the surface.

Exemplary functionalized silicon compounds include compounds of Formula 1 shown below:

##STR00001## wherein R.sub.1 and R.sub.2 are independently a reactive group, such as halide or alkoxy, for example --OCH.sub.3 or --OCH.sub.2CH.sub.3, and R.sub.3 is alkoxy, halide or alkyl; and wherein R.sub.4 is a hydrophobic and/or sterically hindered group. In the functionalized silylating agents of Formula 1, R.sub.4 may be alkyl or haloalkyl, for example, --CH.sub.3, --CH.sub.2CH.sub.3, --CH(CH.sub.3).sub.2, --C(CH.sub.3).sub.3, or --CH(CF.sub.3).sub.2. A hydrophobic and/or sterically hindered R.sub.4 group, such as isopropyl or isobutyl, may be used to increase the hydrolytic stability of the resulting surface layer. Further hydrophobicity may be imparted by the use of a fluorocarbon R.sub.4 group, such as hexafluoroisopropyl ((CF.sub.3).sub.2CH--).

An exemplary compound of Formula 1 is silicon compound I below:

##STR00002##

In general, silicon compounds provide uniform and reproducible coatings. Silicon compounds with one derivatizable functional group can provide a lower concentration of surface derivatizable functional groups at maximum coverage of the substrate than the silicon compounds including multiple derivatizable functional groups. Silicon compounds with one derivatizable functional group, such as silicon compounds of Formula 1, however, which include a hydrophobic and/or sterically hindered R group, such as isopropyl or isobutyl, are advantageous since the hydrophobic or sterically hindered R group increases the hydrolytic stability of the resulting surface layer.

In one embodiment the functionalized silicon compounds of Formula 2 are provided:

##STR00003## In Formula 2, in one embodiment, R.sub.1, R.sub.2 and R.sub.3 are independently a reactive group, such as alkoxy or halide, for example, --OCH.sub.3, or --OCH.sub.2CH.sub.3, and wherein, in one embodiment, R.sub.1, R.sub.2 and R.sub.3 are each --OCH.sub.3. In one embodiment R.sub.1 and R.sub.2 are independently a reactive group, such as alkoxy or halide, for example --OCH.sub.3 or --OCH.sub.2CH.sub.3, and R.sub.3 is an alkoxy or halide group or an alkyl group, such as --CH.sub.3, or substituted alkyl group. In Formula 2, in one embodiment, L.sub.1 and L.sub.2 are independently alkyl, preferably --(CH.sub.2).sub.n--, wherein n=2 to 10, e.g., 3 to 4, or e.g., 2 3. In Formula 2, in one embodiment, A.sub.1 is H or a moiety comprising one or more derivatizable functional groups. In one embodiment, A.sub.1 is a moiety comprising an amino group or a hydroxyl group, such as --CH.sub.2CH.sub.2OH. In another embodiment, A.sub.1 is, for example, a branched hydrocarbon including a plurality of derivatizable functional groups, such as hydroxyl groups. In one embodiment in Formula 2, A.sub.1 is:

##STR00004## wherein p is 1 10,q is 0 or 1, and r is 2 5.

In one embodiment of Formula 2, R.sub.1 and R.sub.2 are independently alkoxy or halide; R.sub.3 is alkoxy, halide or alkyl; L.sub.1 and L.sub.2 are both --(CH.sub.2).sub.n--, wherein n=2 to 10, e.g., 2 to 3; and A.sub.1 is H or a moiety comprising one or more derivatizable functional groups.

Exemplary compounds of Formula 2 include compounds II, III and IV below. Other silicon compounds of Formula 2 that may be used to form functional surface coatings with enhanced hydrolytic stability include silicon compounds IX and X, shown in FIG. 3. In compound VIII, the triethoxysilyl group is shown by way of example, however alternatively, the activated silicon group may be other activated silicon groups or mixtures thereof, such as trimethoxysilyl. In another embodiment, there is provided a compound of Formula 11, wherein n is, for example, 1 to 10, e.g., 1 3, and G is a derivatizable functional group, such as hydroxyl, protected hydroxyl or halide such as Cl or Br, as shown in FIG. 7.

##STR00005##

In another embodiment, silicon compounds of Formula 14 in FIG. 10 are provided wherein, R.sub.1, R.sub.2 are independently a reactive group such as alkoxy, for example --OCH.sub.3 or --OCH.sub.2CH.sub.3, or halide; and R.sub.3 is a reactive group such as alkoxy or halide, or optionally alkyl.

Another embodiment of Formula 2 is as follows: In one embodiment of Formula 2, R.sub.1, R.sub.2, R.sub.3 are independently a reactive group, such as alkoxy or halide, for example, --OCH.sub.3, or --OCH.sub.2CH.sub.3, for example, in one embodiment, R.sub.1, R.sub.2 and R.sub.3 are each --OCH.sub.3; or in another embodiment, R.sub.1 and R.sub.2 are independently a reactive group, such as alkoxy or halide, for example --OCH.sub.3 or --OCH.sub.2CH.sub.3, and R.sub.3 is an alkoxy or halide group or an alkyl group, such as --CH.sub.3, or substituted alkyl group. In one embodiment of Formula 2, L.sub.1 and L.sub.2 are independently alkyl, for example, linear or branched alkyl or heteroalkyl, e.g., C1 C25 alkyl, for example, --(CH.sub.2).sub.n--, wherein n=2 to 10, e.g., 3 to 4, or e.g., 2 3. For example, L.sub.1 and L.sub.2 may optionally comprise a heteroalkyl comprising a heteroatom such as O, S, or N. Each L.sub.1 and L.sub.2 independently comprise one or more derivatizable groups, e.g., 1 4 derivatizable groups, such as hydroxyl, amino or amido.

In Formula 2, in one embodiment, A.sub.1 is H or a moiety comprising one or more derivatizable functional groups. In one embodiment, A.sub.1 is a moiety comprising an amino group or a hydroxyl group, such as --CH.sub.2CH.sub.2OH. In another embodiment, A.sub.1 is, for example, a linear or branched alkyl or heteroalkyl group including a plurality of derivatizable functional groups, for example, 1, 2, or 3 derivatizable groups. In one embodiment, A.sub.1 may comprise a linear or branched alkyl or heteroalkyl, wherein one or more carbon atoms of the alkyl group is functionalized, for example, to comprise an amide.

Examples of compounds include compounds XVIa e shown in FIG. 13, and compounds XVIIIa f shown in FIG. 14. Other examples include compound XII in FIG. 5 and compound XV in FIG. 6, as well as compounds XXIX and XXX in FIG. 19.

In a further embodiment, compounds of Formulas 17, 18, 19, and 20 shown in FIG. 15 are provided. In one embodiment of the compounds of Formulas 17 20, R.sub.1, R.sub.2, R.sub.3 are independently a reactive group, such as alkoxy or halide, for example, --OCH.sub.3, or --OCH.sub.2CH.sub.3, for example, in one embodiment, R.sub.1, R.sub.2 and R.sub.3 are each --OCH.sub.3; or in another embodiment, R.sub.1 and R.sub.2 are independently a reactive group, such as alkoxy or halide, for example --OCH.sub.3 or --OCH.sub.2CH.sub.3, and R.sub.3 is an alkoxy or halide group or an alkyl group, such as --CH.sub.3, or substituted alkyl group. In one embodiment of the compounds of Formulas 17 20, L.sub.1, L.sub.2, and L.sub.3 are independently linear or branched alkyl or heteroalkyl, e.g., C.sub.1-25 alkyl, for example, --(CH.sub.2).sub.n--, wherein n=2 to 10, e.g., 3 to 4, or e.g., 2 3. For example, L.sub.1, L.sub.2, and L.sub.3 may optionally comprise a heteroalkyl comprising a heteroatom such as O, S, or N. Each L.sub.1, L.sub.2, and L.sub.3 independently optionally comprise one or more derivatizable groups, e.g., 1 4 derivatizable groups, such as hydroxyl or an amino group. In one embodiment of Formulas 17 20, A.sub.1 and A.sub.2 may independently comprise H or a moiety comprising one or more derivatizable functional groups. In one embodiment, A.sub.1 and A.sub.2 are independently moieties comprising an amino group or a hydroxyl group, such as --CH.sub.2CH.sub.2OH. In another embodiment, A.sub.1 and A.sub.2 may independently comprise, for example, a linear or branched alkyl or heteroalkyl including a plurality of derivatizable functional groups, for example, 1, 2, or 3 derivatizable groups. In one embodiment, A.sub.1 and A.sub.2 may independently comprise a linear or branched alkyl or heteroalkyl, wherein one or more carbon atoms of the alkyl group is functionalized, for example, to an amide. In one embodiment of the compounds of Formulas 17 20, B.sub.1 and B.sub.2 are independently a branching group, for example alkyl, a heteroatom, or heteroalkyl, for example a C1 12 alkyl. In one embodiment of the compounds of Formulas 17 20, L.sub.4 is a direct bond or a linker, for example, C1 12 alkyl or heteroalkyl optionally comprising one more derivatizable groups. R in one embodiment is H or alkyl or heteroalkyl, for example C1 12 alkyl, or in another embodiment is acyl, for example HC(.dbd.CH.sub.2)CO-- or MeC(.dbd.CH.sub.2)CO--.

Examples of compounds of Formula 17 include compounds XIX, XX and XXIV in FIG. 16. Examples of compounds of Formula 19 include compounds XXI and XXII in FIG. 17. Other examples of compounds include compounds XXVII and XXVIII in FIG. 16.

In one embodiment, compounds of Formula 18 include compounds XXXIa and XXXIb shown in FIG. 20. Compounds of Formula 20 include compounds XXXIIa and XXXIIb shown in FIG. 20.

In another embodiment, the compounds may include a single silicon group, as for example compounds XXXIIIa and XXXIIIb shown in FIG. 20.

In another embodiment, compounds of Formula 21 in FIG. 15 are provided, wherein: In one embodiment of Formula 21, R.sub.1, R.sub.2, R.sub.3 are independently a reactive group, such as alkoxy or halide, for example, --OCH.sub.3, or --OCH.sub.2CH.sub.3, for example, in one embodiment, R.sub.1, R.sub.2 and R.sub.3 are each --OCH.sub.3; or in another embodiment, R.sub.1 and R.sub.2 are independently a reactive group, such as alkoxy or halide, for example --OCH.sub.3 or --OCH.sub.2CH.sub.3, and R.sub.3 is an alkoxy or halide group or an alkyl group, such as --CH.sub.3, or substituted alkyl group. In one embodiment of Formula 21, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 are independently a direct bond, or linear or branched alkyl or heteroalkyl, e.g., C1 25 alkyl, for example, --(CH.sub.2).sub.n--, wherein n=2 to 10, e.g., 3 to 4, or e.g., 2 3. For example, L.sub.1, L.sub.2, L.sub.3 and L.sub.4 may optionally comprise a heteroalkyl comprising a heteroatom such as O, S, or N. Each L.sub.1, L.sub.2, L.sub.3 and L.sub.4 independently optionally comprise one or more derivatizable groups, e.g., 1 4 derivatizable groups, such as hydroxyl or an amino group. In one embodiment, B.sub.1 is a branching group, for example alkyl, a heteroatom, or heteroalkyl, for example a C1 12 alkyl. In one embodiment, A.sub.1, A.sub.2, A.sub.3, and A.sub.4 may independently comprise H or a moiety comprising one or more derivatizable functional groups. In one embodiment, A.sub.1, A.sub.2, A.sub.3, and A.sub.4 are independently a moiety comprising an amino group or a hydroxyl group, such as --CH.sub.2CH.sub.2OH. In another embodiment, A.sub.1, A.sub.2, A.sub.3, and A.sub.4 may independently comprise, for example, an alkyl, such as a linear or branched alkyl, including a plurality of derivatizable functional groups, for example, 1, 2 or 3 derivatizable groups. In one embodiment, A.sub.1, A.sub.2, A.sub.3, and A.sub.4 may independently comprise a linear or branched alkyl or heteroalkyl, wherein one or more carbon atoms of the alkyl group is functionalized, for example, to comprise an amide.

Embodiments of compounds of Formula 21 include compounds XXIII, XXV and XXVI shown in FIG. 18.

In a further embodiment, compounds of Formula 3 are provided:

##STR00006## In Formula 3, in one embodiment, R.sub.1, R.sub.2 and R.sub.3 are independently reactive groups, such as alkoxy or halide, for example, --OCH.sub.3, or --OCH.sub.2CH.sub.3, and wherein, in one embodiment, R.sub.1, R.sub.2 and R.sub.3 are each --OCH.sub.3. In one embodiment R.sub.1 and R.sub.2 are independently a reactive group, such as alkoxy or halide, for example --OCH.sub.3 or --OCH.sub.2CH.sub.3, and R.sub.3 is an alkoxy or halide group or an alkyl group, such as --CH.sub.3, or substituted alkyl group. In Formula 3, in one embodiment, L.sub.1, L.sub.2, and L.sub.3 are independently a linker, for example, a straight chain saturated hydrocarbon, such as --(CH.sub.2).sub.n--, wherein n=1 to 10, or 1 to 5, or, e.g., 2 to 3. In Formula 3, in one embodiment, A.sub.1 and A.sub.2 are independently H or moieties comprising one or more derivatizable functional groups, such as hydroxyl or amino groups, or modified forms thereof, such as protected forms. In another embodiment, A.sub.1 and A.sub.2 each comprise a plurality of derivativizable functional groups. For example, A.sub.1 and A.sub.2 may each comprise a branched moiety including a plurality of derivatizable functional groups, such as hydroxyl groups.

In one embodiment of Formula 3, R.sub.1 and R.sub.2 are independently alkoxy or halide; R.sub.3 is alkoxy, halide or alkyl; L.sub.1, L.sub.2, and L.sub.3 are independently --(CH.sub.2).sub.n--, wherein n is 2 10; and A.sub.1 and A.sub.2 are independently a moiety comprising one or more derivatizable functional groups.

In another embodiment of Formula 3, A.sub.1 is -L.sub.4-G.sub.1 and A.sub.2 is -L.sub.5-G.sub.2; R.sub.1 and R.sub.2 are independently alkoxy or halide; R.sub.3 is alkoxy, halide or alkyl; L.sub.1, L.sub.2, L.sub.3, L.sub.4 and L.sub.5 are --(CH.sub.2).sub.n--, wherein n is 1 to 10, for example 2 to 3; and G.sub.1 and G.sub.2 are independently a moiety comprising one or more derivatizable functional groups. In another embodiment, L.sub.1, L.sub.4, and L.sub.5 are --(CH.sub.2).sub.2--, L.sub.2 and L.sub.3 are --(CH.sub.2).sub.3--, and G.sub.1 and G.sub.2 are --OH.

In another embodiment, silicon compounds of Formula 15 in FIG. 10 are provided, wherein R.sub.1, R.sub.2 are independently alkoxy, for example --OCH.sub.3 or --OCH.sub.2CH.sub.3, or halide; and R.sub.3 is alkoxy, alkyl, or halide.

In a further embodiment, compounds of Formula 6a in FIG. 1 are provided, wherein n is 1 3, for example 2 or 3. Exemplary functionalized silicon compounds include compound V below, and compound VI shown in FIG. 1.

##STR00007##

Another embodiment is illustrated in FIG. 7, which shows a compound of Formula 10, wherein n=1 to 10, e.g., 1 3, and G is a derivatizable functional group, such as hydroxyl, protected hydroxyl, or halide such as Cl or Br.

The hydrolytic stability of the silicon compound coating may be increased by increasing the number of covalent bonds to the surface of the support. For example, silicon compounds II V include two activated silicon groups for binding to a support surface, such as glass. A variety of functionalized silicon compounds including a plurality of activated silicon groups and derivatizable functional groups are useful to form functionalized coatings. A further example is compound VII shown in FIG. 1. Another example is silicon compound VIII shown in FIG. 2, which can form up to three covalent bonds to the surface of a glass support. In compound VIII, the triethoxysilyl group is shown by way of example, however alternatively, the activated silicon group may be other activated silicon groups or mixtures thereof, such as trimethoxysilyl. Similarly, in all of the silicon compounds disclosed herein in which a representative activated silicon group, such as trimethoxysilyl, is substituted on the compound, the compounds in other embodiments also may be substituted with other activated silicon groups known in the art and disclosed herein.

The silicon compounds II VIII having multiple silicon groups enhance potentially by twice as much, or more, the hydrolytic stability in comparison to silicon compounds comprising only a single silicon group, since they possess more trialkoxysilyl groups that can react, and form bonds with, a surface. The number of silicon groups in the silicon compound may be modified for different applications, to increase or decrease the number of bonds to a support such as a glass support. Silcon compounds may be used that form optimally stable surface-bonded films on glass via covalent siloxane bonds. Additionally, the number of derivatizable functional groups may be increased or decreased for different applications, as illustrated by silicon compounds II VIII. Silicon compounds may be selected for use that provide the desired optimum density of surface derivatizable groups, such as hydroxyalkyl groups, for a desired application, such as the synthesis of nucleic acid arrays, or for the optimum stability during use of the array in different applications.

Other embodiments of functionalized silicon compounds include compound XIII shown in FIG. 5.

In another embodiment, polymeric functionalized silicon compounds of Formula 4 are provided:

##STR00008## In Formula 4, in one embodiment, x, y and z are independently 1 3 and, in one embodiment, x, y and z are each 2. In Formula 4, in one embodiment, L.sub.1, L.sub.2 and L.sub.3 are independently linkers, for example, straight chain hydrocarbons, and preferably --(CH.sub.2).sub.m--, wherein m=1 10, e.g., 2 3. In Formula 4, in one embodiment, at least one of A, B and C is --SiR.sub.1R.sub.2R.sub.3, wherein R.sub.1 and R.sub.2 are independently a reactive group, such as alkoxy or halide, for example, --OCH.sub.3, or --OCH.sub.2CH.sub.3 and R.sub.3 is alkoxy, halide or alkyl; and wherein the remainder of A, B and C are independently moieties comprising one or more derivatizable functional groups, such as hydroxyl groups, or amino groups, or modified forms thereof, such as protected forms, for example --OH or a branched molecule comprising one or more hydroxyl groups. In Formula 4, in one embodiment, n is, for example, about 10 to 10,000, or, for example, about 1,000 to 10,000.

In one embodiment of Formula 4, B is --SiR.sub.1R.sub.2R.sub.3, wherein R.sub.1, R.sub.2 and R.sub.3 are independently alkoxy, halide or alkyl; x, y, and z are independently 2 3; L.sub.1, L.sub.2 and L.sub.3 are independently --(CH.sub.2).sub.m--, wherein m is 2 3; A and C are independently moieties comprising derivatizable functional groups; and n is about 10 to 10,000.

In one embodiment of Formula 4, B is --Si(OCH.sub.3).sub.3; x, y, and z are 2; L.sub.1 and L.sub.3 are --(CH.sub.2).sub.2--; L.sub.2 is --(CH.sub.2).sub.3--; A and C are moieties comprising derivatizable functional groups; and n is about 10 to 10,000.

Other embodiments of a polymeric functionalized silicon compound include compounds of Formula 5 and 6 shown in FIG. 4, wherein m is about 0 to 10, e.g., about 1 to 5, and n is about 10 to 10,000. In Formulas 5 and 6, R.sub.1 and R.sub.2 are independently a reactive group, such as alkoxy or halide, for example, --OCH.sub.3 or --OCH.sub.2CH.sub.3, and R.sub.3 is a reactive group, such as alkoxy or halide, or optionally alkyl, for example --CH.sub.3.

Other embodiments include compounds of Formula 7 and 8, shown in FIG. 5, and Formula 9, shown in FIG. 6, wherein n is about 10 to 10,000. In Formulas 7, 8 and 9, R.sub.1 and R.sub.2 are independently a reactive group, such as alkoxy or halide, for example, --OCH.sub.3 or --OCH.sub.2CH.sub.3, and R.sub.3 is a reactive group such as alkoxy or halide or optionally alkyl, for example --CH.sub.3.

Further embodiments include compounds of Formula 12 and 13, shown in FIG. 8, wherein m is about 10 to 10,000, and n is about 1 to 10, e.g., about 5 to 10. In Formulas 12 and 13, R.sub.1 and R.sub.2 are independently a reactive group, such as alkoxy or halide, for example, --OCH.sub.3 or --OCH.sub.2CH.sub.3, and R.sub.3 is a reactive group, such as alkoxy or halide, or optionally alkyl, for example --CH.sub.3. In Formula 12, G is a substitutable leaving group, such as hydroxy, protected hydroxy, or halo, such as --Cl or --Br.

The use of a polymer permits the formation of stable films on surfaces, such as glass, due to the very large number of siloxane bonds that can be formed with the surface. The number of alkoxysilicon groups relative to the number of hydroxyalkyl groups can be selected to provide the desired density of reactive hydroxyl groups.

Synthesis of Functionalized Silicon Compounds

Functionalized silicon compounds for use in the methods described herein are available commercially, or may be synthesized from commercially available starting materials. Commercially available silicon compounds and a review of silicon compounds is provided in Arkles, Ed., "Silicon, Germanium, Tin and Lead Compounds, Metal Alkoxides, Diketonates and Carboxylates, A Survey of Properties and Chemistry," Gelest, Inc., Tullytown, Pa., 1995, the disclosure of which is incorporated herein. Functionalized silicon compounds may be synthesized using methods available in the art of organic chemistry, for example, as described in March, Advanced Organic Chemistry, John Wiley & Sons, New York, 1985, and in R. C. Larock, Comprehensive Organic Transformations, Wiley-VCH, New York, 1989.

Methods of synthesizing compounds of Formula 1 are shown in Scheme I below. Commercially available reagents which may be used in syntheses in accordance with Scheme I include 3-chloro-1-triethoxysilylpropane and ethylene oxide (Aldrich.RTM., Milwaukee, Wis.).

##STR00009##

A method for the conversion of bis(trimethoxysilylpropyl)amine, XIV, which is commercially available from Gelest, Inc. (Tullytown, Pa.) to compound II is illustrated below in Scheme II.

##STR00010##

A method for the conversion of compound XI, bis[3-trimethoxysilyl)propyl]-ethylenediamine, which is commercially available from Gelest, Inc., Tullytown, Pa., to compound V is shown below in Scheme III.

##STR00011##

A method for the synthesis of compound III is shown below in Scheme IV. The reagents shown in Scheme IV are commercially available from Aldrich.RTM. (Milwaukee, Wis.).

##STR00012##

Reaction schemes for the synthesis of functionalized silicon compounds IX and X are provided in FIG. 3. Reaction schemes for the synthesis of compounds of Formulas 5 and 6 are shown in FIG. 4. Polyethyleneimine is available commercially, for example, from Aldrich.RTM.. Polyamines of Formula 5, where R.sub.1, R.sub.2 and R.sub.3 are OMe (trimethoxysilylpropyl modified (polyethyleneimine)), or R.sub.1 is Me and R.sub.2 and R.sub.3 are OMe (dimethoxymethylsilylpropyl modified (polyethylenimine)) are available from Gelest (Tullytown, Pa.). FIG. 9 shows another embodiment of a reaction scheme using commercially available reagents, wherein the compound of Formula 16a is converted to the compound of Formula 16b.

Reaction schemes for the synthesis of compounds XII, XIII, and compounds of Formula 8 are shown in FIG. 5. Synthesis of the reagent, N,N-bis(2-hydroxyethyl)acrylamide is described in U.S. Pat. No. 3,285,886 (1966), the disclosure o