U.S. patent application number 09/832490 was filed with the patent office on 2001-10-04 for functionalized silicon compounds and methods for their synthesis and use.
This patent application is currently assigned to Affymetrix, Inc.. Invention is credited to Forman, Jonathan Eric, McGall, Glenn.
Application Number | 20010027187 09/832490 |
Document ID | / |
Family ID | 22626709 |
Filed Date | 2001-10-04 |
United States Patent
Application |
20010027187 |
Kind Code |
A1 |
McGall, Glenn ; et
al. |
October 4, 2001 |
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.
Inventors: |
McGall, Glenn; (Mountain
View, CA) ; Forman, Jonathan Eric; (San Jose,
CA) |
Correspondence
Address: |
SCHWEGMAN, LUNDBERG, WOESSNER & KLUTH
P.O. BOX 2938
MINNEAPOLIS
MN
55402
US
|
Assignee: |
Affymetrix, Inc.
|
Family ID: |
22626709 |
Appl. No.: |
09/832490 |
Filed: |
April 11, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09832490 |
Apr 11, 2001 |
|
|
|
09418044 |
Oct 13, 1999 |
|
|
|
09418044 |
Oct 13, 1999 |
|
|
|
09172190 |
Oct 13, 1998 |
|
|
|
6262216 |
|
|
|
|
Current U.S.
Class: |
514/100 |
Current CPC
Class: |
C09D 183/08 20130101;
C03C 17/30 20130101; C03C 2218/113 20130101; C08G 73/0206 20130101;
Y10T 428/31663 20150401; C09D 183/14 20130101; C07F 7/1804
20130101; C09D 179/02 20130101 |
Class at
Publication: |
514/100 |
International
Class: |
A61K 031/665; A01N
057/00 |
Claims
What is claimed is:
1. A functionalized silicon compound having a structure of Formula
2: 13wherein R.sub.1 and R.sub.2 are independently selected from
the group consisting of alkoxy and halide, and R.sub.3 is selected
from the group consisting of alkoxy, halide and alkyl; wherein
L.sub.1 and L.sub.2 are both --(CH.sub.2).sub.n--, wherein n=2 to
10; and wherein A.sub.1 is a moiety comprising one or more
derivatizable functional groups.
2. The functionalized silicon compound of claim 1, wherein the
derivatizable functional group is selected from the group
consisting of hydroxyl, amino, amido, carboxyl, thio, halo and
sulfonate.
3. The functionalized silicon compound of claim 2, wherein A.sub.1
comprises a plurality of derivatizable functional groups.
4. The functionalized silicon compound of claim 2, wherein A.sub.1
is a moiety comprising a hydroxyl group.
5. The functionalized silicon compound of claim 2, wherein the
silicon compound is compound II: 14
6. A functionalized silicon compound having a structure of Formula
3: 15wherein R.sub.1 and R.sub.2 are independently selected from
the group consisting of alkoxy or halide, and R.sub.3 is selected
from the group consisting of alkoxy, halide and alkyl; wherein
L.sub.1, L.sub.2, and L.sub.3 are independently
--(CH.sub.2).sub.n--, wherein n is 2-10; and wherein A.sub.1 and
A.sub.2 are independently a moiety comprising one or more
derivatizable functional groups or modified forms thereof.
7. The functionalized silicon compound of claim 6, wherein the one
or more derivatizable functional groups are selected from the group
consisting of hydroxyl, amino, carboxyl, thio, halo, amido and
sulfonate.
8. The functionalized silicon compound of claim 7, wherein A.sub.1
and A.sub.2 each comprise a plurality of derivatizable functional
groups.
9. The functionalized silicon compound of claim 7, wherein A.sub.1
and A.sub.2 each comprise a hydroxyl group.
10. The functionalized silicon compound of claim 7, wherein the
functionalized silicon compound is compound V: 16
11. A functionalized silicon compound having a structure of Formula
4: 17wherein 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;
wherein x, y, and z are independently 2 to 3; wherein L.sub.1,
L.sub.2 and L.sub.3 are independently --(CH.sub.2).sub.m--, wherein
m is 2-10; wherein A and C are independently moieties comprising
derivatizable functional groups; and wherein n is about 10 to
10,000.
12. A functionalized silicon compound of Formula 6: 18wherein
R.sub.1, R.sub.2 and R.sub.3 are independently alkoxy, halide or
alkyl; and wherein m is about 1 to 5, and n is about 10 to
10,000.
13. A fiunctionalized silicon compound of Formula 2 19wherein:
R.sub.1 and R.sub.2 are independently selected from the group
consisting of alkoxy or halide, and R.sub.3 is selected from the
group consisting of alkoxy, halide and alkyl; L.sub.1, and L.sub.2
are independently alkyl or heteroalkyl optionally comprising one or
more derivatizable groups; A.sub.1 is H or a moiety comprising one
or more derivatizable functional groups.
14. The silicon compound of claim 13, wherein the derivatizable
functional groups are independently selected from the group
consisting of hydroxyl, amino, carboxyl, thio, halo and
sulfonate.
15. The silicon compound of claim 13 selected from the group
consisting of 20
16. A functionalized silicon compound of Formulas 17, 18, 19, or
20: 21wherein: 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 alkyl or 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.
17. The silicon compound of claim 16, wherein the derivatizable
functional groups are independently selected from the group
consisting of hydroxyl, amino, carboxyl, thio, halo, amido and
sulfonate.
18. The silicon compound of claim 16, wherein the compound is
selected from the group consisting of 22
19. A functionalized silicon compound of Formula 21: 23wherein:
R.sub.1 and R.sub.2 are independently selected from the group
consisting of alkoxy or halide, and R.sub.3 is selected from the
group consisting of alkoxy, halide and alkyl; L.sub.1, L.sub.2,
L.sub.3, and L.sub.4 are independently alkyl or heteroalkyl
optionally comprising one or more derivatizable groups; B.sub.1 is
alkyl, a heteroatom, or heteroalkyl; A.sub.1, A.sub.2, A.sub.3, and
A.sub.4 are independently H or a moiety comprising one or more
derivatizable functional groups.
20. The silicon compound of claim 19, wherein the derivatizable
functional groups are independently selected from the group
consisting of hydroxyl, amino, carboxyl, thio, halo amido and
sulfonate.
21. A silicon compound of claim 19, selected from the group
consisting of 24
22. A method of flnctionalizing 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.
23. The method of claim 22, wherein the method comprises 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.
24. The method of claim 22, wherein the silicon compound is a
compound having a structure of Formula 2: 25wherein R.sub.1 and
R.sub.2 are independently selected from the group consisting of
alkoxy and halide, and R.sub.3 is selected from the group
consisting of alkoxy, halide and alkyl; wherein L.sub.1 and L.sub.2
are both --(CH.sub.2).sub.n--, wherein n=2 to 10; and wherein
A.sub.1 is H or a moiety comprising one or more derivatizable
functional groups.
25. The method of claim 24, wherein the derivatizable functional
group is a hydroxyl group or modified form thereof.
26. The method of claim 24, wherein A.sub.1 comprises a plurality
of derivatizable functional groups.
27. The method of claim 24, wherein the silicon compound is
compound II: 26
28. The method of claim 22, wherein the method comprising
covalently attaching to the surface a functionalized silicon
compound having a structure of Formula 3: 27wherein R.sub.1 and
R.sub.2 are independently alkoxy or halide, and R.sub.3 is selected
from the group consisting of alkoxy, halide and alkyl; wherein
L.sub.1, L.sub.2, and L.sub.3 are --(CH.sub.2).sub.n--, wherein n
is 2 to 10; and wherein A.sub.1 and A.sub.2 are independently H or
a moiety comprising one or more derivatizable functional
groups.
29. The method of claim 28, wherein the one or more derivatizable
functional groups are hydroxyl groups or modified forms
thereof.
30. The method of claim 28, wherein A.sub.1 and A.sub.2 each
comprise a plurality of derivatizable functional groups.
31. The method of claim 28, wherein the functionalized silicon
compound is compound V: 28
32. The method of claim 22, wherein the method comprises covalently
attaching to the surface a functionalized silicon compound having a
structure of Formula 4: 29wherein 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; wherein x, y, and z are independently 2 to 3;
wherein L.sub.1, L.sub.2 and L.sub.3 are independently
--(CH.sub.2).sub.m--, wherein m is 2 to 10; wherein A and C are
independently moieties comprising derivatizable functional groups;
and wherein n is about 10 to 10,000.
33. A method of functionalizing a surface, the method comprising
covalently attaching to the surface a functionalized silicon
compound according to claim 12.
34. A method of functionalizing a surface, the method comprising
covalently attaching to the surface a functionalized silicon
compound of claim 13.
35. The method of claim 34, wherein the method comprises 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.
36. The method of claim 34, wherein the derivatizable groups are
hydroxyl groups or modified forms thereof.
37. The method of claim 34, wherein the compound is selected from
the group consisting of 30
38. A method of functionalizing a surface, the method comprising
covalently attaching to the surface a functionalized silicon
compound of claim 16.
39. The method of claim 38, wherein the method comprises 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.
40. The method of claim 38, wherein the derivatizable groups are
hydroxyl groups or modified forms thereof.
41. The method of claim 38, wherein the compound is selected from
the group consisting of 31
42. A method of functionalizing a surface, the method comprising
covalently attaching to the surface a functionalized silicon
compound of claim 19.
43. The method of claim 42, wherein the method comprises 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.
44. The method of claim 42, wherein the derivatizable groups are
hydroxyl groups or modified forms thereof.
45. The method of claim 42, wherein the compound is selected from
the group consisting of 32
46. The method of claim 22, wherein the surface is the surface of a
substrate comprising silica.
47. The method of claim 24, wherein the surface is the surface of a
substrate comprising silica.
48. The method of claim 28, wherein the surface is the surface of a
substrate comprising silica.
49. The method of claim 32 wherein the surface is the surface of a
substrate comprising silica.
50. The method of claim 34, wherein the surface is the surface of a
substrate comprising silica.
51. The method of claim 38, wherein the surface is the surface of a
substrate comprising silica.
52. The method of claim 42, wherein the surface is the surface of a
substrate comprising silica.
53. The method of claim 22, wherein the substrate is in a form
selected from the group consisting of particles, films and chips.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 09/172,190, filed Oct. 13, 1998, the
disclosure of which is incorporated herein by reference.
TECHNICAL FIELD
[0002] 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
[0003] 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.
[0004] 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
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.
[0005] 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-triethoxysila- ne 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.
[0006] 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 etal., 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.
[0007] 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
[0008] 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
[0009] FIG. 1 shows the structure of the functionalized silicon
compounds VI and VII and compounds of Formula 6a.
[0010] FIG. 2 shows the structure of the functionalized silicon
compound VIII.
[0011] FIG. 3 shows schemes for the synthesis of compounds IX and
X.
[0012] FIG. 4 is a scheme showing the synthesis of compounds of
Formula 5 or 6.
[0013] FIG. 5 show schemes for the synthesis of compounds XII, XIII
and compounds of Formula 8.
[0014] FIG. 6 shows schemes for the synthesis of compounds XV, VI
and compounds of Formula 9.
[0015] FIG. 7 shows schemes showing the synthesis of compounds of
Formula 10 or 11.
[0016] FIG. 8 shows schemes showing the synthesis of compounds of
Formula 12 or 13.
[0017] FIG. 9 is a scheme of the synthesis of compounds of Formula
16b.
[0018] FIG. 10 illustrates the structure of compounds of Formula 14
and 15.
[0019] FIG. 11 is a graph of stability of silicon compound bonded
phases vs. time.
[0020] FIG. 12 is a graph of hybridization fluorescence intensity
vs. silane.
[0021] FIG. 13 is a scheme showing the synthesis of silicon
compounds XVIa-e.
[0022] FIG. 14 is a scheme showing the synthesis of silicon
compounds XVIIa-f.
[0023] FIG. 15 shows the structure of compounds of the general
Formulas 17-21.
[0024] FIG. 16 shows the structure of some exemplary silicon
compounds.
[0025] FIG. 17 shows another embodiment of exemplary silicon
compounds XXI and XXII.
[0026] FIG. 18 shows the structure of exemplary silicon compounds
XXIII, XXV and XXVI.
[0027] FIG. 19 shows the structure of exemplary silicon compounds
XXIX and XXX.
[0028] FIG. 20 shows the structure of exemplary silicon compounds
XXIa-b, XXIIa-b and XXIIIa-b.
[0029] FIG. 21 is a scheme showing the synthesis of silicon
compounds XIX and XX.
[0030] FIG. 22 is a scheme showing the synthesis of silicon
compounds XXI and XXII.
[0031] FIG. 23 is a scheme showing the synthesis of silicon
compound XXIII.
[0032] FIG. 24 is a scheme showing the synthesis of silicon
compound XXIV.
[0033] FIG. 25 is a scheme showing the synthesis of silicon
compound XXVIII.
[0034] FIG. 26 is a scheme showing the synthesis of silicon
compounds XXVI and XXV.
[0035] FIG. 27 is a scheme showing the synthesis of silicon
compounds XXIX and XXX.
[0036] FIG. 28 is a scheme showing the synthesis of silicon
compounds XXXIa-b and XXXIIIa-b.
[0037] FIG. 29 is a scheme showing the synthesis of silicon
compounds XXXIIa-b.
[0038] FIG. 30 is a graph of normalized intensity vs. silane for
silicon compounds bound to a solid substrate.
[0039] FIG. 31 is a graph of normalized hybridization fluorescence
intensity vs. silane.
MODES FOR CARRYING OUT THE INVENTION
[0040] 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.
[0041] Functionalized Silicon Compounds
[0042] 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.
[0043] 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.
[0044] 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.
[0045] 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.
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] Exemplary functionalized silicon compounds include compounds
of Formula 1 shown below: 1
[0051] 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--).
[0052] An exemplary compound of Formula 1 is silicon compound I
below: 2
[0053] 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.
[0054] In one embodiment the functionalized silicon compounds of
Formula 2 are provided: 3
[0055] 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.
[0056] In Formula 2, in one embodiment, L.sub.1 and L.sub.2 are
independently alkyl, preferably --(CH2).sub.n--, wherein n=2 to 10,
e.g., 3 to 4, or e.g., 2-3.
[0057] In Formula 2, in one embodiment, A.sub.1 is H or a moiety
comprising one or more derivatizable functional groups. In one
embodiment, Al 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: 4
wherein p is 1-10, q is 0 or 1, and r is 2-5.
[0058] 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.
[0059] 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.
5
[0060] 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.
[0061] Another embodiment of Formula 2 is as follows:
[0062] 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.
[0063] 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.
[0064] 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.
[0065] 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.
[0066] In a further embodiment, compounds of Formulas, 17, 18, 19,
and 20 shown in FIG. 15 are provided.
[0067] 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.
[0068] 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., 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, 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.
[0069] 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.
[0070] 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.
[0071] 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.
[0072] 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--.
[0073] 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.
[0074] 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.
[0075] In another embodiment, the compounds may include a single
silicon group, as for example compounds XXXIIIa and XXXIIIb shown
in FIG. 20.
[0076] In another embodiment, compounds of Formula 21 in FIG. 15
are provided, wherein:
[0077] 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.
[0078] 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.
[0079] In one embodiment, B.sub.1 is a branching group, for example
alkyl, a heteroatom, or heteroalkyl, for example a C1-12 alkyl.
[0080] 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.20H.
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.
[0081] Embodiments of compounds of Formula 21 include compounds
XXIII, XXV and XXVI shown in FIG. 18.
[0082] In a further embodiment, compounds of Formula 3 are
provided: 6
[0083] 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.
[0084] 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.
[0085] 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.
[0086] 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.
[0087] 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.
[0088] 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.
[0089] 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. 7
[0090] 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.
[0091] 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.
[0092] 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.
[0093] Other embodiments of functionalized silicon compounds
include compound XIII shown in FIG. 5.
[0094] In another embodiment, polymeric functionalized silicon
compounds of Formula 4 are provided: 8
[0095] 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.
[0096] 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.
[0097] 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.
[0098] In Formula 4, in one embodiment, n is, for example, about 10
to 10,000, or, for example, about 1,000 to 10,000.
[0099] 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.
[0100] 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.
[0101] 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.
[0102] 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.
[0103] 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.
[0104] 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.
[0105] Synthesis of Functionalized Silicon Compounds
[0106] 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.
[0107] 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.). 9
[0108] 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. 10
[0109] A method for the conversion of compound XI,
bis[3-trimethoxysilyl)p- ropyl]-ethylenediamine, which is
commercially available from Gelest, Inc., Tullytown, Pa., to
compound V is shown below in Scheme III. 11
[0110] 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.). 12
[0111] 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 1 6a is converted to the
compound of Formula 1 6b.
[0112] 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 of which is incorporated
herein.
[0113] Reaction schemes for the synthesis of functionalized silicon
compounds XV, VI and compounds of Formula 9 are shown in FIG. 6.
Use of the reagent N,N-bis(2-hydroxyethyl)-2-chloro-ethylamine is
described in Okubo et al., Deutsches Patent 2144759 (1971), the
disclosure of which is incorporated herein.
[0114] FIG. 7 illustrates reaction schemes for the synthesis of
compounds of Formulas 10 and 11. The use of the reagent,
4-chlorobutanoyl chloride, is described in Njoroge et al., PCT
US97/15899 (1998), the disclosure of which is incorporated herein.
Other reagents include lactones, such as .gamma.-butyrolactone,
.delta.-valerolactone, and .epsilon.-caprolactone (Aldrich.RTM.).
Compounds XI and XIV are commercially available from Gelest.
[0115] FIG. 8 illustrates reaction schemes for compounds of
Formulas 12 and 13. In FIG. 8, G is a substitutable leaving group
such as halo. Reagents in addition to those discussed above that
may be used in syntheses which may be conducted as shown in FIG. 8
include diethanolamine and N,N-bis(2-hydroxyethyl)glycine, which
are commercially available, for example, from Aldrich.RTM..
[0116] Further exemplary reaction schemes are shown in FIGS. 13 and
14. FIGS. 13 and 14 illustrate examples of methods of synthesis of
compounds of Formula 2, compounds XVIa-e, and XVIIa-f. FIG. 13
illustrates examples of methods of synthesis where a compound
containing a primary amino function reacts with two equivalents of
(3-glycidoxypropyl)trimethoxysila- ne (available from Gelest, Inc.,
Tullytown, Pa.) to provide tertiary amine containing compounds
XVIa-XVIe. FIG. 14 illustrates examples of methods of synthesis
where a compound containing a primary amino function reacts with a
carbamoyl chloride (produced by reaction of XIV with triphosgene or
other phosgene synthon) to produce the substituted urea compounds
XVIIa-XVIIf.
[0117] FIG. 21 provides an exemplary reaction scheme for compounds
of Formula 17. The exemplary compounds XIX and XX are synthesized
through the common intermediate 4-amino-1,6-heptadiene. This
intermediate is prepared from ethylformimidate and methylmagnesium
bromide (both available from Aldrich, Milwaukee, Wis.); the
preparation is described in Barbot, F.; Tetrahedron Lett. 1989, 30,
185 and Barber, H. J.; J. Chem. Soc. 1943, 10. Hydrosilylation of
4-amino-1,6-heptadiene followed by reaction with ethylene oxide
provides XIX. Acylation of 4-amino-1,6-heptadiene with
4-chlorobutyryl chloride (Aldrich, Milwaukee, Wis.) followed by
hydrosilylation, and subsequent reaction of the resulting disilane
with diethanol amine affords XX.
[0118] FIG. 22 provides an exemplary reaction scheme for compounds
of Formula 19. The exemplary compounds XXI and XXII are synthesized
through the common intermediate 4-allyl-4-amino-1,6-heptadiene.
This intermediate can be prepared, for example, from
ethylformimidate and methylmagnesium bromide (both available from
Aldrich, Milwaukee, Wis.); the preparation is described in Barbot,
F., Tetrahedron Lett. 1989, 30, 185; and Barber, H. J., J. Chem.
Soc. 1943, 101. The intermediate also may be prepared from
triallylborane as described in Bubnov, Y. N., et al., Russian Chem.
Bull. 1996, 45, 2598. Hydrosilylation of
4-ally-4-amino-1,6-heptadiene followed by reaction with ethylene
oxide provides XXI. Acylation of 4-5 allyl-4-amino-1,6-heptadiene
with 4-chlorobutyryl chloride (Aldrich, Milwaukee, Wis.) followed
by hydrosilylation, and subsequent reaction of the resulting
trisilane with diethanol amine affords XXII.
[0119] FIG. 23 provides an exemplary reaction scheme for compounds
of Formula 21, including structure XXIII. This compound is prepared
by catalytic hydrogen reduction of
4,4-dicyano-1,6-bis(triethoxysilyl)heptan- e followed by reaction
of the resultant diamine with ethylene oxide. The dicyano
intermediate is prepared, for example, by a malonitrile synthesis
of 4,4-dicyano-1,6-heptadiene followed by hydrosilylation, or by
alkylation of malonitrile with 2 equivalents of a
3-halo-1-(triethoxysily- l)propane compound.
[0120] FIG. 24 provides an exemplary reaction scheme for a compound
of Formula 17, including structure XXIV. This compound is prepared
by catalytic hydrogen reduction of
4-cyano-1,6-bis(triethoxysilyl)heptane followed by reaction of the
resultant amine with ethylene oxide. The cyano intermediate is
prepared starting from a malonitrile synthesis of
4,4-dicyano-1,6-heptadiene followed by conversion to
4-cyano-1,6-heptadiene by reduction with an organotin reagent as
described in Curran, D. P.; et al., Synthesis, 1991, 107.
Hydrosilylation of this diene provides
4-cyano-1,6-bis(triethoxysilyl)heptane.
[0121] FIG. 25 provides an exemplary reaction scheme for compounds
of Formula 19, for example, structures XXVII and XXVIII. These
compounds are prepared by reaction of appropriate primary (XXVIII)
or secondary (XXVII) amines with ethyl 1
,7-bis(triethoxysilyl)-4-heptanoate. The ester precursor is
synthesized starting from diethyl 2,2-diallylmalonate (Aldrich,
Milwaukee, Wis.); the diester is decarboxylated following the
method of Beckwith, A. C. J.; et al., J. Chem. Soc., Perkin Trans.
II, 1975, 1726, to produce ethyl 4-hepta-1,6-dieneoate; the
triethoxysilyl moieties are introduced by hydrosilylation.
[0122] FIG. 26 provides an exemplary reaction scheme for compounds
of Formula 21, for example, structures XXV and XXVI. These
compounds are prepared by reaction of appropriate primary (XXVI) or
secondary (XXV) amines with diethyl
2,2-bis(3-triethoxysilylpropyl)malonate. The diester precursor can
be synthesized by hydrosilylation of diethyl 2,2-diallylmalonate
(Aldrich, Milwaukee, Wis.).
[0123] Another embodiment of a synthesis of a compound of Formula 2
is shown in FIG. 27, wherein the synthesis of compounds XXIX and
XXX is shown. The general method illustrated here is to N-alkylate
(XXX) or N-acylate (XXIX) bis(3-trimethoxypropyl)amine (compound
XI, Gelest, Inc., Tullytown, Pa.) with a alkyl chain containing an
ester of a carboxylic acid that can be subjected to aminolysis with
dihydroxyethylamine (Aldrich, Milwaukee, Wis.). The alkylating
agent, methyl 4-chlorobutyrate, and the acylating agent, methyl
4-chloro-4-oxobutyrate, are available from Aldrich (Milwaukee,
Wis.).
[0124] FIG. 28 provides an exemplary reaction scheme for compounds
of Formula 18, for example, structures XXXIa and XXXIb. Preparation
begins with the formation of 1,6-heptadiene-4-ol from reaction of
ethyl formate with allylmagnesium bromide (Aldrich, Milwaukee,
Wis.). Reaction of the alcohol with zinc chloride by the method of
Reeve, W., J. Org. Chem. 1969,34, 1921 affords a halo diene which
after hydrosilylation can be used to alkylate dihydroxyethylamine
(Aldrich, Milwaukee, Wis.). N-acylation with acroyl chloride
(XXXIa) or methacroyl chloride (XXXIb) (Aldrich, Milwaukee, Wis.)
following the procedure of Yokota, M., et al., European patent
Application 97309882.5, 1998 affords the desired compounds.
[0125] FIG. 29 provides an exemplary reaction scheme for compounds
of Formula 20, including structures XXXIIa and XXXIIb. Preparation
begins with the formation of triallylmethanol from reaction of
diethyl carbonate with allylmagnesium bromide by the method of
Dreyfuss, M. P., J. Org Chem. 1963,28, 3269. Reaction of the
alcohol with zinc chloride by the method of Reeve, W., J. Org.
Chem. 1969, 34, 192 affords a halo diene which after
hydrosilylation can be used to alkylate dihydroxyethylamine
(Aldrich, Milwaukee, Wis.). N-acylation with acroyl chloride
(XXXIa) or methacroyl chloride (XXXIb) (Aldrich, Milwaukee, Wis.)
following the procedure of Yokota, M.; et al., European patent
Application 97309882.5, 1998 affords the desired compounds.
[0126] FIG. 28 provides an exemplary reaction scheme for structures
XXXIIIa and XXXIIIb, compounds which contain a single silicon atom.
Preparation begins with the alkylation of dihydroxyethylamine
(Aldrich, Milwaukee, Wis.) with an appropriate
3-halo-1-trialkoxylsilylproane reagent, followed by N-acylation
with acroyl chloride (XXXIIIa) or methacroyl chloride (XXXIIIb)
(Aldrich, Milwaukee, Wis.) following the procedure of Yokota, M.,
et al., European patent Application 97309882.5, 1998 affords the
desired compounds.
[0127] Functionalized silicon compounds within the scope of the
invention that may be used to form functionalized covalent coatings
on surfaces that are useful in a variety of applications and assays
further include amine compounds such as compound XI, as well as
reaction products formed therefrom as disclosed herein.
[0128] Applications
[0129] The methods and compositions disclosed herein may be used in
a variety of applications. The functionalized silicon compounds may
be covalently attached to a variety of materials, to provide
derivatizable functional groups on the materials. Exemplary
materials include materials that comprise a functional group that
is capable of reacting with the activated silicon group of the
silicon compound. For example, the material may comprise a silica
material comprising surface silanols capable of reacting with the
activated silicon group to form a siloxane bond between the silicon
atom on the silicon compound and the silicon atom on the surface.
Thus, the functionalized silicon compounds may be attached to, for
example, materials comprising silica, such as glass, chromatography
material, and solid supports used for solid phase synthesis, such
as nucleic acid synthesis. The functionalized silicon compounds
further may be attached to materials comprising oxides such as
titanium(IV) dioxide and zirconium dioxide, aluminum oxide and
indium-tin oxides, as well as nitrides, such as silicon
nitride.
[0130] Solid substrates which may be coated by the silicon
compounds include any of a variety of fixed organizational support
matrices. In some embodiments, the substrate is substantially
planar. In some embodiments, the substrate may be physically
separated into regions, for example, with trenches, grooves, wells
and the like. Examples of substrates include slides, beads and
solid chips. The solid substrates may be, for example, biological,
nonbiological, organic, inorganic, or a combination thereof, and
may be in forms including particles, strands, gels, sheets, tubing,
spheres, containers, capillaries, pads, slices, films, plates, and
slides depending upon the intended use.
[0131] The functionalized silicon compounds used advantageously may
be selected with selecte properties for a particular application.
Functionalized silicon compounds may be selected which can form
silicon compound surface coatings that have good stability to
hydrolysis. Functionalized silicon compounds may be selected which
have a selected reactivity with the substrate and a selected
derivatizable functional group depending on the intended use.
[0132] In one embodiment, the functionalized silicon compounds may
be covalently attached to the surface of a solid substrate to
provide a coating comprising derivatizable functional groups on the
substrate, thus permitting arrays of immobilized oligomers to be
covalently attached to the substrate via covalent reaction with the
derivatizable functional groups. The immobilized oligomers, such as
polypeptides, or nucleic acids can be used in a variety of binding
assays including biological binding assays. In one embodiment, high
density arrays of immobilized nucleic acid probes may be formed on
the substrate, and then one or more target nucleic acids comprising
different target sequences may be screened for binding to the high
density array of nucleic acid probes comprising a diversity of
different potentially complementary probe sequences. For example,
methods for light-directed synthesis of DNA arrays on glass
substrates is described in McGall et al., J. Am. Chem. Soc.,
119:5081-5090 (1997), the disclosure of which is incorporated
herein.
[0133] Silanation of glass substrates with the silicon compounds
described herein can be conducted, for example by dip-, or
spin-application with a 1% -10% solution of silicon compound in an
aqueous or organic solvent or mixture thereof, for example in 95%
EtOH, followed by thermal curing. See, for example, Arkles,
Chemtech, 7:766-778 (1997); Leyden, Ed., "Silanes Surfaces and
Interfaces, Chemically Modified Surfaces," Vol. 1, Gordon &
Breach Science, 1986; and Plueddemann, E. P., Ed., "Silane Coupling
Reagents", Plenum Pub. Corp., 1991, the disclosures of which are
incorporated herein. Methods for screening target molecules for
specific binding to arrays of polymers, such as nucleic acids,
immobilized on a solid substrate, are disclosed, for example, in
U.S. Pat. No. 5,510,270, the disclosure of which is incorporated
herein. The fabrication of arrays of polymers, such as nucleic
acids, on a solid substrate, and methods of use of the arrays in
different assays, are also described in: U.S. Pat. Nos. 5,677,195,
5,624,711, 5,599,695, 5,445,934, 5,451,683, 5,424,186, 5,412,087,
5,405,783, 5,384,261, 5,252,743 and 5,143,854; PCT WO 92/10092; and
U.S. application Ser. No. 08/388,321, filed Feb. 14, 1995, the
disclosures of each of which are incorporated herein. Accessing
genetic information using high density DNA arrays is further
described in Chee, Science 274:610-614 (1996), the disclosure of
which is incorporated herein by reference. The combination of
photolithographic and fabrication techniques allows each probe
sequence to occupy a very small site on the support. The site may
be as small as a few microns or even a small molecule. Such probe
arrays may be of the type known as Very Large Scale Immobilized
Polymer Synthesis (VLSIPS.RTM.) arrays, as described in U.S. Pat.
No. 5,631,734, the disclosure of which is incorporated herein.
[0134] In the embodiment wherein solid phase chemistry, photolabile
protecting groups and photolithography are used to create light
directed spatially addressable parallel chemical synthesis of a
large array of polynucleotides on the substrate, as described in
U.S. Pat. No. 5,527,681, the disclosure of which is incorporated
herein, computer tools may be used for forming arrays. For example,
a computer system may be used to select nucleic acid or other
polymer probes on the substrate, and design the layout of the array
as described in U.S. Pat. No. 5,571,639, the disclosure of which is
incorporated herein.
[0135] Substrates having a surface to which arrays of
polynucleotides are attached are referred to herein as "biological
chips". The substrate may be, for example, silicon or glass, and
can have the thickness of a microscope slide or glass cover slip.
Substrates that are transparent to light are useful when the assay
involves optical detection, as described, e.g., in U.S. Pat. No.
5,545,531, the disclosure of which is incorporated herein. Other
substrates include Langmuir Blodgett film, germanium,
(poly)tetrafluorethylene, polystyrene, (poly)vinylidenedifluoride,
polycarbonate, gallium arsenide, gallium phosphide, silicon oxide,
silicon nitride, and combinations thereof. In one embodiment, the
substrate is a flat glass or single crystal silicon surface with
relief features less than about 10 Angstoms.
[0136] The surfaces on the solid substrates will usually, but not
always, be composed of the same material as the substrate. Thus,
the surface may comprise any number of materials, including
polymers, plastics, resins, polysaccharides, silica or silica based
materials, carbon, metals, inorganic glasses, membranes, or any of
the above-listed substrate materials. Preferably, the surface will
contain reactive groups, such as carboxyl, amino, and hydroxyl. In
one embodiment, the surface is optically transparent and will have
surface Si--OH functionalities such as are found on silica
surfaces.
[0137] In the embodiment wherein arrays of nucleic acids are
immobilized on a surface, the number of nucleic acid sequences may
be selected for different applications, and may be, for example,
about 100 or more, or, e.g., in some embodiments, more than
10.sup.5 or 10.sup.8. In one embodiment, the surface comprises at
least 100 probe nucleic acids each preferably having a different
sequence, each probe contained in an area of less than about 0.1
cm.sup.2, or, for example, between about 1 .mu.m.sup.2 and 10,000
.mu.m.sup.2, and each probe nucleic acid having a defined sequence
and location on the surface. In one embodiment, at least 1,000
different nucleic acids are provided on the surface, wherein each
nucleic acid is contained within an area less than about 10.sup.-3
cm.sup.2, as described, for example, in U.S. Pat. No. 5,510,270,
the disclosure of which is incorporated herein.
[0138] Arrays of nucleic acids for use in gene expression
monitoring are described in PCT WO 97/10365, the disclosure of
which is incorporated herein. In one embodiment, arrays of nucleic
acid probes are immobilized on a surface, wherein the array
comprises more than 100 different nucleic acids and wherein each
different nucleic acid is localized in a predetermined area of the
surface, and the density of the different nucleic acids is greater
than about 60 different nucleic acids per 1 cm.sup.2.
[0139] Arrays of nucleic acids immobilized on a surface which may
be used also are described in detail in U.S. Pat. No. 5,744,305,
the disclosure of which is incorporated herein. As disclosed
therein, on a substrate, nucleic acids with different sequences are
immobilized each in a predefined area on a surface. For example,
10, 50, 60, 100, 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7,
or 10.sup.8 different monomer sequences may be provided on the
substrate. The nucleic acids of a particular sequence are provided
within a predefined region of a substrate, having a surface area,
for example, of about 1 cm.sup.2 to 10.sup.-10 cm.sup.2. In some
embodiments, the regions have areas of less than about 10.sup.-1,
10.sup.-2, 10.sup.-3, 10.sup.-4, 10.sup.-5, 10.sup.-6, 10.sup.-7,
10.sup.-8, 10.sup.-9, or 10.sup.-10 cm.sup.2. For example, in one
embodiment, there is provided a planar, non-porous support having
at least a first surface, and a plurality of different nucleic
acids attached to the first surface at a density exceeding about
400 different nucleic acids/cm.sup.2, wherein each of the different
nucleic acids is attached to the surface of the solid support in a
different predefined region, has a different determinable sequence,
and is, for example, at least 4 nucleotides in length. The nucleic
acids may be, for example, about 4 to 20 nucleotides in length. The
number of different nucleic acids may be, for example, 1000 or
more. In the embodiment where polynucleotides of a known chemical
sequence are synthesized at known locations on a substrate, and
binding of a complementary nucleotide is detected, and wherein a
fluorescent label is detected, detection may be implemented by
directing light to relatively small and precisely known locations
on the substrate. For example, the substrate is placed in a
microscope detection apparatus for identification of locations
where binding takes place. The microscope detection apparatus
includes a monochromatic or polychromatic light source for
directing light at the substrate, means for detecting fluoresced
light from the substrate, and means for determining a location of
the fluoresced light. The means for detecting light fluoresced on
the substrate may in some embodiments include a photon counter. The
means for determining a location of the fluoresced light may
include an x/y translation table for the substrate. Translation of
the substrate and data collection are recorded and managed by an
appropriately programmed digital computer, as described in U.S.
Pat. No. 5,510,270, the disclosure of which is incorporated
herein.
[0140] Devices for concurrently processing multiple biological chip
assays may be used as described in U.S. Pat. No. 5,545,531, the
disclosure of which is incorporated herein. Methods and systems for
detecting a labeled marker on a sample on a solid support, wherein
the labeled material emits radiation at a wavelength that is
different from the excitation wavelength, which radiation is
collected by collection optics and imaged onto a detector which
generates an image of the sample, are disclosed in U.S. Pat. No.
5,578,832, the disclosure of which is incorporated herein. These
methods permit a highly sensitive and resolved image to be obtained
at high speed. Methods and apparatus for detection of fluorescently
labeled materials are further described in U.S. Pat. Nos. 5,631,734
and 5,324,633, the disclosures of which are incorporated
herein.
[0141] The methods and compositions described herein may be used in
a range of applications including biomedical and genetic research
and clinical diagnostics. Arrays of polymers such as nucleic acids
may be screened for specific binding to a target, such as a
complementary nucleotide, for example, in screening studies for
determination of binding affinity and in diagnostic assays. In one
embodiment, sequencing of polynucleotides can be conducted, as
disclosed in U.S. Pat. No. 5,547,839, the disclosure of which is
incorporated herein. The nucleic acid arrays may be used in many
other applications including detection of genetic diseases such as
cystic fibrosis, diabetes, and acquired diseases such as cancer, as
disclosed in U.S. patent application Ser. No. 08/143,312, the
disclosure of which is incorporated herein. Genetic mutations may
be detected by sequencing by hydridization. In one embodiment,
genetic markers may be sequenced and mapped using Type-IIs
restriction endonucleases as disclosed in U.S. Pat. No. 5,710,000,
the disclosure of which is incorporated herein.
[0142] Other applications include chip based genotyping, species
identification and phenotypic characterization, as described in
U.S. patent application Ser. No. 08/797,812, filed Feb. 7, 1997,
and U.S. application Ser. No. 08/629,031, filed Apr. 8, 1996, the
disclosures of which are incorporated herein.
[0143] Gene expression may be monitored by hybridization of large
numbers of mRNAs in parallel using high density arrays of nucleic
acids in cells, such as in microorganisms such as yeast, as
described in Lockhart et al., Nature Biotechnology, 14:1675-1680
(1996), the disclosure of which is incorporated herein. Bacterial
transcript imaging by hybridization of total RNA to nucleic acid
arrays may be conducted as described in Saizieu et al., Nature
Biotechnology, 16:45-48 (1998), the disclosure of which is
incorporated herein.
[0144] All publications cited herein are incorporated herein by
reference in their entirety.
[0145] The invention will be further understood by the following
non-limiting examples.
EXAMPLES
Example 1
[0146] Silicon compounds were obtained commercially or synthesized
from commercially available starting materials. Silicon compounds
N,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane, and
N-(2-hydroxyethyl)-N-methyl-3-aminopropyltriethoxysilane (compound
I) were purchased from Gelest, Inc. (Tullytown, Pa.).
[0147] Silicon compounds II and V and were prepared as shown in
Schemes II and III. Solutions of the starting materials,
bis(trimethoxysilyl-propyl)- amine and
bis[(3-trimethoxysilyl)propyl]ethylenediamine in methanol (62 wt %,
from Gelest, Inc.) were combined with 1.1-2.2 theoretical
equivalents of ethylene oxide at room temperature under a dry
ice-acetone condenser. The resulting solutions were analyzed by
.sup.1H--NMR, which indicated 95% conversion in some runs to the
hydroxyethylated products, and these were used without further
purification. In other runs, a 60-65% conversion was obtained for
compound V and also was used without further purification.
[0148] .sup.1H--NMR(CDCl.sub.3) data are provided below:
[0149] (II): 0.55-0.70 (br m, 4H), 1.55-1.65 (br m, 4H), 2.40-2.50
(br m, 2H), 2.55-2.60 (br m, 4H), 3.50 (s, MeOH), 3.55 (d, 9H),
3.75 (t, 2H);
[0150] (V): 0.55-0.70 (br m, 4H), 1.50-1.65 (br m, 4H), 2.40-2.80
(br m, 12H), 3.50 (s, MeOH), 3.40-3.60 (m-s, .about.20H), 3.75 (m,
.about.3-4H);
[0151] Substrates were treated by a silanation procedure as
follows. Glass substrates (borosilicate float glass, soda lime or
fused silica, 2".times.3".times.0.027", obtained from U.S.
Precision Glass (Santa Rosa, Calif.) were cleaned by soaking
successively in Nanostrip (Cyantek, Fremont, Calif.) for 15
minutes, 10% aqueous NaOHI/70.degree. C. for 3 minutes, and then 1%
aqueous HCl for 1 minute (rinsing thoroughly with deionized water
after each step). Substrates were then spin-dried for 5 minutes
under a stream of nitrogen at 35.degree. C. Silanation was carried
out by soaking under gentle agitation in a freshly prepared 1-2%
(wt/vol) solution of the silicon compound in 95:5 ethanol-water for
15 minutes. The substrates were rinsed thoroughly with 2-propanol,
then deionized water, and finally spin-dried for 5 minutes at
90.degree.-110.degree. C.
[0152] The stability of silicon compound bonded phase was
evaluated. The surface hydroxyalkylsilane sites on the resulting
substrates were "stained" with fluorescein in a checkerboard
pattern by first coupling a MeNPOC--HEG linker phosphoramidite,
image-wise photolysis of the surface, then coupling to the
photo-deprotected linker sites a 1:20 mixture of fluorescein
phosphoramidite and DMT--T phosphoramidite (Amersham-Pharmacia
Biotech, Piscataway, N.J.), and then deprotecting the surface
molecules in 1:1 ethylenediamine-ethanol for 4 hr. The steps were
conducted using standard protocols, as described in McGall et al.,
J. Am. Chem. Soc., 119:5081-5090 (1997), the disclosure of which is
incorporated herein.
[0153] The pattern and intensity of surface fluorescence was imaged
with a scanning laser confocal fluorescence microscope, which
employed excitation with a 488 nm argon ion laser beam focused to a
2 micron spot size at the substrate surface. Emitted light was
collected through confocal optics with a 530(+15) nm bandpass
filter and detected with a PMT equipped with photon counting
electronics. Output intensity values (photon counts/second) are
proportional to the amount of surface-bound fluorescein, so that
relative yields of free hydroxyl groups within different regions of
the substrate could be determined by direct comparison of the
observed surface fluorescence intensities. All intensity values
were corrected for nonspecific background fluorescence, taken as
the surface fluorescence within the non-illuminated regions of the
substrate.
[0154] The relative surface reactive site density was measured. For
each silicon compound tested, the number of available surface
synthesis sites achieved per unit area was estimated, relative to
N,N-bis(2-hydroxyethyl)- -3-aminopropyltriethoxysilane, by
comparison of the observed initial surface fluorescence intensities
of the various substrates immediately after deprotection in
ethanolic diaminoethane. 1 Site Density ( % rel . ) =
Intensity(silicon compound X ) .times. 100 Intensity (silicon
compound II)
[0155] To determine the relative stability of the silicon compound
coatings, substrates were gently agitated on a rotary shaker at
45.degree. C. in 5xSSPE or 6xSSPE aqueous buffer (BioWhittacker
Products, Walkersville, Md.) at pH 7.3. Periodically, the
substrates were removed from the buffer and re-scanned to measure
the amount of fluorescein remaining bound to the surface.
[0156] The results are shown in FIG. 11. As shown in FIG. 11, more
of the fluorescein tag remained bound to the substrate after
prolonged exposure to the aqueous buffer in the case of substrates
silanated with II or V, than remained bound to the substrates
silanated with N,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane
or compound I. This demonstrates that the surface bonded phase
obtained with silicon compounds II or V is much more stable towards
hydrolysis than that obtained with
N,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane.
[0157] The hybridization performance of silanated substrates was
evaluated. A comparison was made between substrates silanated with
N,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane and compound V
in terms of performance under typical hybridization assay
conditions. A nucleic acid probe sequence (5'-GTC AAG ATG CTA CCG
TTC AG-3') (SEQ. ID NO. 1) was synthesized photolithographically in
a checkerboard array pattern (400.times.400 micron features) on the
substrates that had been derivatized with either
N,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysil- ane or silicon
compound V. After deprotection in ethanolic diaminoethane, the
arrays were hybridized with a fluorescein-labeled complementary
"target" nucleic acid (5'-fluorescein-CTG AAC GGT AGC ATC TTG
AC-3') (SEQ. ID NO. 2) at a concentration of 250 pM in 6xSSPE
buffer (0.9 M NaCl, 60 mM NaH.sub.2PO.sub.4, 6 mM EDTA, pH 7.5) for
16 hours at 45.degree. C. After cooling to room temperature, the
target nucleic acid solution was removed, and the array was washed
briefly with 6x SSPE buffer and then scanned on a confocal imaging
system. The relative amount of bound target was determined from the
fluorescence signal intensity. The hybridization signal intensities
obtained with the more stable substrates, derivatized with silicon
compound V, were at least four times higher than those obtained
with substrates that were derivatized with
N,N-bis(2-hydroxyethyl)-3-aminopropyltriethoxysilane. A graph of
fluorescence signal intensity vs. silane is shown in FIG. 12.
Example 2
[0158] Silicon compound XVIb was prepared as shown in FIG. 13. An
oven dried 25 ml single-neck flask was charged with 1.07 g (1 ml,
0.0066 mol) N,N-bis(2-hydroxyethyl)-1,3-diaminopropane (TCI
America, Portland, Oreg.) and 5 ml anhydrous methanol (Aldrich
Chemical; Milwaukee, Wis.). To this stirring solution was added
2.92 ml (3.12 g, 0.0132 mol) (3-glycidoxypropyl)trimethoxysilane
(Gelest, Inc., Tullytown, Pa.) dropwise over a 30 minute period.
The solution was allowed to stir under an Ar atmosphere until
.sup.1H NMR indicated that the triplet signal for the
(CH.sub.2)CH.sub.2(NH.sub.2) protons of the starting amine
(.delta.=2.86 ppm) had shifted completely away (41 hours).
Molecular ions corresponding to the starting materials were absent
in the mass spectrum (positive ion mode) which was dominated by the
[M+H].sup.+ peak of m/z=635.5.
Example 3
[0159] An alternate preparation of silicon compound XVIb was
conducted as shown in FIG. 13. An oven dried 25 ml single-neck
flask was charged with 1.07 g (1 ml, 0.0066 mol)
N,N-bis(2-hydroxyethyl)-1,3-diaminopropane (TCI America, Portland,
Oreg.) and 5 ml anhydrous methanol (Aldrich Chemical; Milwaukee,
Wis.). To this stirring solution was added 2.92 ml (3.12 g, 0.0132
mol) (3-glycidoxypropyl)trimethoxysilane (Gelest, Inc., Tullytown,
Pa.) dropwise over a 30 minute period. The solution was brought to
reflux and allowed to stir under an Ar atmosphere until .sup.1H NMR
indicated that the triplet signal for the
(CH.sub.2)CH.sub.2(NH.sub.2) protons of the starting amine
(.delta.=2.86 ppm) had shifted completely away (17 hours).
[0160] Molecular ions corresponding to the starting materials were
absent in the mass spectrum (positive ion mode), an M+H.sup.+ peak
of m/z=635.5 was observed but it did not dominate the spectrum as
seen in the room temperature preparation of Example 2.
Example 4
[0161] Silicon compound XVIe was prepared as shown in FIG. 13. An
oven dried 25 ml single-neck flask was charged with 2.74 g (0.026
mol) tris(hydroxymethyl)-methylamine (Aldrich Chemical; Milwaukee,
Wis.), 10 ml (10.70 g, 0.0452 mol)
(3-glycidoxypropyl)trimethoxysilane (Gelest, Inc., Tullytown, Pa.),
and 11 ml anhydrous methanol (Aldrich Chemical; Milwaukee, Wis.).
The suspension was allowed to stir under an Ar atmosphere. After
all the solids dissolved (6 days), .sup.13C NMR indicated that the
singlet signal for the C(CH.sub.2OH).sub.3 carbon of the starting
amine (.delta.=55.38 ppm) had shifted completely away. .sup.13C NMR
(.delta., ppm): 73.12 (CH--OH), 71.03 (C--N), 61.94 (CH.sub.2OH),
50.62 (C--[OC]), 50.06 ([C]--C--O), 49.39 (CH.sub.3OSi), 43.85
(N--C--[CH.sub.2OH].sub.3), 22.34 (C--[CSi]), 4.81 (C--Si). Mass
Spectrum: M.sup.+ (m/z 593.4) and [M--CH.sub.3OH].sup.+ (m/z
561.5).
Example 5
[0162] A solution of 104 g triphosgene (0.102 mol) (Aldrich
Chemical; Milwaukee, Wis.) and 1 liter anhydrous THF (Aldrich
Chemical; Milwaukee, Wis.) was prepared under Ar in a 2-1
oven-dried flask. To the stirring solution was slowly added 42.5 ml
(30.8 g, 0.304 mol) triethylamine (Aldrich Chemical; Milwaukee,
Wis.) and a white precipitate formed. The stirring suspension was
cooled in ice and 100 ml N,N-bis(3-trimethoxysily- lpropyl)amine
(104 g, 0.304 mol) (compound XIV, Gelest, Inc., Tullytown, Pa.) was
added dropwise over an hour. The reaction mixture was allowed to
stir for 21.5 hours then filtered under an Ar blanket and the
solids washed with anhydrous THF. The filtrate was concentrated
under reduced pressure to an orange oil and dried under vacuum to
give 116 g of product (95% crude yield). .sup.1H NMR (.delta.,
ppm): 3.57 (s, CH.sub.3OSi), 3.32 (dd, CH.sub.2--NC.dbd.O, 4 H),
1.70 (q, [CH.sub.2]--CH.sub.2--[CH.su- b.2], 4 H), 0.58 (t,
CH.sub.2Si, 4 H). .sup.13C NMR (.delta., ppm): 149.14 (C.dbd.O),
53.53 (C--N--C.dbd.O), 52.25 (C--N--C.dbd.O), 50.66 (CH.sub.3OSi),
21.83 (C--C--N), 20.82 (C--C--N), 6.50 (C--Si).
Example 6
[0163] Silicon compound XVIIb was prepared as shown in FIG. 14. An
oven dried 25 ml single-neck flask was charged with 0.21 ml (0.15
g, 0.0015 mol) triethylamine (Aldrich Chemical; Milwaukee, Wis.),
0.61 g of a carbamoyl chloride prepared as per Example 5 (0.0015
mol), and 5 ml anhydrous THF (Aldrich Chemical; Milwaukee, Wis.).
After addition of 0.20 g (0.0015 mol) diethanolamine (Aldrich
Chemical; Milwaukee, Wis.) a precipitate formed. After 5 days of
stirring under Ar at room temperature, the mixture was filtered and
concentrated to a brown oil. After drying under vacuum, 0.62 g (87%
crude yield) of material was obtained. The oil was dissolved in 0.6
ml methanol and stored. .sup.13C NMR (.delta., ppm): 166.29
(C.dbd.O), 60.50 (C--OH), 57.28 (N--C--COH), 46.14 (C--C--N), 21.01
(C--[CSi]), 6.27 (C--Si). Mass Spectrum: [M+H].sup.+ (m/z 473) and
[M--CH.sub.3O].sup.+ (m/z 441).
Example 7
[0164] Silicon compound XVIIf was prepared as shown in FIG. 14. An
oven dried 25 ml single-neck flask was charged with 42.4 ml (30.8
g, 0.304 mol) triethylamine (Aldrich Chemical; Milwaukee, Wis.),
116 g of a carbamoyl chloride prepared as per Example 5 (0.304
mol), and 500 ml anhydrous THF (Aldrich Chemical; Milwaukee, Wis.).
After addition of 1.07 g (1 ml, 0.0066 mol)
N,N-Bis(2-hydroxyethyl)-1,3-diaminopropane (TCI America, Portland,
Oreg.), 500 ml anhydrous CH.sub.2Cl.sub.2 (Aldrich Chemical;
Milwaukee, Wis.) was added. The mixture was stirred under Ar at
room temperature, during which time an insoluble orange oil formed.
After 18 hours, .sup.1H NMR indicated that the triplet signal for
the (CH.sub.2)CH.sub.2(NH.sub.2) protons of the starting amine
(.delta.=2.86 ppm) had shifted completely away. The mixture was
decanted and the filtrate concentrated. The concentrate was
combined with 400 ml anhydrous THF (Aldrich Chemical; Milwaukee,
Wis.). The resultant suspension was filtered, concentrated under
reduced pressure, and dried under vacuum to yield 145 g (95% crude
yield) of a faint orange oil. The oil was dissolved in 150 ml
methanol and stored. Mass Spectrum: [M+H].sup.+ (m/z 530).
Example 8
[0165] Coated substrates with XVIb, XVIe, XVIIb, and XVIIf were
prepared by a modified version of the silanation procedure of
Example 1. In this case, the silane content of the 95:5
ethanol:water bath was 1-2% by volume of the methanol/silane
compound solutions described in Examples 2-4 and 6-7 and curing was
done at 50.degree. C. for 2 minutes.
Example 9
[0166] Evaluation of stability of the silicon bonded phase,
relative surface reactive site density, and hybridization
performance of substrates coated with XVIb, XVIe, XVIIb, and XVIIf
was carried out in a single experimental protocol. First,
MeNPOC--HEG linker phosphoramidite is coupled to the surface, then
image-wise photolysis is used to pattern the surface into a series
of stripes. Two stripes, 400 .mu.m.times.12800 .mu.m, were stained
with fluorescein label to evaluate stability of the silicon bonded
phase and relative surface reactive site density. A nucleic acid
probe sequence was synthesized-photolithographically into two
additional stripes, 1600 .mu.m.times.12800 .mu.m. After patterning,
the surface is deprotected in 1:1 ethylenediamine ethanol for 4
hours. The steps were conducted using standard protocols, as
described in McGall et al., J. Am. Chem. Soc., 119:5081-5090
(1997), the disclosure of which is incorporated herein. The pattern
and intensity of surface fluorescence from both fluorescein labeled
patterns and adsorbed fluorescein labeled target oligonucleotide is
imaged with a scanning fluorescence microscope as described in
Example 1.
[0167] In the assay the surface was imaged after a period of 1 hour
exposure to 5 nM target in 6X SSPE buffer (0.9 M NaCl, 60 mM
NaH.sub.2PO.sub.4, 6 mM EDTA, pH 7.5) at 25.degree. C. and again
after 16 hours at 45.degree. C. Except as noted herein, the method
employed for the assay is the same as described in Example 1. The
fluorescence intensity was averaged and evaluated at both the 1
hour and 16 hour time points. Relative surface reactive site
density was determined by normalization of the signal to that
obtained from compound I as described in Example 1. FIG. 30 shows
the normalized data at the 1 and 16 hour time points. The
percentage of remaining fluorescence intensity is noted for the
16-hour time point for each silane coating.
[0168] Hybridization performance of substrates for matched probes
is illustrated in FIG. 31, where the 1 hour, 25.degree. C.
intensities of the averaged background corrected fluorescence
signals of the matched sequence stripes are shown. Signals are
normalized to 1=intensity of identical probes synthesized on
N,N-bis(2-hydroxyethyl)-3-trimethoxypropy- lamine (compound I)
surfaces.
* * * * *