U.S. patent application number 09/897340 was filed with the patent office on 2001-12-13 for functionalization of substrate surfaces with silane mixtures.
Invention is credited to Dellinger, Douglas J., Fulcrand, Geraldine, Hotz, Charles Z., Lefkowitz, Steven M..
Application Number | 20010051221 09/897340 |
Document ID | / |
Family ID | 22511220 |
Filed Date | 2001-12-13 |
United States Patent
Application |
20010051221 |
Kind Code |
A1 |
Lefkowitz, Steven M. ; et
al. |
December 13, 2001 |
Functionalization of substrate surfaces with silane mixtures
Abstract
Low surface energy functionalized surfaces on solid supports are
provided by treating a solid support having hydrophilic moieties on
its surface with a derivatizing composition containing a mixture of
silanes. A first silane provides the desired reduction in surface
energy, while the second silane enables functionalization with
molecular moieties of interest, such as small molecules, initial
monomers to be used in the solid phase synthesis of oligomers, or
intact oligomers. Molecular moieties of interest may be attached
through cleavable sites. Derivatizing compositions for carrying out
the surface functionalization process are provided as well.
Inventors: |
Lefkowitz, Steven M.;
(Millbrae, CA) ; Fulcrand, Geraldine; (Sunnyvale,
CA) ; Dellinger, Douglas J.; (Sunnyvale, CA) ;
Hotz, Charles Z.; (San Mateo, CA) |
Correspondence
Address: |
AGILENT TECHNOLOGIES, INC.
Legal Department, DL429
Intellectual Property Administration
P.O. Box 7599
Loveland
CO
80537-0599
US
|
Family ID: |
22511220 |
Appl. No.: |
09/897340 |
Filed: |
July 2, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09897340 |
Jul 2, 2001 |
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09145015 |
Sep 1, 1998 |
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6258454 |
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Current U.S.
Class: |
427/387 |
Current CPC
Class: |
B82Y 30/00 20130101;
B05D 1/185 20130101; Y10T 428/261 20150115; B82Y 40/00 20130101;
Y10T 428/31612 20150401; B32B 17/10688 20130101; G01N 33/54353
20130101; Y10T 428/31667 20150401; Y10T 428/31663 20150401 |
Class at
Publication: |
427/387 |
International
Class: |
B05D 003/02 |
Claims
1. A process for preparing a low surface energy functionalized
surface on a substrate, comprising: contacting a substrate having
reactive hydrophilic moieties on the surface thereof with a
derivatizing composition comprising a first silane
R.sup.1--Si(R.sup.LR.sup.xR.sup.y) and a second silane
R.sup.2--(L).sub.n--Si(R.sup.LR.sup.xR.sup.y) under reaction
conditions effective to couple the silanes to the substrate surface
and provide --Si--R.sup.1 groups and --Si--(L).sub.n--R.sup.2
groups thereon, wherein the R.sup.L, moieties, which may be the
same or different, are leaving groups, the R.sup.x and R.sup.y are
independently lower alkyl or leaving groups, R.sup.1 is a
chemically inert moiety that upon binding to the substrate surface
lowers the surface energy thereof, n is 0 or 1, L is a linking
group, and R.sup.2 is a functional group enabling covalent binding
of a molecular moiety or a modifiable group that may be converted
to such a functional group.
2. The process of claim 1, wherein the reactive hydrophilic
moieties are selected from the group consisting of hydroxyl,
carboxyl, thiol, amino, and combinations thereof.
3. The process of claim 2, wherein the reactive hydrophilic
moieties are hydroxyl groups.
4. The process of claim 1, wherein the R.sup.L are selected from
the group consisting of halogen and alkoxy.
5. The process of claim 4, wherein the R.sup.L are selected from
the group consisting of chloro and lower alkoxy.
6. The process of claim 1, wherein R.sup.1 is an alkyl group.
7. The process of claim 6, wherein R.sup.1 is C.sub.1-C.sub.24
alkyl.
8. The process of claim 1, wherein n is 1.
9. The process of claim 8, wherein L is a C.sub.1-C.sub.24
hydrocarbylene linking group substituted with 0 to 6 substituents
selected from the group consisting of lower alkyl, hydroxyl,
halogen and amino, optionally containing 1 to 6 --O--, --S--,
--NR--, --CONH--, --(CO)-- or --COO-- linkages wherein R is
hydrogen or lower alkyl.
10. The process of claim 9, wherein L is C.sub.1-C.sub.24
alkylene.
11. The process of claim 10, wherein L is C.sub.10-C.sub.18
alkylene.
12. The process of claim 1, wherein R.sup.2 is
--CH.dbd.CH.sub.2.
13. The process of claim 1, wherein, in the first silane, R.sup.x
and R.sup.y are lower alkyl.
14. The process of claim 1, wherein, in the second silane, R.sup.x
and R.sup.y are lower alkyl.
15. A process for preparing a low surface energy functionalized
surface on a substrate, comprising: contacting a substrate having
reactive hydrophilic moieties on the surface thereof with a
derivatizing composition comprising a first silane
R.sup.1--Si(R.sup.LR.sup.xR.sup.y) and a second silane
R.sup.2--L--Si(R.sup.LR.sup.xR.sup.y) under reaction conditions
effective to couple the silanes to the substrate surface and
provide --Si--L--R.sup.1 groups and --Si--L--R.sup.2 groups
thereon, wherein the R.sup.L moieties are independently selected
from the group consisting of halogen and lower alkoxy, R.sup.x and
R.sup.y are independently selected from the group consisting of
halogen, lower alkoxy and lower alkyl, R.sup.1 is C.sub.1-C.sub.24
alkyl, L is C.sub.1-C.sub.24 alkylene, and R.sup.2 is
--CH.dbd.CH.sub.2.
16. The process of claim 15, further comprising converting R.sup.2
to a hydroxyl group by boration and oxidation.
17. A process for preparing support-bound cleavable ligands on a
low surface energy substrate, comprising: contacting a substrate
having reactive hydrophilic moieties on the surface thereof with a
derivatizing composition comprising a first silane
R.sup.1--Si(R.sup.LR.sup.xR.sup.y) and a second silane
R.sup.2--(L).sub.n--Si(R.sup.LR.sup.xR.sup.y) under reaction
conditions effective to couple the silanes to the substrate surface
and provide --Si--R.sub.1 groups and --Si--(L).sub.n--R.sup.2
groups thereon, wherein the R.sup.L, moieties, which may be the
same or different, are leaving groups, the R.sup.x and R.sup.y are
independently lower alkyl or leaving groups, R.sup.1 is a
chemically inert moiety that upon binding to the substrate surface
lowers the surface energy thereof, n is 0 or 1, L is a linking
group, and R.sup.2 is a functional group or a modifiable group that
may be converted to such a functional group; if R.sup.2 is a
modifiable group, converting it to a functional group; and coupling
a ligand to R.sup.2 through a linking moiety containing a
chemically cleavable site.
18. The process of claim 17, wherein the chemically cleavable site
is base-cleavable, acid-cleavable, or nucleophile-cleavable.
19. The process of claim 18, wherein the cleavable site is a
base-cleavable site.
20. The process of claim 19, wherein the cleavable site is an ester
linkage.
21. The process of claim 17, wherein R.sup.2 is a modifiable
group.
22. The process of claim 21, wherein R.sup.2 is
--CH.dbd.CH.sub.2.
23. The process of claim 22, wherein prior to coupling the ligand,
R.sup.2 is converted to a hydroxyl group by boration and
oxidation.
24. The process of claim 17, wherein the ligand is an intact
oligomer.
25. The process of claim 24, wherein the oligomer is an
oligopeptide or an oligosaccharide.
26. The process of claim 17, wherein the ligand is a first monomer
to be used as the starting point for solid phase synthesis of an
oligomer.
27. The process of claim 26, wherein the ligand is an amino
acid.
28. The process of claim 26, wherein the ligand is a
nucleotide.
29. A derivatizing composition for preparing a low surface energy
functionalized surface on a substrate, comprising: a first silane
R.sup.1--Si(R.sup.LR.sup.xR.sup.y) and a second silane
R.sup.2--(L).sub.n--Si(R.sup.LR.sup.xR.sup.y), wherein the R.sup.L
are independently leaving groups, the R.sup.x and R.sup.y may be
the same or different and are either lower alkyl or leaving groups,
R.sup.1 is a chemically inert moiety that upon binding to a
substrate surface lowers the surface energy thereof, n is 0 or 1, L
is a linking group, and R.sup.2 is a functional group enabling
covalent binding of a molecular moiety or a modifiable group that
can be converted to such a functional group.
30. A derivatizing composition for preparing a low surface energy
functionalized surface on a substrate, comprising a first silane
R.sup.1--Si(R.sup.LR.sup.xR.sup.y) and a second silane
R.sup.2--L--Si(R.sup.LR.sup.xR.sup.y), wherein the R.sup.L moieties
are independently selected from the group consisting of halogen and
lower alkoxy, R.sup.x and R.sup.y are independently selected from
the group consisting of halogen, lower alkoxy and lower alkyl,
R.sup.1 is C.sub.1-C.sub.24 alkyl, L is C.sub.1-C.sub.24 alkylene,
and R.sup.2 is --CH.dbd.CH.sub.2.
31. A substrate having a low surface energy functionalized surface,
comprising: a solid support having a plurality of surface
hydrophilic, nucleophilic groups, a first fraction of which are
covalently bound to an --Si--R.sup.1 moiety and a second fraction
of which are covalently bound to an --Si--(L).sub.n--R.sup.2
moiety, wherein R.sup.1 is a chemically inert moiety that lowers
the surface energy of the solid support, n is 0 or 1, L is a
linking group, R.sup.2 is a functional group enabling covalent
binding of a molecular moiety or a modifiable group that can be
converted to such a functional group, and the solid support is
comprised of a material selected from the group consisting of
polystyrene, agarose, dextran, cellulosic polymers,
polyacrylamides, and glass.
32. A substrate having a low surface energy functionalized surface,
comprising: a solid support having a plurality of surface
hydrophilic, nucleophilic groups, a first fraction of which are
covalently bound to an --Si--R.sup.1 moiety and a second fraction
of which are covalently bound to an
--Si(R.sup.xR.sup.y)--L--R.sup.2 moiety, wherein R.sup.x and
R.sup.y are independently selected from the group consisting of
halogen, lower alkoxy and lower alkyl, R.sup.1 is C.sub.1-C.sub.24
alkyl, L is C.sub.1-C.sub.24 alkylene, and R.sup.2 is
--CH.dbd.CH.sub.2.
Description
TECHNICAL FIELD
[0001] This invention relates generally to chemical
functionalization of surfaces to modify the properties thereof.
More particularly, the invention relates to functionalization of a
substrate with a silane mixture to reduce surface energy and thus
constrain droplets of liquid that are applied to the substrate
surface. A primary use of the invention is in the field of solid
phase chemical synthesis, particularly solid phase synthesis of
oligomer arrays.
BACKGROUND
[0002] Chemically modified, "functionalized," solid surfaces are
necessary in many laboratory procedures involved in chemistry and
biotechnology. One important application is in solid phase chemical
synthesis, wherein initial derivatization of a substrate surface
enables synthesis of polymers such as oligonucleotides and peptides
on the substrate itself. Support-bound oligomer arrays,
particularly oligonucleotide arrays, may be used in screening
studies for determination of binding affinity and in diagnostic
applications, i.e., to detect the presence of a nucleic acid
containing a specific, known oligonucleotide sequence. Modification
of surfaces for use in chemical synthesis has been described, for
example, in U.S. Pat. Nos. 5,624,711 to Sundberg et al., in
5,266,222 to Willis et al., in 5,137,765 to Farnsworth, and in
numerous other patents and publications.
[0003] In modifying siliceous or metal oxide surfaces, one
technique that has been used is derivatization with bifunctional
silanes, i.e., silanes having a first functional group enabling
covalent binding to the surface (often an Si-halogen or Si-alkoxy
group, as in --SiCl.sub.3 or --Si(OCH.sub.3).sub.3, respectively)
and a second functional group that can impart the desired chemical
and/or physical modifications to the surface. A problem with this
type of surface modification, however, is that incorporation of a
desirable surface chemical functionality--provided by the second
functional group--may result in a surface with undesirable physical
properties. For example, there is currently a great deal of
interest in synthesizing arrays of different oligonucleotides on
siliceous surfaces, and a high density of array features is
generally considered desirable. The various array features can be
independently created by the planar separation of individual
phosphoramidite coupling reactions as the oligonucleotides are
synthesized; a simple way to achieve this separation is by spotting
the phosphoramidite solutions onto the surface. Feature density is
then determined by the spread of the solution droplet, which is in
turn uniquely determined by both the volume of the droplet and the
contact angle between the droplet and the surface. However, to
covalently couple the first nucleotide phosphoramidite to the
substrate surface requires hydroxyl moieties on the surface, which
makes the surface wettable by the phosphoramidite solutions and
thus creates droplet spread; for a given droplet volume, then,
relatively large array features are provided, limiting feature
density.
[0004] The aforementioned problem can be overcome using a variety
of techniques to constrain the droplets as they are applied to the
substrate surface. Permanent wells can be formed by micromachining
the substrate, with the active surfaces subsequently modified,
constraining the droplet by capillary action. Temporary wells can
also be formed using either a pre-formed "stencil" or by applying a
coating to the substrate and patterning the coating. These wells
could constrain the droplet by either capillary action and/or by
using a relatively unwettable coating. Alternatively, as described
in U.S. Pat. No. 5,474,796 to Brennan, a pattern of two different
surface-bound silanes can be formed by physically masking the
surface, depositing the first silane, and then removing the mask
and depositing the second silane. This procedure can be used to
constrain a droplet by surrounding a reactive spot on the surface,
formed by one of the two silanes, with a lower surface energy spot,
formed by the other of the two silanes.
[0005] All of these procedures, however, require considerable
processing and thus add substantially to the time and cost required
to fabricate an array. Also, the existence of a pattern on the
substrate requires that the array writing apparatus be aligned with
the surface pattern, a non-trivial issue for small array
features.
[0006] The present invention is directed to the aforementioned need
in the art, and provides a way of functionalizing substrate
surfaces to reduce surface energy and thus constrain droplets of
liquid that are applied to the substrate surface, while avoiding
the aforementioned problems and difficulties associated with the
procedures of the prior art.
SUMMARY OF THE INVENTION
[0007] Accordingly, it is a primary object of the invention to
address the aforementioned need in the art and provide a relatively
simple, straightforward process for preparing a low surface energy
functionalized surface on a substrate.
[0008] It is an additional object of the invention to provide such
a process by coupling a mixture of silanes to hydrophilic moieties
present on a substrate surface.
[0009] It is another object of the invention to provide a process
for preparing support-bound cleavable ligands on a low surface
energy substrate, wherein the ligands may be small molecules,
oligonucleotides, oligopeptides, or the like.
[0010] It is another object of the invention to provide a
derivatizing composition for preparing a low surface energy
functionalized surface on a substrate.
[0011] It is still another object of the invention to provide such
a derivatizing composition comprising a mixture of silanes.
[0012] It is yet another object of the invention to provide such a
derivatizing composition comprising a first silane that upon
binding to a substrate reduces the surface energy thereof, and a
second silane that upon binding to a substrate provides a means for
covalently binding molecular moieties to the substrate surface.
[0013] It is a further object of the invention to provide
substrates having low surface energy functionalized surfaces.
[0014] Additional objects, advantages and novel features of the
invention will be set forth in part in the description which
follows, and in part will become apparent to those skilled in the
art upon examination of the following, or may be learned by
practice of the invention.
[0015] In one embodiment of the invention, then, a process is
provided for preparing a low surface energy functionalized surface
on a substrate, which comprises contacting a substrate having
reactive hydrophilic moieties on its surface with a derivatizing
composition comprising a first silane
R.sup.1--Si(R.sup.LR.sup.xR.sup.y) and a second silane
R.sup.2--(L).sub.n--Si(R.sup.LR.sup.xR.sup.y) under reaction
conditions effective to couple the silanes to the substrate surface
and provide --Si--R.sup.1 groups and --Si--(L).sub.n--R.sup.2
groups thereon. The R.sup.L, which may be the same or different,
are leaving groups, the R.sup.x and R.sup.y, which may also be the
same or different, are either leaving groups, like R.sup.L, or are
lower alkyl, R.sup.1 is a chemically inert moiety that upon binding
to the substrate surface lowers the surface energy thereof, n is 0
or 1, L is a linking group, and R.sup.2 comprises either a
functional group enabling covalent binding of a molecular moiety or
a group that may be modified to provide such a functional group.
The ratio of the silanes in the derivatizing composition determines
the surface energy of the functionalized substrate and the density
of molecular moieties that can ultimately be bound to the substrate
surface.
[0016] In another embodiment, a process is provided for preparing
support-bound cleavable ligands on a low surface energy substrate.
The process involves contacting a substrate having reactive
hydrophilic moieties on the surface thereof with a derivatizing
composition comprising a first silane
R.sup.1--SiR.sup.LR.sup.xR.sup.y) and a second silane
R.sup.2--(L).sub.n--Si(R.sup.LR.sup.xR.sup.y) as described above,
under reaction conditions effective to couple the silanes to the
substrate surface and provide --Si--R.sup.1 groups and
--Si--(L).sub.n--R.sup.2 groups thereon. A ligand is then coupled
to the surface at R.sup.2, through a linking moiety containing a
cleavable site. The ligand may be, for example, a small molecule, a
first monomer in the solid phase synthesis of an oligomer, an
intact oligomer, or the like.
[0017] In an additional embodiment, a derivatizing composition is
provided for carrying out the aforementioned processes. The
derivatizing composition comprises a mixture of silanes, including
a first silane R.sup.1--Si(R.sup.LR.sup.xR.sup.y) and a second
silane R.sup.2--(L).sub.n--Si(R.sup.LR.sup.xR.sup.y), wherein
R.sup.1, R.sup.2, R.sup.L, R.sup.x, R.sup.y and n are as defined
above.
[0018] Finally, the functionalized substrates provided using the
presently disclosed and claimed processes and compositions
represent a further embodiment of the invention. The substrates
have surface-bound --Si--R.sup.1 groups and
--Si--(L).sub.n--R.sup.2 groups, wherein the R.sup.1 moieties
reduce surface energy and the R.sup.2 moieties comprise either
functional groups enabling covalent attachment of a molecular
moiety of interest or modifiable groups that can be converted to
such functional groups.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 schematically illustrates the functionalization of a
substrate surface with a derivatizing composition comprising 97.5
wt. % n-decyltrichlorosilane ("NDS") and 2.5 wt. %
undecenyltrichlorosilane ("UTS"), as described in Example 1.
[0020] FIG. 2 is a graph showing the dependence of surface hydroxyl
content on the mole ratio of UTS in the UTS/NTS derivatizing
composition, evaluated as described in Example 2.
[0021] FIG. 3 illustrates, in graph form, the increase in contact
angle for various derivatizing compositions, including a
derivatizing composition of the invention, as described in Example
2.
[0022] FIG. 4 is a graph showing the general relationship between
spot diameter and contact angle, again, as described in Example
2.
DETAILED DESCRIPTION OF THE INVENTION
[0023] Overview and Definitions:
[0024] Before describing the present invention in detail, it is to
be understood that this invention is not limited to specific
compositions, reagents, process steps, or equipment, as such may
vary. It is also to be understood that the terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to be limiting.
[0025] It must be noted that, as used in this specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, reference to "a first silane having the structural formula
R.sup.1--Si(R.sup.LR.sup- .xR.sup.y)" includes mixtures of silanes
having the recited structure, while, similarly, a second silane
having the structural formula
R.sup.2--(L).sub.n--Si(R.sup.LR.sup.xR.sup.y)" includes mixtures of
such silanes, "a cleavable site" includes a multiplicity of
cleavable sites, and the like.
[0026] In describing and claiming the present invention, the
following terminology will be used in accordance with the
definitions set out below.
[0027] The term "functionalization" as used herein relates to
modification of a solid substrate to provide a plurality of
functional groups on the substrate surface. By a "functionalized
surface" as used herein is meant a substrate surface that has been
modified so that a plurality of functional groups are present
thereon.
[0028] The terms "reactive hydrophilic site" or "reactive
hydrophilic group" refer to hydrophilic moieties that can be used
as the starting point in a synthetic organic process. This is
contrast to "inert" hydrophilic groups that could also be present
on a substrate surface, e.g., hydrophilic sites associated with
polyethylene glycol, a polyamide or the like.
[0029] The "surface energy" .gamma. (measured in ergs/cm.sup.2) of
a liquid or solid substance pertains to the free energy of a
molecule on the surface of the substance, which is necessarily
higher than the free energy of a molecule contained in the in the
interior of the substance; surface molecules have an energy roughly
25% above that of interior molecules. The term "surface tension"
refers to the tensile force tending to draw surface molecules
together, and although measured in different units (as the rate of
increase of surface energy with area, in dynes/cm), is numerically
equivalent to the corresponding surface energy. By modifying a
substrate surface to "reduce" surface energy is meant lowering the
surface energy below that of the unmodified surface.
[0030] The term "monomer" as used herein refers to a chemical
entity that can be covalently linked to one or more other such
entities to form an oligomer. Examples of "monomers" include
nucleotides, amino acids, saccharides, peptoids, and the like. In
general, the monomers used in conjunction with the present
invention have first and second sites (e.g., C-termini and
N-termini, or 5' and 3' sites) suitable for binding to other like
monomers by means of standard chemical reactions (e.g.,
condensation, nucleophilic displacement of a leaving group, or the
like), and a diverse element which distinguishes a particular
monomer from a different monomer of the same type (e.g., an amino
acid side chain, a nucleotide base, etc.). The initial
substrate-bound monomer is generally used as a building-block in a
multi-step synthesis procedure to form a complete ligand, such as
in the synthesis of oligonucleotides, oligopeptides, and the
like.
[0031] The term "oligomer" is used herein to indicate a chemical
entity that contains a plurality of monomers. As used herein, the
terms "oligomer" and "polymer" are used interchangeably, as it is
generally, although not necessarily, smaller "polymers" that are
prepared using the functionalized substrates of the invention,
particularly in conjunction with combinatorial chemistry
techniques. Examples of oligomers and polymers include
polydeoxyribonucleotides, polyribonucleotides, other
polynucleotides which are -- or C-glycosides of a purine or
pyrimidine base, polypeptides, polysaccharides, and other chemical
entities that contain repeating units of like chemical structure.
In the practice of the instant invention, oligomers will generally
comprise about 2-50 monomers, preferably about 2-20, more
preferably about 3-10 monomers.
[0032] The term "ligand" as used herein refers to a moiety that is
capable of covalently or otherwise chemically binding a compound of
interest. Typically, when the present substrates are used in solid
phase synthesis, they are used so that "ligands" are synthesized
thereon. These solid-supported ligands can then be used in
screening or separation processes, or the like, to bind a component
of interest in a sample. The term "ligand" in the context of the
invention may or may not be an "oligomer" as defined above.
However, the term "ligand" as used herein may also refer to a
compound that is not synthesized on the novel functionalized
substrate, but that is "pre-synthesized" or obtained commercially,
and then attached to the substrate.
[0033] The term "sample" as used herein relates to a material or
mixture of materials, typically, although not necessarily, in fluid
form, containing one or more components of interest.
[0034] The terms "nucleoside" and "nucleotide" are intended to
include those moieties which contain not only the known purine and
pyrimidine bases, but also other heterocyclic bases that have been
modified. Such modifications include methylated purines or
pyrimidines, acylated purines or pyrimidines, or other
heterocycles. In addition, the terms "nucleoside" and "nucleotide"
include those moieties that contain not only conventional ribose
and deoxyribose sugars, but other sugars as well. Modified
nucleosides or nucleotides also include modifications on the sugar
moiety, e.g., wherein one or more of the hydroxyl groups are
replaced with halogen atoms or aliphatic groups, or are
functionalized as ethers, amines, or the like.
[0035] As used herein, the term "amino acid" is intended to include
not only the L-, D- and nonchiral forms of naturally occurring
amino acids (alanine, arginine, asparagine, aspartic acid,
cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine,
leucine, lysine, methionine, phenylalanine, proline, serine,
threonine, tryptophan, tyrosine, valine), but also modified amino
acids, amino acid analogs, and other chemical compounds which can
be incorporated in conventional oligopeptide synthesis, e.g.,
4-nitrophenylalanine, isoglutamic acid, isoglutamine,
.epsilon.-nicotinoyl-lysine, isonipecotic acid,
tetrahydroisoquinoleic acid, .alpha.-aminoisobutyric acid,
sarcosine, citrulline, cysteic acid, t-butylglycine,
t-butylalanine, phenylglycine, cyclohexylalanine, .beta.-alanine,
4-aminobutyric acid, and the like.
[0036] The terms "protection" and "deprotection" as used herein
relate, respectively, to the addition and removal of chemical
protecting groups using conventional materials and techniques
within the skill of the art and/or described in the pertinent
literature; for example, reference may be had to Greene et al.,
Protective Groups in Organic Synthesis, 2nd Ed., New York: John
Wiley & Sons, 1991. Protecting groups prevent the site to which
they are attached from participating in the chemical reaction to be
carried out.
[0037] The term "alkyl" as used herein refers to a branched or
unbranched saturated hydrocarbon group of 1 to 24 carbon atoms,
such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,
t-butyl, octyl, decyl, tetradecyl, hexadecyl, eicosyl, tetracosyl
and the like, as well as cycloalkyl groups such as cyclopentyl,
cyclohexyl and the like. The term "lower alkyl" intends an alkyl
group of 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms.
[0038] The term "alkoxy" as used herein refers to a substituent
--O--R wherein R is alkyl as defined above. The term "lower alkoxy"
refers to such a group wherein R is lower alkyl.
[0039] The term "alkylene" as used herein refers to a difunctional
saturated branched or unbranched hydrocarbon chain containing from
1 to 24 carbon atoms, and includes, for example, methylene
(--CH.sub.2--), ethylene (--CH.sub.2--CH.sub.2--), propylene
(--CH.sub.2--CH.sub.2--CH.su- b.2--), 2-methylpropylene
(--CH.sub.2--CH(CH.sub.3)--CH.sub.2--), hexylene
(--(CH.sub.2).sub.6--), and the like. "Lower alkylene" refers to an
alkylene group of 1 to 6, more preferably 1 to 4, carbon atoms.
[0040] The terms "alkenyl" and "olefinic" as used herein refer to a
branched or unbranched hydrocarbon group of 2 to 24 carbon atoms
containing at least one carbon-carbon double bond, such as ethenyl,
n-propenyl, isopropenyl, n-butenyl, isobutenyl, t-butenyl, octenyl,
decenyl, tetradecenyl, hexadecenyl, eicosenyl, tetracosenyl and the
like.
[0041] The terms "halogen" or "halo" are used in the conventional
sense to refer to a chloro, bromo, fluoro or iodo substituent.
[0042] "Optional" or "optionally" means that the subsequently
described circumstance may or may not occur, so that the
description includes instances where the circumstance occurs and
instances where it does not. For example, the phrase "optionally
substituted" means that a non-hydrogen substituent may or may not
be present, and, thus, the description includes structures wherein
a non-hydrogen substituent is present and structures wherein a
non-hydrogen substituent is not present.
[0043] Accordingly, the invention in a first embodiment is directed
to a process for preparing a low surface energy functionalized
surface on a substrate. The functionalized surface prepared using
this process has functional groups enabling covalent binding of
molecular moieties, such as in solid phase chemical synthesis or
the like, but nevertheless has lowered surface energy so that
wettability is reduced and liquid droplets applied to the substrate
surface are constrained (i.e., do not spread to the extent that
they would in the absence of the presently disclosed and claimed
surface modification process).
[0044] The inventive process involves contacting the surface of a
solid substrate with a derivatizing composition that contains a
mixture of silanes, under reaction conditions effective to couple
the silanes to the substrate surface via reactive hydrophilic
moieties present on the substrate surface. The reactive hydrophilic
moieties on the substrate surface are typically hydroxyl groups,
carboxyl groups, thiol groups, and/or substituted or unsubstituted
amino groups, although, preferably, the reactive hydrophilic
moieties are hydroxyl groups. The substrate may comprise any
material that has a plurality of reactive hydrophilic sites on its
surface, or that can be treated or coated so as to have a plurality
of such sites on its surface. Suitable materials include, but are
not limited to, supports that are typically used for solid phase
chemical synthesis, e.g., cross-linked polymeric materials (e.g.,
divinylbenzene styrene-based polymers), agarose (e.g.,
Sepharose.RTM.), dextran (e.g., Sephadex.RTM.), cellulosic
polymers, polyacrylamides, silica, glass (particularly controlled
pore glass, or "CPG"), ceramics, and the like. The supports may be
obtained commercially and used as is, or they may be treated or
coated prior to functionalization.
[0045] The derivatizing composition contains two types of silanes,
a first silane that may be represented as
R.sup.1--SiR.sup.LR.sup.xR.sup.y) and a second silane having the
formula R.sup.2--(L).sub.n--Si(R.sup.LR.sup.xR.s- up.y). In these
formulae, the R.sup.L, which may be the same or different, are
leaving groups, the R.sup.x and R.sup.y, which may be the same or
different, are either lower alkyl or leaving groups like R.sup.L,
R.sup.1 is a chemically inert moiety that upon binding to the
substrate surface lowers the surface energy thereof, n is 0 or 1, L
is a linking group, and R.sup.2 is a functional group enabling
covalent binding of a molecular moiety or a group that may be
modified to provide such a functional group. Reaction of the
substrate surface with the derivatizing composition is carried out
under reaction conditions effective to couple the silanes to the
surface hydrophilic moieties and thereby provide --Si--R.sup.1
groups and --Si--(L).sub.n--R.sup.2 groups on the substrate
surface.
[0046] More specifically, the R.sup.L moieties, which are leaving
groups, are such that they enable binding of the silanes to the
surface. Typically, the leaving groups are hydrolyzable so as to
form a silanol linkage to surface hydroxyl groups. Examples of
suitable leaving groups include, but are not limited to, halogen
atoms, particularly chloro, and alkoxy moieties, particularly lower
alkoxy moieties. The R.sup.x and R.sup.y are either lower alkyl,
e.g., methyl, ethyl, isopropyl, n-propyl, t-butyl, or the like, or
leaving groups as just described with respect to R.sup.L. Thus,
each type of silane will generally contain a trichlorosilyl
functionality, a tri(lower)alkoxysilyl functionality such as
trimethoxysilyl, mixed functionalities such as
diisopropylchlorosilyl, dimethylchlorosilyl, ethyldichlorosilyl,
methylethylchlorosilyl or the like.
[0047] The first silane is the derivatizing agent that reduces
surface energy as desired, while the second silane provides the
surface functionalization necessary for covalent attachment of an
additional molecular moiety, e.g., a ligand, a monomer, an
oligomer, etc. Thus, with respect to the first silane, coupling to
the substrate yields surface --Si--R.sup.1 groups as explained
above, wherein R.sup.1 is a chemically inert moiety that upon
binding to the substrate surface lowers surface energy. By
"chemically inert" is meant that R.sup.1 will not be cleaved or
modified when the functionalized substrate is used for its intended
purpose, e.g., in solid phase chemical synthesis, hybridization
assays, or the like. Typically, R.sup.1 is an alkyl group,
generally although not necessarily containing in the range of 2 to
24 carbon atoms, preferably in the range of 10 to 18 carbon atoms.
R.sup.1 may also be benzyl, either unsubstituted or substituted
with 1 to 5, typically 1 to 3, halogen, preferably fluoro,
atoms.
[0048] The second silane, upon coupling, provides surface
--Si--(L).sub.n--R.sup.2 groups. Of course, if the R.sup.x and
R.sup.y are not leaving groups, the surface moieties provided will
actually be "--SiR.sup.xR.sup.y--(L).sub.n--R.sup.2" groups, which
applicants intend to encompass by the more generic representation
"--Si--(L).sub.n--R.sup.2- ". R.sup.2 comprises either a functional
group that can bind directly to an additional molecular species of
interest, or a modifiable group that can be converted to such a
functional group under conditions that do not substantially affect
any other chemical species that are present. That is, R.sup.2 may
be a functional group such as hydroxyl, carboxyl, amino, or the
like, or it may be a modifiable group such an olefinic moiety,
e.g., a terminal --CH.dbd.CH.sub.2 group, which can readily be
converted to a reactive hydroxyl group by boration and oxidation
using procedures known in the art. L represents a linker and n is 0
or 1, such that a linker may or may not be present. If a linker is
present, it will generally be a C.sub.1-C.sub.24 hydrocarbylene
linking group. Normally, L is C.sub.1-C.sub.24 alkylene, preferably
C.sub.10-C.sub.18 alkylene.
[0049] The density of R.sup.2 groups on the substrate surface,
following reaction with the derivatizing composition, is determined
by the relative proportions of the first and second silanes in the
derivatizing composition. That is, a higher proportion of the
second silane in the derivatizing composition will provide a
greater density of R.sup.2 groups, while a higher proportion of the
first silane will give rise to a lower density of R.sup.2 groups.
Optimally, the first silane represents in the range of
approximately 0.5 wt. % to 50 wt. % of the derivatization
composition, preferably in the range of approximately 1.0 wt. % to
10 wt. % of the composition, while the second silane
correspondingly represents in the range of approximately 50 wt. %
to 99.5 wt. % of the derivatization composition, preferably in the
range of approximately 90 wt. % to 99 wt. % of the composition.
[0050] Functionalized substrates prepared using the aforementioned
procedures are believed to be novel and are claimed as such herein.
The surface of the functionalized substrates contain both
--Si--R.sup.1 and Si--(L).sub.n--R.sup.2 groups, present at a
predetermined ratio, with the ratio determining both surface energy
and density of functional groups. These substrates may be used, for
example, in any of a number of known chemical and biological
procedures, such as in solid phase chemical synthesis, e.g., of
oligonucleotides, oligopeptides, and oligosaccharides, in the
preparation of combinatorial libraries, in chemical separation
procedures, in screening processes, and the like. Such procedures
are in current use and will thus be known to those skilled in the
art and/or described in the pertinent literature and texts. For
example, synthesis of polynucleotide libraries using now
conventional phosphoramidite or phosphotriester chemistry is
described by Beaucage et al. (1981) Tetrahedron Lett. 22:1859-62,
and Itakura et al. (1975) J. Biol. Chem. 250:4592 (1975). Houghten
(1985) Proc. Natl. Acad. Sci. USA 82:5131-5135), describes the
preparation of a combinatorial library of peptides using a
modification of the Merrifield method (Merrifield (1963) J. Am.
Chem. Soc. 85:2149-2154; Tam et al., The Peptides (New York:
Academic Press, 1975), at pp. 185-249); and Oligonucleotide
Synthesis, M. J. Gait, Ed. (New York: IRL Press, 1990).
[0051] For example, synthesis of support-bound oligonucleotides is
normally conducted by successive addition of protected nucleotides
to a growing oligonucleotide chain, wherein the terminal 5'
hydroxyl group is caused to react with a
deoxyribonucleoside-3'-O-(N,N-diisopropylamino)pho- sphoramidite
protected at the 5' position with dimethoxytrityl or the like, the
5' protecting group is removed after the coupling reaction, and the
procedure is repeated with additional protected nucleotides until
synthesis of the desired oligonucleotide is complete.
[0052] Additionally, and as will be appreciated by those skilled in
the art, oligopeptide synthesis on a support--as may be carried out
herein by virtue of the support-bound R.sup.2 substituent--involves
sequential addition of carboxyl-protected amino acids to a growing
peptide chain, with each additional amino acid in the sequence
similarly protected and coupled to the terminal amino acid of the
oligopeptide under conditions suitable for forming an amide
linkage. After oligopeptide synthesis is complete, acid is used to
remove the remaining terminal protecting groups. The support-bound
oligopeptides thus provided can then be used in any number of ways,
e.g., in screening procedures involved in combinatorial processes,
in chromatographic methods, and the like.
[0053] In an alternative embodiment, the method and reagents of the
invention are used to provide oligomers bound to the support via a
chemically cleavable site. That is, in this alternative process,
following reaction of the substrate surface with the first and
second silanes, a further reaction is conducted at R.sup.2. This
reaction involves reaction of R.sup.2 with a linking group
containing a cleavable site, such as an ester group, and the free
terminus of the bound linking group is then used for solid phase
synthesis. Conversion of R.sup.2 to a different moiety may or may
not be desired prior to attaching the linking group. For example,
R.sup.2 may be an alkylamino substituent, in which case the amino
moiety serves as the reactive site for binding the linking group,
or R.sup.2 may be bromo, in which case it is desirable to convert
R.sup.2 to a primary or secondary amino substituent, and then carry
out the reaction to the linking group. In this way, the bound
ligand, monomer, oligomer, or the like may be cleaved from the
solid support by treatment of the surface with an appropriate
reagent.
[0054] Suitable cleavable sites include, but are not limited to,
the following: base-cleavable sites such as esters, particularly
succinates (cleavable by, for example, ammonia or trimethylamine),
quaternary ammonium salts (cleavable by, for example,
diisopropylamine) and urethanes (cleavable by aqueous sodium
hydroxide); acid-cleavable sites such benzyl alcohol derivatives
(cleavable using trifluoroacetic acid), teicoplanin aglycone
(cleavable by trifluoroacetic acid followed by base), acetals and
thioacetals (also cleavable by trifluoroacetic acid), thioethers
(cleavable, for example, by HF or cresol) and sulfonyls (cleavable
by trifluoromethane sulfonic acid, trifluoroacetic acid,
thioanisole, or the like); nucleophile-cleavable sites such as
phthalamide (cleavable with substituted hydrazines), esters
(cleavable with, for example, aluminum trichloride) and Weinreb
amide (cleavable with lithium aluminum hydride); and other types of
chemically cleavable sites, including phosphorothioate (cleavable
with silver or mercuric ions) and diisopropyldialkoxysilyl
(cleavable with fluoride ion). Other cleavable sites will be
apparent to those skilled in the art or are described in the
pertinent literature and texts (e.g., Brown (1997) Contemporary
Organic Synthesis 4(3):216-237).
[0055] It is to be understood that while the invention has been
described in conjunction with the preferred specific embodiments
thereof, the foregoing description as well as the example that
follows are intended to illustrate and not limit the scope of the
invention. Other aspects, advantages and modifications within the
scope of the invention will be apparent to those skilled in the art
to which the invention pertains.
[0056] All patents and publications mentioned herein, both supra
and infra, are hereby incorporated by reference.
EXAMPLE 1
[0057] Preparation of Functionalized Surfaces:
[0058] This example describes functionalization of a glass
substrate with a derivatizing composition comprising 97.5 wt. %
n-decyltrichlorosilane ("NTS") as a first silane and 2.5 wt. %
undecenyltrichlorosilane ("UTS") as a second silane, followed by
boration and oxidation to convert the terminal olefinic moiety of
the surface-bound UTS to a hydroxyl group. This procedure is shown
schematically in FIG. 1. Evaluation of the functionalized surface
is also described.
[0059] (a) Silylation:
[0060] Under moisture-free conditions, 14 ml NTS and 0.4 ml UTS
were added to 800 ml of anhydrous toluene, and swirled to mix.
Cleaned glass substrates were placed into a ca. 1 liter reactor
equipped for inert gas purging, heating and stirring, and purging
was conducted for 30 minutes. Moisture-free conditions were
maintained, and the NTS/UTS solution was added to the reactor. The
solution was heated to 100.degree. C. for 4 hours, while stirring
and continuing to maintain moisture-free conditions. The silane
solution was removed from the reactor and replaced with anhydrous
toluene. This step was repeated twice.
[0061] The substrates were then removed from the reactor and rinsed
rigorously with an appropriate solvent. The bulk solvent was
removed from the substrates by blowing with clean inert gas. The
substrates were placed in a vacuum oven preheated to 150.degree. C.
and heated under vacuum for 1 hour. The substrates were removed and
allowed to cool to ambient temperature.
[0062] (b) Boration and Oxidation:
[0063] The silylated substrates prepared in part (a) were placed in
a ca. 1 liter reactor equipped for inert gas purging and stirring,
and purging was conducted for 30 minutes. Under moisture-free
conditions, 800 ml of 1.0 M borane-tetrahydrofuran complex was
transferred to the reactor. The substrates were incubated while
stirring, for two hours. Then, while maintaining moisture-free
conditions, the boration solution was removed and replaced with 800
ml anhydrous tetrahydrofuran. The substrates were removed and
rinsed rigorously with an appropriate solvent. Bulk solvent was
removed by blowing with clean inert gas.
[0064] To a 1 liter vessel equipped for stirring, 800 ml of 0.1 N
NaOH in 30% hydrogen peroxide (aqueous) was added. The oxidized
substrates were immersed therein, and incubated, with stirring, for
10 minutes. The substrates were removed and rinsed rigorously with
an appropriate solvent, then dried by blowing with clean inert
gas.
[0065] The processes of steps (a) and (b) were repeated using
different mole ratios of NTS and UTS, 100% UTS, and a mixture of
glycidoxypropyl trimethoxysilane and hexaethylene glycol
(GOPS-HEG). This hydroxyl silane-linker was prepared following the
procedure of Maskos et al. (Maskos et al. (1992) Nucleic Acids Res.
20:1679) who demonstrated it to be useful for both oligonucleotide
synthesis and hybridization.
EXAMPLE 2
[0066] Evaluation of Functionalized Surfaces:
[0067] Surface hydroxyl density (molecules/.mu.m.sup.2) of the
functionalized surfaces prepared in Example 1 was evaluated
spectrophotometrically, and FIG. 2 shows the dependence of surface
hydroxyl content on the mole ratio of UTS in the UTS/NTS
derivatizing composition. FIG. 3 shows the increase in contact
angle for several UTS mole ratios and two solvents of interest,
acetonitrile and adiponitrile, in comparison to a GOPS-ethanol
mixture. Contact angles reported are static contact angle
measurements as described in the literature (Chan, Chi-Ming,
Polymer Surface Modification and Characterization, chapter 2 (New
York: Hansa Publishers, 1993). Measurements were performed on 25
.mu.l aliquots of the appropriate solvent using an FTA200
instrument (First Ten Angstroms, South San Francisco, Calif.). FIG.
4 shows the general relationship between the spot diameter and
contact angle. For 100 nl drops, the following spot diameters were
observed for the GOPS-ethanol mixture and the 2.5% UTS/NTS
derivatizing composition:
1 GOPS-ethanol 2.5% UTS/NTS acetonitrile >5 mm 1.4 mm
adiponitrile 2.7 mm 0.9 mm
[0068] Thus, the derivatizing composition of the invention
significantly reduces spot diameter for a droplet of a given
volume.
* * * * *