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The file of this patent contains drawings executed in color. Copies of this
patent with color drawings will be provided by the Patent and Trademark
Office upon request and payment of the necessary fee.
COPYRIGHT NOTICE
A portion of the disclosure of this patent document contains material which
is subject to copyright protection. The copyright owner has no objection
to the facsimile reproduction by anyone of the patent document or the
patent disclosure as it appears in the Patent and Trademark Office patent
file or records, but otherwise reserves all copyright rights whatsoever.
BACKGROUND OF THE INVENTION
The present inventions relate to the synthesis and placement of materials
at known locations. In particular, one embodiment of the inventions
provides a method and associated apparatus for preparing diverse chemical
sequences at known locations on a single substrate surface. The inventions
may be applied, for example, in the field of preparation of oligomer,
peptide, nucleic acid, oligosaccharide, phospholipid, polymer, or drug
congener preparation, especially to create sources of chemical diversity
for use in screening for biological activity.
The relationship between structure and activity of molecules is a
fundamental issue in the study of biological systems. Structure-activity
relationships are important in understanding, for example, the function of
enzymes, the ways in which cells communicate with each other, as well as
cellular control and feedback systems.
Certain macromolecules are known to interact and bind to other molecules
having a very specific three-dimensional spatial and electronic
distribution. Any large molecule having such specificity can be considered
a receptor, whether it is an enzyme catalyzing hydrolysis of a metabolic
intermediate, a cell-surface protein mediating membrane transport of ions,
a glycoprotein serving to identify a particular cell to its neighbors, an
IgG-class antibody circulating in the plasma, an oligonucleotide sequence
of DNA in the nucleus, or the like. The various molecules which receptors
selectively bind are known as ligands.
Many assays are available for measuring the binding affinity of known
receptors and ligands, but the information which can be gained from such
experiments is often limited by the number and type of ligands which are
available. Novel ligands are sometimes discovered by chance or by
application of new techniques for the elucidation of molecular structure,
including x-ray crystallographic analysis and recombinant genetic
techniques for proteins.
Small peptides are an exemplary system for exploring the relationship
between structure and function in biology. A peptide is a sequence of
amino acids. When the twenty naturally occurring amino acids are condensed
into polymeric molecules they form a wide variety of three-dimensional
configurations, each resulting from a particular amino acid sequence and
solvent condition. The number of possible pentapeptides of the 20
naturally occurring amino acids, for example, is 20.sup.5 or 3.2 million
different peptides. The likelihood that molecules of this size might be
useful in receptor-binding studies is supported by epitope analysis
studies showing that some antibodies recognize sequences as short as a few
amino acids with high specificity. Furthermore, the average molecular
weight of amino acids puts small peptides in the size range of many
currently useful pharmaceutical products.
Pharmaceutical drug discovery is one type of research which relies on such
a study of structure-activity relationships. In most cases, contemporary
pharmaceutical research can be described as the process of discovering
novel ligands with desirable patterns of specificity for biologically
important receptors. Another example is research to discover new compounds
for use in agriculture, such as pesticides and herbicides.
Sometimes, the solution to a rational process of designing ligands is
difficult or unyielding. Prior methods of preparing large numbers of
different polymers have been painstakingly slow when used at a scale
sufficient to permit effective rational or random screening. For example,
the "Merrifield" method (J. Am. Chem. Soc. (1963) 85:2149-2154, which is
incorporated herein by reference for all purposes) has been used to
synthesize peptides on a solid support. In the Merrifield method, an amino
acid is covalently bonded to a support made of an insoluble polymer.
Another amino acid with an alpha protected group is reacted with the
covalently bonded amino acid to form a dipeptide. After washing, the
protective group is removed and a third amino acid with an alpha
protective group is added to the dipeptide. This process is continued
until a peptide of a desired length and sequence is obtained. Using the
Merrifield method, it is not economically practical to synthesize more
than a handful of peptide sequences in a day.
To synthesize larger numbers of polymer sequences, it has also been
proposed to use a series of reaction vessels for polymer synthesis. For
example, a tubular reactor system may be used to synthesize a linear
polymer on a solid phase support by automated sequential addition of
reagents. This method still does not enable the synthesis of a
sufficiently large number of polymer sequences for effective economical
screening.
Methods of preparing a plurality of polymer sequences are also known in
which a porous container encloses a known quantity of reactive particles,
the particles being larger in size than pores of the container. The
containers may be selectively reacted with desired materials to synthesize
desired sequences of product molecules. As with other methods known in the
art, this method cannot practically be used to synthesize a sufficient
variety of polypeptides for effective screening.
Other techniques have also been described. These methods include the
synthesis of peptides on 96 plastic pins which fit the format of standard
microtiter plates. Unfortunately, while these techniques have been
somewhat useful, substantial problems remain. For example, these methods
continue to be limited in the diversity of sequences which can be
economically synthesized and screened.
From the above, it is seen that an improved method and apparatus for
synthesizing a variety of chemical sequences at known locations is
desired.
SUMMARY OF THE INVENTION
An improved method and apparatus for the preparation of a variety of
polymers is disclosed.
In one preferred embodiment, linker molecules are provided on a substrate.
A terminal end of the linker molecules is provided with a reactive
functional group protected with a photoremovable protective group. Using
lithographic methods, the photoremovable protective group is exposed to
light and removed from the linker molecules in first selected regions. The
substrate is then washed or otherwise contacted with a first monomer that
reacts with exposed functional groups on the linker molecules. In a
preferred embodiment, the monomer is an amino acid containing a
photoremovable protective group at its amino or carboxy terminus and the
linker molecule terminates in an amino or carboxy acid group bearing a
photoremovable protective group.
A second set of selected regions is, thereafter, exposed to light and the
photoremovable protective group on the linker molecule/protected amino
acid is removed at the second set of regions. The substrate is then
contacted with a second monomer containing a photoremovable protective
group for reaction with exposed functional groups. This process is
repeated to selectively apply monomers until polymers of a desired length
and desired chemical sequence are obtained. Photolabile groups are then
optionally removed and the sequence is, thereafter, optionally capped.
Side chain protective groups, if present, are also removed.
By using the lithographic techniques disclosed herein, it is possible to
direct light to relatively small and precisely known locations on the
substrate. It is, therefore, possible to synthesize polymers of a known
chemical sequence at known locations on the substrate.
The resulting substrate will have a variety of uses including, for example,
screening large numbers of polymers for biological activity. To screen for
biological activity, the substrate is exposed to one or more receptors
such as antibodies whole cells, receptors on vesicles, lipids, or any one
of a variety of other receptors. The receptors are preferably labeled
with, for example, a fluorescent marker, radioactive marker, or a labeled
antibody reactive with the receptor. The location of the marker on the
substrate is detected with, for example, photon detection or
autoradiographic techniques. Through knowledge of the sequence of the
material at the location where binding is detected, it is possible to
quickly determine which sequence binds with the receptor and, therefore,
the technique can be used to screen large numbers of peptides. Other
possible applications of the inventions herein include diagnostics in
which various antibodies for particular receptors would be placed on a
substrate and, for example, blood sera would be screened for immune
deficiencies. Still further applications include, for example, selective
"doping" of organic materials in semiconductor devices, and the like.
In connection with one aspect of the invention an improved reactor system
for synthesizing polymers is also disclosed. The reactor system includes a
substrate mount which engages a substrate around a periphery thereof. The
substrate mount provides for a reactor space between the substrate and the
mount through or into which reaction fluids are pumped or flowed. A mask
is placed on or focused on the substrate and illuminated so as to
deprotect selected regions of the substrate in the reactor space. A
monomer is pumped through the reactor space or otherwise contacted with
the substrate and reacts with the deprotected regions. By selectively
deprotecting regions on the substrate and flowing predetermined monomers
through the reactor space, desired polymers at known locations may be
synthesized.
Improved detection apparatus and methods are also disclosed. The detection
method and apparatus utilize a substrate having a large variety of polymer
sequences at known locations on a surface thereof. The substrate is
exposed to a fluorescently labeled receptor which binds to one or more of
the polymer sequences. 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 slide and data
collection are recorded and managed by an appropriately programmed digital
computer.
A further understanding of the nature and advantages of the inventions
herein may be realized by reference to the remaining portions of the
specification and the attached drawings.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 illustrates masking and irradiation of a substrate at a first
location. The substrate is shown in cross-section;
FIG. 2 illustrates the substrate after application of a monomer "A";
FIG. 3 illustrates irradiation of the substrate at a second location;
FIG. 4 illustrates the substrate after application of monomer "B";
FIG. 5 illustrates irradiation of the "A" monomer;
FIG. 6 illustrates the substrate after a second application of "B";
FIG. 7 illustrates a completed substrate;
FIGS. 8A and 8B illustrate alternative embodiments of a reactor system for
forming a plurality of polymers on a substrate;
FIG. 9 illustrates a detection apparatus for locating fluorescent markers
on the substrate;
FIGS. 10A-10M illustrate the method as it is applied to the production of
the trimers of monomers "A" and "B";
FIGS. 11A and 11B are fluorescence traces for standard fluorescent beads;
FIGS. 12A and 12B are fluorescence curves for NVOC
(6-nitroveratryloxycarbonyl) slides not exposed and exposed to light
respectively;
FIGS. 13A to 13D are fluorescence plots of slides exposed through 100
.mu.m, 50 .mu.m, 20 .mu.m, and 10 .mu.m masks; 14A and 14B illustrate
formation of YGGFL (a peptide of sequence
H2N-tyrosine-glycine-glycine-phenylalanine-leucine-CO.sub.2 H) and GGFL (a
peptide of sequence H.sub.2
N-glycine-glycine-phenylalanine-leucine-CO.sub.2 H), followed by exposure
to labeled Herz antibody (an antibody that recognizes YGGFL but not GGFL);
FIGS. 15A and 15B fluorescence plots of a slide with a checkerboard pattern
of YGGFL and GGFL exposed to labeled Herz antibody; FIG. 15A illustrates a
500.times.500 .mu.m mask which has been focused on the substrate according
to FIG. 8A while FIG. 15B illustrates a 50.times.50 .mu.m mask placed in
direct contact with the substrate in accord with FIG. 8B;
FIG. 16 is a fluorescence plot of YGGFL and PGGFL synthesized in a 50 .mu.m
checkerboard pattern;
FIG. 17 is a fluorescence plot of YPGGFL and YGGFL synthesized in a 50
.mu.m checkerboard pattern;
FIGS. 18A and 18B illustrate the mapping of sixteen sequences synthesized
on two different glass slides;
FIG. 19 is a fluorescence plot of the slide illustrated in FIG. 18A; and
FIG. 20 is a fluorescence plot of the slide illustrated in FIG. 10B.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
CONTENTS
I. Glossary
II. General
III. Polymer Synthesis
IV. Details of One Embodiment of a Reactor System
V. Details of One Embodiment of a Fluorescent Detection Device
VI. Determination of Relative Binding Strength of Receptors
VII. Examples
A. Slide Preparation
B. Synthesis of Eight Trimers of "A" and "B"
C. Synthesis of a Dimer of an Aminopropyl Group and a Fluorescent Group
D. Demonstration of Signal Capability
E. Determination of the Number of Molecules Per Unit Area
F. Removal of NVOC and Attachment of a Fluorescent Marker
G. Use of a Mask in Removal of NVOC
H. Attachment of YGGFL and Subsequent Exposure to Herz Antibody and Goat
Antimouse
I. Monomer-by-Monomer Formation of YGGFL and Subsequent Exposure to Labeled
Antibody
J. Monomer-by-Monomer Synthesis of YGGFL and PGGFL
K. Monomer-by Monomer Synthesis of YGGFL and YPGGFL
L. Synthesis of an Array of Sixteen Different Amino Acid Sequences and
Estimation of Relative Binding Affinity to Herz Antibody
VIII. Illustrative Alternative Embodiment
IX. Conclusion
I. Glossary
The following terms are intended to have the following general meanings as
they are used herein:
1. Complementary: Refers to the topological compatibility or matching
together of interacting surfaces of a ligand molecule and its receptor.
Thus, the receptor and its ligand can be described as complementary, and
furthermore, the contact surface characteristics are complementary to each
other.
2. Epitope: The portion of an antigen molecule which is delineated by the
area of interaction with the subclass of receptors known as antibodies.
3. Ligand: A ligand is a molecule that is recognized by a particular
receptor. Examples of ligands that can be investigated by this invention
include, but are not restricted to, agonists and antagonists for cell
membrane receptors, toxins and venoms, viral epitopes, hormones (e.g.,
steroids, etc.), hormone receptors, peptides, enzymes, enzyme substrates,
cofactors, drugs (e.g., opiates, etc), lectins, sugars, oligonucleotides,
nucleic acids, oligosaccharides, proteins, and monoclonal antibodies.
4. Monomer: A member of the set of small molecules which can be joined
together to form a polymer. The set of monomers includes but is not
restricted to, for example, the set of common L-amino acids, the set of
D-amino acids, the set of synthetic amino acids, the set of nucleotides
and the set of pentoses and hexoses. As used herein, monomers refers to
any member of a basis set for synthesis of a polymer. For example, dimers
of L-amino acids form a basis set of 400 monomers for synthesis of
polypeptides. Different basis sets of monomers may be used at successive
steps in the synthesis of a polymer.
5. Peptide: A polymer in which the monomers are alpha amino acids and which
are joined together through amide bonds and alternatively referred to as a
polypeptide. In the context of this specification it should be appreciated
that the amino acids may be the L-optical isomer or the D-optical isomer.
Peptides are more than two amino acid monomers long, and often more than
20 amino acid monomers long. Standard abbreviations for amino acids are
used (e.g., P for proline). These abbreviations are included in Stryer,
Biochemstry, Third Ed., 1988, which is incorporated herein by reference
for all purposes.
6. Radiation: Energy which may be selectively applied including energy
having a wavelength of between 10.sup.-14 and 10.sup.4 meters including,
for example, electron beam radiation, gamma radiation, x-ray radiation,
ultraviolet radiation, visible light, infrared radiation, microwave
radiation, and radio waves. "Irradiation" refers to the application of
radiation to a surface.
7. Receptor: A molecule that has an affinity for a given ligand. Receptors
may be naturally-occuring or manmade molecules. Also, they can be employed
in their unaltered state or as aggregates with other species. Receptors
may be attached, covalently or noncovalently, to a binding member, either
directly or via a specific binding substance. Examples of receptors which
can be employed by this invention include, but are not restricted to,
antibodies, cell membrane receptors, monoclonal antibodies and antisera
reactive with specific antigenic determinants (such as on viruses, cells
or other materials), drugs, polynucleotides, nucleic acids, peptides,
cofactors, lectins, sugars, polysaccharides, cells, cellular membranes,
and organelles. Receptors are sometimes referred to in the art as
anti-ligands. As the term receptors is used herein, no difference in
meaning is intended. A "Ligand Receptor Pair" is formed when two
macromolecules have combined through molecular recognition to form a
complex.
Other examples of receptors which can be investigated by this invention
include but are not restricted to:
a) Microorganism receptors: Determination of ligands which bind to
receptors, such as specific transport proteins or enzymes essential to
survival of microorganisms, is useful in a new class of antibiotics. Of
particular value would be antibiotics against opportunistic fungi,
protozoa, and those bacteria resistant to the antibiotics in current use.
b) Enzymes: For instance, the binding site of enzymes such as the enzymes
responsible for cleaving neurotransmitters; determination of ligands which
bind to certain receptors to modulate the action of the enzymes which
cleave the different neurotransmitters is useful in the development of
drugs which can be used in the treatment of disorders of
neurotransmission.
c) Antibodies: For instance, the invention may be useful in investigating
the ligand-binding site on the antibody molecule which combines with the
epitope of an antigen of interest; determining a sequence that mimics an
antigenic epitope may lead to the development of vaccines of which the
immunogen is based on one or more of such sequences or lead to the
development of related diagnostic agents or compounds useful in
therapeutic treatments such as for auto immune diseases (e.g., by blocking
the binding of the "self" antibodies).
d) Nucleic Acids: Sequences of nucleic acids may be synthesized to
establish DNA or RNA binding sequences.
e) Catalytic Polypeptides: Polymers, preferably polypeptides, which are
capable of promoting a chemical reaction involving the conversion of one
or more reactants to one or more products. Such polypeptides generally
include a binding site specific for at least one reactant or reaction
intermediate and an active functionality proximate to the binding site,
which functionality is capable of chemically modifying the bound reactant.
Catalytic polypeptides are described in, for example, U.S. application
Ser. No. 404,920, which is incorporated herein by reference for all
purposes.
f) Hormone receptors: For instance, the receptors for insulin and growth
hormone. Determination of the ligands which bind with high affinity to a
receptor is useful in the development of, for example, an oral replacement
of the daily injections which diabetics must take to relieve the symptoms
of diabetes, and in the other case, a replacement for the scarce human
growth hormone which can only be obtained from cadavers or by recombinant
DNA technology. Other examples are the vasoconstrictive hormone receptors;
determination of those ligands which bind to a receptor may lead to the
development of drugs to control blood pressure.
g) Opiate receptors: Determination of ligands which bind to the opiate
receptors in the brain is useful in the development of less-addictive
replacements for morphine and related drugs.
8. Substrate: A material having a rigid or semi-rigid surface. In many
embodiments, at least one surface of the substrate will be substantially
flat, although in some embodiments it may be desirable to physically
separate synthesis regions for different polymers with, for example,
wells, raised regions, etched trenches, or the like. According to other
embodiments, small beads may be provided on the surface which may be
released upon completion of the synthesis.
9. Protective Group: A material which is bound to a monomer unit and which
may be spatially removed upon selective exposure to an activator such as
electromagnetic radiation. Examples of protective groups with utility
herein include Nitroveratryloxy carbonyl, Nitrobenzyloxy carbonyl,
Dimethyl dimethoxybenzyloxy carbonyl, 5-Bromo-7-nitroindolinyl,
o-Hydroxy-.alpha.-methyl cinnamoyl, and 2-Oxymethylene anthraquinone.
Other examples of activators include ion beams, electric fields, magnetic
fields, electron beams, x-ray, and the like.
10. Predefined Region: A predefined region is a localized area on a surface
which is, was, or is intended to be activated for formation of a polymer.
The predefined region may have any convenient shape, e.g., circular,
rectangular, elliptical, wedge-shaped, etc. For the sake of brevity
herein, "predefined regions" are sometimes referred to simply as
"regions."
11. Substantially Pure: A polymer is considered to be "substantially pure"
within a predefined region of a substrate when it exhibits characteristics
that distinguish it from other predefined regions. Typically, purity will
be measured in terms of biological activity or function as a result of
uniform sequence. Such characteristics will typically be measured by way
of binding with a selected ligand or receptor.
II. General
The present invention provides methods and apparatus for the preparation
and use of a substrate having a plurality of polymer sequences in
predefined regions. The invention is described herein primarily with
regard to the preparation of molecules containing sequences of amino
acids, but could readily be applied in the preparation of other polymers.
Such polymers include, for example, both linear and cyclic polymers of
nucleic acids, polysaccharides, phospholipids, and peptides having either
.alpha.-, .beta.-, or .omega.-amino acids, heteropolymers in which a known
drug is covalently bound to any of the above, polyurethanes, polyesters,
polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene
sulfides, polysiloxanes, polyimides, polyacetates, or other polymers which
will be apparent upon review of this disclosure. In a preferred
embodiment, the invention herein is used in the synthesis of peptides.
The prepared substrate may, for example, be used in screening a variety of
polymers as ligands for binding with a receptor, although it will be
apparent that the invention could be used for the synthesis of a receptor
for binding with a ligand. The substrate disclosed herein will have a wide
variety of other uses. Merely by way of example, the invention herein can
be used in determining peptide and nucleic acid sequences which bind to
proteins, finding sequence-specific binding drugs, identifying epitopes
recognized by antibodies, and evaluation of a variety of drugs for
clinical and diagnostic applications, as well as combinations of the
above.
The invention preferably provides for the use of a substrate "S" with a
surface. Linker molecules "L" are optionally provided on a surface of the
substrate. The purpose of the linker molecules, in some embodiments, is to
facilitate receptor recognition of the synthesized polymers.
Optionally, the linker molecules may be chemically protected for storage
purposes. A chemical storage protective group such as t-BOC
(t-butoxycarbonyl) may be used in some embodiments. Such chemical
protective groups would be chemically removed upon exposure to, for
example, acidic solution and would serve to protect the surface during
storage and be removed prior to polymer preparation.
On the substrate or a distal end of the linker molecules, a functional
group with a protective group P.sub.0 is provided. The protective group
P.sub.0 may be removed upon exposure to radiation, electric fields,
electric currents, or other activators to expose the functional group.
In a preferred embodiment, the radiation is ultraviolet (UV), infrared
(IR), or visible light. As more fully described below, the protective
group may alternatively be an electrochemically-sensitive group which may
be removed in the presence of an electric field. In still further
alternative embodiments, ion beams, electron beams, or the like may be
used for deprotection.
In some embodiments, the exposed regions and, therefore, the area upon
which each distinct polymer sequence is synthesized are smaller than about
1 cm.sup.2 or less than 1 mm.sup.2. In preferred embodiments the exposed
area is less than about 10,000 .mu.m.sup.2 or, more preferably, less than
100 .mu.m.sup.2 and may, in some embodiments, encompass the binding site
for as few as a single molecule. Within these regions, each polymer is
preferably synthesized in a substantially pure form.
Concurrently or after exposure of a known region of the substrate to light,
the surface is contacted with a first monomer unit M.sub.1 which reacts
with the functional group which has been exposed by the deprotection step.
The first monomer includes a protective group P.sub.1. P.sub.1 may or may
not be the same as P.sub.0.
Accordingly, after a first cycle, known first regions of the surface may
comprise the sequence:
S-L-M.sub.1 -P.sub.1
while remaining regions of the surface comprise the sequence:
S-L-P.sub.0.
Thereafter, second regions of the surface (which may include the first
region) are exposed to light and contacted with a second monomer M.sub.2
(which may or may not be the same as M.sub.1) having a protective group
P.sub.2. P.sub.2 may or may not be the same as P.sub.0 and P.sub.1. After
this second cycle, different regions of the substrate may comprise one or
more of the following sequences:
S-L-M.sub.1 -M.sub.2 -P.sub.2 S-L-M.sub.2 -P.sub.2 S-L-M.sub.1 -P.sub.1
and/or S-L-P.sub.0.
The above process is repeated until the substrate includes desired polymers
of desired lengths. By controlling the locations of the substrate exposed
to light and the reagents exposed to the substrate following exposure, the
location of each sequence will be known.
Thereafter, the protective groups are removed from some or all of the
substrate and the sequences are, optionally, capped with a capping unit C.
The process results in a substrate having a surface with a plurality of
polymers of the following general formula:
S-[L]-(M.sub.i)-(M.sub.j)-(M.sub.k) . . . (M.sub.x)-[C]
where square brackets indicate optional groups, and M.sub.i . . . M.sub.x
indicates any sequence of monomers. The number of monomers could cover a
wide variety of values, but in a preferred embodiment they will range from
2 to 100.
In some embodiments a plurality of locations on the substrate polymers are
to contain a common monomer subsequence. For example, it may be desired to
synthesize a sequence S-M.sub.1 -M.sub.2 -M.sub.3 at first locations and a
sequence S-M.sub.4 -M.sub.2 -M.sub.3 at second locations. The process
would commence with irradiation of the first locations followed by
contacting with M.sub.1 -P, resulting in the sequence S-M.sub.1 -P at the
first location. The second locations would then be irradiated and
contacted with M.sub.4 -P, resulting in the sequence S-M.sub.4 -P at the
second locations. Thereafter both the first and second locations would be
irradiated and contacted with the dimer M.sub.2 -M.sub.3, resulting in the
sequence S-M.sub.1 -M.sub.2 -M.sub.3 at the first locations and S-M.sub.4
-M.sub.2 -M.sub.3 at the second locations. Of course, common subsequences
of any length could be utilized including those in a range of 2 or more
monomers, 2 to 100 monomers, 2 to 20 monomers, and a most preferred range
of 2 to 3 monomers.
According to other embodiments, a set of masks is used for the first
monomer layer and, thereafter, varied light wavelengths are used for
selective deprotection. For example, in the process discussed above, first
regions are first exposed through a mask and reacted with a first monomer
having a first protective group P.sub.1, which is removable upon exposure
to a first wavelength of light (e.g., IR). Second regions are masked and
reacted with a second monomer having a second protecive group P.sub.2,
which is removable upon exposure to a second wavelength of light (e.g.,
UV). Thereafter, masks become unnecessary in the synthesis because the
entire substrate may be exposed alternatively to the first and second
wavelengths of light in the deprotection cycle.
The polymers prepared on a substrate according to the above methods will
have a variety of uses including, for example, screening for biological
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