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CROSS REFERENCE TO RELATED APPLICATIONS
Application Ser. No. 07/624,120 was a continuation-in-part of application
Ser. No. 492,462, filed Mar. 7, 1990, allowed as U.S. Pat. No. 5,143,854,
which is a continuation-in-part of pending application Ser. No. 362,901,
filed Jun. 7, 1989, abandoned, assigned to the assignee of the present
application, and incorporated herein by reference for all purposes. This
application is related to applications with Ser. Nos. 07,626,730 and
07/642,114, abandoned, respectively filed on the same day as the present
application, and also incorporated herein by reference for all purposes.
Microfiche appendices A and B are attached, including 3 sheets of
microfiche comprising 226 frames.
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 invention relates to the field of polymer synthesis. More
specifically, the invention provides a reactor system, a masking strategy,
photoremovable protective groups, data collection and processing
techniques, and applications for light directed synthesis of diverse
polymer sequences on substrates.
SUMMARY OF THE INVENTION
Methods, apparatus, and compositions for synthesis and use of diverse
polymer sequences on a substrate are disclosed, as well as applications
thereof.
According to one aspect of the invention, an improved reactor system for
synthesis of diverse polymer sequences on a substrate is provided.
According to this embodiment the invention provides for a reactor for
contacting reaction fluids to a substrate; a system for delivering
selected reaction fluids to the reactor; a translation stage for moving a
mask or substrate from at least a first relative location relative to a
second relative location; a light for illuminating the substrate through a
mask at selected times; and an appropriately programmed digital computer
for selectively directing a flow of fluids from the reactor system,
selectively activating the translation stage, and selectively illuminating
the substrate so as to form a plurality of diverse polymer sequences on
the substrate at predetermined locations.
The invention also provides a technique for selection of linker molecules
in a very large scale immobilized polymer synthesis VLSIPS.TM.method.
According to this aspect of the invention, the invention provides a method
of screening a plurality of linker polymers for use in binding affinity
studies. The invention includes the steps of forming a plurality of linker
polymers on a substrate in selected regions, the linker polymers formed by
the steps of recursively: on a surface of a substrate, irradiating a
portion of the selected regions to remove a protective group, and
contacting the surface with a monomer; contacting the plurality of linker
polymers with a ligand; and contacting the ligand with a labeled receptor.
According to another aspect of the invention, improved photoremovable
protective groups are provided. According to this aspect of the invention
a compound having the formula:
##STR1##
wherein n=0 or 1; Y is selected from the group consisting of an oxygen of
the carboxyl group of a natural or unnatural amino acid, an amino group of
a natural or unnatural amino acid, or the C--5' oxygen group of a natural
or unnatural deoxyribonucleic or ribonucleic acid; R.sup.1 and R.sup.2
independently are a hydrogen atom, a lower alkyl, aryl, benzyl, halogen,
hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate,
formamido, sulfido, or phosphido group; and R.sup.3 is a alkoxy, alkyl,
aryl, hydrogen, or alkenyl group is provided.
The invention also provides improved masking techniques for the VLSIPS.TM.
methodology. According to one aspect of the masking technique, the
invention provides an ordered method for forming a plurality of polymer
sequences by sequential addition of reagents comprising the step of
serially protecting and deprotecting portions of the plurality of polymer
sequences for addition of other portions of the polymer sequences using a
binary synthesis strategy.
Improved data collection equipment and techniques are also provided.
According to one embodiment, the instrumentation provides a system for
determining affinity of a receptor to a ligand comprising: means for
applying light to a surface of a substrate, the substrate comprising a
plurality of ligands at predetermined locations, the means for providing
simultaneous illumination at a plurality of the predetermined locations;
and an array of detectors for detecting light fluoresced at the plurality
of predetermined locations. The invention further provides for improved
data analysis techniques including the steps of exposing fluorescently
labelled receptors to a substrate, the substrate comprising a plurality of
ligands in regions at known locations; at a plurality of data collection
points within each of the regions, determining an amount of light
fluoresced from the data collection points; removing the data collection
points deviating from a predetermined statistical distribution; and
determining a relative binding affinity of the receptor to remaining data
collection points.
Protected amino acid N-carboxy anhydrides for use in polymer synthesis are
also disclosed. According to this aspect, the invention provides a
compound having the formula:
##STR2##
where R is a side chain of a natural or unnatural amino acid and X is a
photoremovable protecting group.
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 DRAWINGS
FIG. 1 schematically illustrates light-directed spatially-addressable
parallel chemical synthesis;
FIG. 2 schematically illustrates one example of light-directed peptide
synthesis;
FIG. 3 is a three-dimensional representation of a portion of the
checkerboard array of YGGFL and PGGFL;
FIG. 4 schematically illustrates an automated system for synthesizing
diverse polymer sequences;
FIG. 5a and 5b illustrate operation of a program for polymer sythesis;
FIG. 6 is a schematic illustration of a "pure" binary masking strategy;
FIG. 7 is a schematic illustration of a gray code binary masking strategy;
FIG. 8 is a schematic illustration of a modified gray code binary masking
strategy;
FIG. 9a schematically illustrates a masking scheme for a four step
synthesis;
FIG. 9b schematically illustrates synthesis of all 400 peptide dimers;
FIG. 10 is a coordinate map for the ten-step binary synthesis;
FIG. 11 schematically illustrates a data collection system;
FIG. 12 is a block diagram illustrating the architecture of the data
collection system;
FIG. 13 is a flow chart illustrating operation of software for the data
collection/analysis system; and
FIG. 14 illustrates a three-dimensional plot of intensity versus position
for light directed synthesis of a dinucleotide.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
CONTENTS
I. Definitions
II. General
A. Deprotection and Addition
1. Example
2. Example
B. Antibody recognition
1. Example
III. Synthesis
A. Reactor System
B. Binary Synthesis Strategy
1. Example
2. Example
3. Example
4. Example
5. Example
6. Example
C. Linker Selection
D. Protecting Groups
1. Use of Photoremovable Groups During Solid-Phase Synthesis of Peptides
2. Use of Photoremovable Groups During Solid-Phase Synthesis of
Oligonucleotides
E. Amino Acid N-Carboxy Anhydrides Protected with a Photoremovable Group
IV. Data Collection
A. Data Collection System
B. Data Analysis
V. Other Representative Applications
A. Oligonucleotide Synthesis
1. Example
VI. Conclusion
I. Definitions
Certain terms used herein are intended to have the following general
definitions:
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 hormone
receptors, peptides, enzymes, enzyme substrates, cofactors, drugs (e.g.,
opiates, steroids, 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 the 20 naturally occurring 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.
Furthermore, each of the sets may include protected members which are
modified after synthesis.
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 often two or more 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, Biochemistry, 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,
ultra-violet 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-occurring 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 developing 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 one type of receptor includes, 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. Pat. No.
4,215,899, which is incorporated herein by reference for all purposes.
f) Hormone receptors: Examples of hormone receptors include, e.g., 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 chemically bound to a monomer unit
and which may be removed upon selective exposure to an activator such as
electromagnetic radiation. Examples of protective groups with utility
herein include those comprising nitropiperonyl, pyrenylmethoxy-carbonyl,
nitroveratryl, nitrobenzyl, dimethyl dimethoxybenzyl,
5-bromo-7-nitroindolinyl, .omicron.-hydroxy-.alpha.-methyl cinnamoyl, and
2-oxymethylene anthraquinone.
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, wedgeshaped, 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.
12. Activator refers to an energy source adapted to render a group active
and which is directed from a source to a predefined location on a
substrate. A primary illustration of an activator is light. Other examples
of activators include ion beams, electric fields, magnetic fields,
electron beams, x-ray, and the like.
13. Binary Synthesis Strategy refers to an ordered strategy for parallel
synthesis of diverse polymer sequences by sequential addition of reagents
which may be represented by a reactant matrix, and a switch matrix, the
product of which is a product matrix. A reactant matrix is a 1.times.n
matrix of the building blocks to be added. The elements of the switch
matrix are binary numbers. In preferred embodiments, a binary strategy is
one in which at least two successive steps illuminate half of a region of
interest on the substrate. In most preferred embodiments, binary synthesis
refers to a synthesis strategy which also factors a previous addition
step. For example, a strategy in which a switch matrix for a masking
strategy halves regions that were previously illuminated, illuminating
about half of the previously illuminated region and protecting the
remaining half (while also protecting about half of previously protected
regions and illuminating about half of previously protected regions). It
will be recognized that binary rounds may be interspersed with non-binary
rounds and that only a portion of a substrate may be subjected to a binary
scheme, but will still be considered to be a binary masking scheme within
the definition herein. A binary "masking" strategy is a binary synthesis
which uses light to remove protective groups from materials for addition
of other materials such as amino acids. In preferred embodiments, selected
columns of the switch matrix are arranged in order of increasing binary
numbers in the columns of the switch matrix.
14. Linker refers to a molecule or group of molecules attached to a
substrate and spacing a synthesized polymer from the substrate for
exposure/binding to a receptor.
II. General
The present invention provides synthetic strategies and devices for the
creation of large scale chemical diversity. Solid-phase chemistry,
photolabile protecting groups, and photolithography are brought together
to achieve light-directed spatially-addressable parallel chemical
synthesis in preferred embodiments.
The invention is described herein for purposes of illustration primarily
with regard to the preparation of peptides and nucleotides, 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, hetero-polymers 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. It will be recognized
further, that illustrations herein are primarily with reference to C- to
N-terminal synthesis, but the invention could readily be applied to N- to
C-terminal synthesis without departing from the scope of the invention.
A. Deprotection and Addition
The present invention uses a masked light source or other activator to
direct the simultaneous synthesis of many different chemical compounds.
FIG. 1 is a flow chart illustrating the process of forming chemical
compounds according to one embodiment of the invention. Synthesis occurs
on a solid support 2. A pattern of illumination through a mask 4a using a
light source 6 determines which regions of the support are activated for
chemical coupling. In one preferred embodiment activation is accomplished
by using light to remove photolabile protecting groups from selected areas
of the substrate.
After deprotection, a first of a set of building blocks (indicated by "A"
in FIG. 1), each bearing a photolabile protecting group (indicated by "X")
is exposed to the surface of the substrate and it reacts with regions that
were addressed by light in the preceding step. The substrate is then
illuminated through a second mask 4b, which activates another region for
reaction with a second protected building block "B". The pattern of masks
used in these illuminations and the sequence of reactants define the
ultimate products and their locations, resulting in diverse sequences at
predefined locations, as shown with the sequences ACEG and BDFH in the
lower portion of FIG. 1. Preferred embodiments of the invention take
advantage of combinatorial masking strategies to form a large number of
compounds in a small number of chemical steps.
A high degree of miniaturization is possible because the density of
compounds is determined largely with regard to spatial addressability of
the activator, in one case the diffraction of light. Each compound is
physically accessible and its position is precisely known. Hence, the
array is spatially-addressable and its interactions with other molecules
can be assessed.
In a particular embodiment shown in FIG. 1, the substrate contains amino
groups that are blocked with a photolabile protecting group. Amino acid
sequences are made accessible for coupling to a receptor by removal of the
photoprotective groups.
When a polymer sequence to be synthesized is, for example, a polypeptide,
amino groups at the ends of linkers attached to a glass substrate are
derivatized with nitroveratryloxycarbonyl (NVOC), a photoremovable
protecting group. The linker molecules may be, for example, aryl
acetylene, ethylene glycol oligomers containing from 2-10 monomers,
diamines, diacids, amino acids, or combinations thereof. Photodeprotection
is effected by illumination of the substrate through, for example, a mask
wherein the pattern has transparent regions with dimensions of, for
example, less than 1 cm.sup.2, 10.sup.-1 cm.sup.2, 10.sup.-2 cm.sup.2,
10.sup.-3 cm.sup.2, 10.sup.-4 cm.sup.2, 10.sup.-5 cm.sup.2, 10.sup.-6
cm.sup.2, 10.sup.-7 cm.sup.2, 10.sup.-8 cm.sup.2, or 10.sup.-10 cm.sup.2.
In a preferred embodiment, the regions are between about 10.times.10 .mu.m
and 500.times.500 .mu.m. According to some embodiments, the masks are
arranged to produce a checkerboard array of polymers, although any one of
a variety of geometric configurations may be utilized.
1. Example
In one example of the invention, free amino groups were fluorescently
labelled by treatment of the entire substrate surface with fluorescein
isothiocynate (FITC) after photodeprotection. Glass microscope slides were
cleaned, aminated by treatment with 0.1% aminopropyltriethoxysilane in 95%
ethanol, and incubated at 110.degree. C. for 20 min. The aminated surface
of the slide was then exposed to a 30 mM solution of the
N-hydroxysuccinimide ester of NVOC-GABA
(nitroveratryloxycarbonyl-.tau.-amino butyric acid) in DMF. The NVOC
protecting group was photolytically removed by imaging the 365 nm output
from a Hg arc lamp through a chrome on glass 100 .mu.m checkerboard mask
onto the substrate for 20 min at a power density of 12 mW/cm.sup.2. The
exposed surface was then treated with 1 mM FITC in DMF. The substrate
surface was scanned in an epi-fluorescence microscope (Zeiss Axioskop 20)
using 488 nm excitation from an argon ion laser (Spectra-Physics model
2025). The fluorescence emission above 520 nm was detected by a cooled
photomultiplier (Hamamatsu 943-02) operated in a photon counting mode.
Fluorescence intensity was translated into a color display with red in the
highest intensity and black in the lowest intensity areas. The presence of
a high-contrast fluorescent checkerboard pattern of 100.times.100 .mu.m
elements revealed that free amino groups were generated in specific
regions by spatially-localized photodeprotection.
2. Example
FIG. 2 is a flow chart illustrating another example of the invention.
Carboxy-activated NVOC-leucine was allowed to react with an aminated
substrate. The carboxy activated HOBT ester of leucine and other amino
acids used in this synthesis was formed by mixing 0.25 mmol of the NVOC
amino protected amino acid with 37 mg HOBT (1-hydroxybenzotriazole), 111
mg BOP (benzotriazolyl-n-oxy-tris
(dimethylamino)-phosphoniumhexa-fluorophosphate) and 86 .mu.l DIEA
(diisopropylethylamine) in 2.5 ml DMF. The NVOC protecting group was
removed by uniform illumination. Carboxy-activated NVOC-phenylalanine was
coupled to the exposed amino groups for 2 hours at room temperature, and
then washed with DMF and methylene chloride. Two unmasked cycles of
photodeprotection and coupling with carboxy-activated NVOC-glycine were
carried out. The surface was then illuminated through a chrome on glass 50
.mu.m checkerboard pattern mask. Carboxy-activated
N.alpha.-tBOC-O-tButyl-L-tyrosine was then added. The entire surface was
uniformly illuminated to photolyze the remaining NVOC groups. Finally,
carboxy-activated NVOC-L-proline was added, the NVOC group was removed by
illumination, and the t-BOC and t-butyl protecting groups were removed
with TFA. After removal of the protecting groups, the surface consisted of
a 50 .mu.m checkerboard array of Tyr-Gly-Gly-Phe-Leu (YGGFL) (SEQ ID NO:
1) and Pro-Gly-Gly-Phe-Leu (PGGFL) (SEQ ID NO: 2).
B. Antibody Recognition
In one preferred embodiment the substrate is used to determine which of a
plurality of amino acid sequences is recognized by an antibody of
interest.
1. Example
In one example, the array of pentapeptides in the example illustrated in
FIG. 2 was probed with a mouse monoclonal antibody directed against
.beta.-endorphin. This antibody (called 3E7) is known to bind YGGFL and
YGGFM (SEQ ID NO: 21) with nanomolar affinity and is discussed in Meo et
al., Proc. Natl. Acad. Sci. USA (1983) 80:4084, which is incorporated by
reference herein for all purposes. This antibody requires the amino
terminal tyrosine for high affinity binding. The array of peptides formed
as described in FIG. 2 was incubated with a 2 .mu.g/ml mouse monoclonal
antibody (3E7) known to recognize YGGFL. 3E7 does not bind PGGFL. A second
incubation with fluoresceinated goat anti-mouse antibody labeled the
regions that bound 3E7. The surface was scanned with an epi-fluorescence
microscope. The results showed alternating bright and dark 50 .mu.m
squares indicating that YGGFL and PGGFL were synthesized in geometric
array determined by the mask. A high contrast (>12:1 intensity ratio)
fluorescence checkerboard image shows that (a) YGGFL and PGGFL were
synthesized in alternate 50 .mu.m squares, (b) YGGFL attached to the
surface is accessible for binding to antibody 3E7, and (c) antibody 3E7
does not bind to PGGFL.
A three-dimensional representation of the fluorescence intensity data in a
portion of the checkboard is shown in FIG. 3. This figure shows that the
border between synthesis sites is sharp. The height of each spike in this
display is linearly proportional to the integrated fluorescence intensity
in a 2.5 .mu.m pixel. The transition between PGGFL and YGGFL occurs within
two spikes (5 .mu.m). There is little variation in the fluorescence
intensity of different YGGFL squares. The mean intensity of sixteen YGGFL
synthesis sites was 2.03.times.10.sup.5 counts and the standard deviation
was 9.6.times.10.sup.3 counts.
III. Synthesis
A. Reactor System
FIG. 4 schematically illustrates a device used to synthesize diverse
polymer sequences on a substrate. The device includes an automated peptide
synthesizer 401. The automated peptide synthesizer is a device which flows
selected reagents through a flow cell 402 under the direction of a
computer 404. In a preferred embodiment the synthesizer is an ABI Peptide
Synthesizer, model no. 431A. The computer may be selected from a wide
variety of computers or discrete logic including for, example, an IBM
PC-AT or similar computer linked with appropriate internal control systems
in the peptide synthesizer. The PC is provided with signals from the board
computer indicative of, for example, the end of a coupling cycle.
Substrate 406 is mounted on the flow cell, forming a cavity between the
substrate and the flow cell. Selected reagents flow through this cavity
from the peptide synthesizer at selected times, forming an array of
peptides on the face of the substrate in the cavity. Mounted above the
substrate, and preferably in contact with the substrate is a mask 408.
Mask 408 is transparent in selected regions to a selected wavelength of
light and is opaque in other regions to the selected wavelength of light.
The mask is illuminated with a light source 410 such as a UV light source.
In one specific embodiment the light source 410 is a model no. 82420 made
by Oriel. The mask is held and translated by an x-y-z translation stage
412 such as an x-y translation stage made by Newport Corp. The computer
coordinates action of the peptide synthesizer, x-y translation stage, and
light source. Of course, the invention may be used in some embodiments
with translation of the substrate instead of the mask.
In operation, the substrate is mounted on the reactor cavity. The slide,
with its surface protected by a suitable photo removable protective group,
is exposed to light at selected locations by positioning the mask and
illuminating the light source for a desired period of time (such as, for
example, 1 sec to 60 min in the case of peptide synthesis). A selected
peptide or other monomer/polymer is pumped through the reactor cavity by
the peptide synthesizer for binding at the selected locations on the
substrate. After a selected reaction time (such as about 1 sec to 300 min
in the case of peptide reactions) the monomer is washed from the system,
the mask is appropriately repositioned or replaced, and the cycle is
repeated. In most embodiments of the invention, reactions may be conducted
at or near ambient temperature.
FIGS. 5a and 5b are flow charts of the software used in operation of the
reactor system. At step 502 the peptide synthesis software is initialized.
At step 504 the system calibrates positioners on the x-y translation stage
and begins a main loop. At step 506 the system determines which, if any,
of the function keys on the computer have been pressed. If F1 has been
pressed, the system prompts the user for input of a desired synthesis
process. If the user enters F2, the system allows a user to edit a file
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