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BACKGROUND OF THE INVENTION
The present invention relates to the field of solid phase polymer synthesis. More specifically, the invention provides methods and reagents for solid phase synthesis of oligomer arrays which may be used, for example, in screening studies for
determination of binding affinity.
The synthesis of biological polymers such as peptides and oligonucleotides has been evolving in dramatic fashion from the earliest stages of solution synthesis to solid phase synthesis of a single polymer to the more recent preparations of
libraries having large numbers of diverse oligonucleotide sequences on a single solid support or chip.
The evolution of solid phase synthesis of biological polymers began with the early "Merrifield" solid phase peptide synthesis, described in Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963), incorporated herein by reference for all purposes.
Solid-phase synthesis techniques have also been provided for the synthesis of several peptide sequences on, for example, a number of "pins." See e.g., Geysen et al., J. Immun. Meth. 102:259-274 (1987), incorporated herein by reference for all purposes. Other solid-phase techniques involve, for example, synthesis of various peptide sequences on different cellulose disks supported in a column. See Frank and Doting, Tetrahedron 44:6031-6040 (1988), incorporated herein by reference for all purposes.
Still other solid-phase techniques are described in U.S. Pat. No. 4,728,502 issued to Hamill and WO 90/00626 (Beattie, inventor).
Each of the above techniques produces only a relatively low density array of polymers. For example, the technique described in Geysen et al. is limited to producing 96 different polymers on pins spaced in the dimensions of a standard microtiter
plate.
Improved methods of forming large arrays of oligonucleotides, peptides and other polymer sequences in a short period of time have been devised. Of particular note, Pirrung et al., U.S. Pat. No. 5,143,854 (see also PCT Application No. WO
90/15070) and Fodor et al., PCT Publication No. WO 92/10092, all incorporated herein by reference, disclose methods of forming vast arrays of peptides, oligonucleotides and other polymer sequences using, for example, light-directed synthesis techniques.
See also, Fodor et al., Science, 251:767-777 (1991), also incorporated herein by reference for all purposes. These procedures are now referred to as VLSIPS.TM. procedures.
In the above-referenced Fodor et al., PCT application, an elegant method is described for using a computer-controlled system to direct a VLSIPS.TM. procedure. Using this approach, one heterogenous array of polymers is converted, through
simultaneous coupling at a number of reaction sites, into a different heterogenous array. See U.S. Pat. No. 5,384,261 and application Ser. No. 07/980,523, now abandoned, the disclosures of which are incorporated herein for all purposes.
The development of VLSIPS.TM. technology as described in the above-noted U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and 92/10092, is considered pioneering technology in the fields of combinatorial synthesis and
screening of combinatorial libraries. More recently, patent application Ser. No. 08/082,937, filed Jun. 25, 1993, describes methods for making arrays of oligonucleotide probes that can be used to provide a partial or complete sequence of a target
nucleic acid and to detect the presence of a nucleic acid containing a specific oligonucleotide sequence.
SUMMARY OF THE INVENTION
The present invention provides new compounds, compositions and methods which find application in solid phase synthesis including the preparation of high-density arrays of diverse polymer sequences such as diverse peptides and oligonucleotides as
well as in preparation of arrays of small ligand molecules. The compounds of the present invention are those which are typically referred to as linking groups, linkers or spacers.
According to a first aspect of the invention, novel compounds are provided which are useful as linking groups in solid phase polymer synthesis. Additionally, these compounds when used in the solid phase preparation of peptides and
oligonucleotides provide improved presentation of the polymers in subsequent assays and diagnostic applications. In one group of embodiments, the compounds are polyether derivatives which vary not only in their hydrophilicities, but also provide varying
degrees of solvation-flexibility-"bindability". In another group of embodiments, the compounds are useful as linking groups which are photochemically cleavable.
According to another aspect of the invention, improvements to the coupling chemistry used in the light-directed methods of the VLSIPS.TM. process are provided. In one embodiment, additional reagents are provided which provide increased yields
of the desired products.
According to yet another aspect of the invention, methods are provided for determining the fidelity of polymer synthesis which occurs on a solid support.
A farther 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 is a bar graph illustration of streptavidin binding to a biotin moiety which is tethered to a surface via multiple 15-ATOM-PEG linking groups.
FIGS. 2A-2D illustrate the results achieved by removal of two thiazolidinones from a resin via photolysis. The thiazolidinones were synthesized on a resin having a photocleavable linking group. FIGS. 2A and 2B show the reactions which produce
the two thiazolidinones. FIGS. 2C and 2D show the HPLC chromatograms of the resulting thiazolidinones and illustrate the purity of each.
DETAILED DESCRIPTION OF THE INVENTION
CONTENTS
I. Glossary
II. General
III. Novel Linking Groups (Hydrophilic and Photochemical)
IV. Improvements to Solid Phase Coupling Sequences
V. Assays for Determination of Synthesis Fidelity
VI. Examples
VII. Conclusion
I. Glossary
The following abbreviations are used herein: AcOH, acetic acid; ALLOC, allyloxycarbonyl; BOC, t-butoxycarbonyl; BOP, benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate; CAP, .epsilon.-aminocaproic acid; CHC,
4-aminomethylcyclohexane carboxylic acid; DDZ, 3,5-dimethoxy-.alpha..alpha.-dimethylbenzyl; DIEA diisopropylethylamine; DIGLY, glycylglycine; DMF, dimethylformamide; DMT, dimethoxytrityl; DTT, dithiothreitol; EtOAc, ethyl acetate; FMOC,
fluorenylmethoxycarbonyl; MeNPOC, .alpha.-methylnitro-piperonyloxycarbonyl; MeNVOC, .alpha.-methylnitroveratryloxycarbonyl; MP, melting point; NVOC, nitroveratryloxycarbonyl; OBt, hydroxybenzotriazole radical; PBS, phosphate buffered saline; TFA,
trifluoroacetic acid; TRIGLY, glycylglycylglycine; UND, .omega.-aminoundecanoic acid. The abbreviations 15-, 19-, 20- and 24-ATOM-PEG are used to refer to the amino acids (in unprotected form) which are shown in Table I.
The following terms are intended to have the following general meanings as they are used herein:
Chemical Terms
As used herein, the term "alkyl" refers to a saturated hydrocarbon radical which may be straight-chain or branched-chain (for example, ethyl, isopropyl, t-amyl, or 2,5-dimethylhexyl). When "alkyl" or "alkylene" is used to refer to a linking
group or a spacer, it is taken to be a group having two available valences for covalent attachment, for example, --CH.sub.2 CH.sub.2 --, --CH.sub.2 CH.sub.2 CH.sub.2 --, --CH.sub.2 CH.sub.2 CH(CH.sub.3)CH.sub.2 -- and --CH.sub.2 (CH.sub.2 CH.sub.2).sub.2
CH.sub.2 --. Preferred alkyl groups as substituents are those containing 1 to 10 carbon atoms, with those containing 1 to 6 carbon atoms being particularly preferred. Preferred alkyl or alkylene groups as linking groups are those containing 1 to 20
carbon atoms, with those containing 3 to 6 carbon atoms being particularly preferred. The term "polyethylene glycol" is used to refer to those molecules which have repeating units of ethylene glycol, for example, hexaethylene glycol (HO--(CH.sub.2
CH.sub.2 O).sub.5 --CH.sub.2 CH.sub.2 OH). When the term "polyethylene glycol" is used to refer to linking groups and spacer groups, it would be understood by one of skill in the art that other polyethers or polyols could be used as well (i.e,
polypropylene glycol or mixtures of ethylene and propylene glycols).
The term "aryl" as used herein, refers to an aromatic substituent which may be a single ring or multiple rings which are fused together, linked covalently or linked to a common group such as an ethylene or methylene moiety. The aromatic rings
may each contain heteroatoms, for example, phenyl, naphthyl, biphenyl, diphenylmethyl, 2,2-diphenyl-1-ethyl, thienyl, pyridyl and quinoxalyl. The aryl moieties may also be optionally substituted with halogen atoms, or other groups such as nitro,
carboxyl, alkoxy, phenoxy and the like. Additionally, the aryl radicals may be attached to other moieties at any position on the aryl radical which would otherwise be occupied by a hydrogen atom (such as, for example, 2-pyridyl, 3-pyridyl and
4-pyridyl). As used herein, the term "aralkyl" refers to an alkyl group bearing an aryl substituent (for example, benzyl, phenylethyl, 3-(4-nitrophenyl)propyl, and the like).
The term "protecting group" as used herein, refers to any of the groups which are designed to block one reactive site in a molecule while a chemical reaction is carded out at another reactive site. More particularly, the protecting groups used
herein can be any of those groups described in Greene, et al., Protective Groups In Organic Chemistry, 2nd Ed., John Wiley & Sons, New York, N.Y., 1991, incorporated herein by reference. The proper selection of protecting groups for a particular
synthesis will be governed by the overall methods employed in the synthesis. For example, in "light-directed" synthesis, discussed below, the protecting groups will be photolabile protecting groups such as dimethoxybenzoin, NVOC, MeNPOC, and those
disclosed in co-pending Application PCT/15S93/10162 (filed Oct. 22, 1993), incorporated herein by reference. In other methods, protecting groups may be removed by chemical methods and include groups such as FMOC, DMT and others known to those of skill
in the art.
The term "protected amino acid" refers to an amino acid, typically an .alpha.-amino acid having either or both the amine functionality and the carboxylic acid functionality suitably protected by one of the groups described above. Additionally,
for those amino acids having reactive sites or functional groups on a side chain (i.e., serine, tyrosine, glutamic acid), the term "protected amino acid" is meant to refer to those compounds which optionally have the side chain functionality protected as
well.
The term "activating agent" refers to those groups which, when attached to a particular functional group or reactive site, render that site more reactive toward covalent bond formation with a second functional group or reactive site. For
example, the group of activating groups which are useful for a carboxylic acid include simple ester groups and anhydrides. The ester groups include alkyl, aryl and alkenyl esters and in particular such groups as 4-nitrophenyl, N-hydroxylsuccinimide and
pentafluorophenol. Other activating agents are known to those of skill in the art.
Ligand
A ligand is a molecule that is recognized by a receptor. Examples of ligands which can be synthesized using the methods and compounds of this invention include, but are not restricted to, agonists and antagonists for cell membrane receptors,
toxins and venoms, viral epitopes, hormones, opiates, steroids, peptides, enzyme substrates, cofactors, drugs, lectins, sugars, oligonucleotides, nucleic acids, oligosaccharides, and proteins.
Monomer
A monomer is a member of the set of small molecules which are or can be joined together to form a polymer or a compound composed of two or more members. 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 and/or natural amino acids, the set of nucleotides and the set of pentoses and hexoses. The particular ordering of monomers within a polymer is referred to herein as the "sequence" of the
polymer. 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. The invention is described herein primarily with regard to the preparation of
molecules containing sequences of monomers such as 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, polynucleotides, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines,
polyarylene sulfides, polysiloxanes, polyirnides, polyacetates, or other polymers which will be apparent upon review of this disclosure. Such polymers are "diverse" when polymers having different monomer sequences are formed at different predefined
regions of a substrate. Methods of cyclization and polymer reversal of polymers are disclosed in U.S. Pat. No. 5,550,215 which is a CIP of U.S. Pat. No. 5,242,974 entitled "POLYMER REVERSAL ON SOLID SURFACES," incorporated herein by reference for
all purposes.
Peptide
A peptide is a polymer in which the monomers are amino acids and are joined together through amide bonds, alternatively referred to as a polypeptide. When the amino acids are .alpha.-amino acids, either the L-optical isomer or the D-optical
isomer may be used. Additionally, unnatural amino acids, for example, .beta.-alanine, phenylglycine and homoarginine are also meant to be included. Peptides are two or more amino acid monomers long and are 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.
Receptor
A receptor is a molecule that has an affinity for a ligand. Receptors may be naturally-occurring or manmade molecules. 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, viruses, cells, drugs, polynucleotides, nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides, 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 molecules have combined through molecular recognition to form a complex.
Specific examples of receptors which can be investigated using ligands and libraries prepared using the methods and compounds of this invention include but are not restricted to:
a) Microorganism receptors: Determination of ligands that bind to microorganism receptors such as specific transport proteins or enzymes essential to survival of microorganisms would be a useful tool for discovering new classes of antibiotics.
Of particular value would be antibiotics against opportunistic fungi, protozoa, and bacteria resistant to antibiotics in current use.
b) Enzymes: For instance, a receptor can comprise a binding site of an enzyme such as an enzyme responsible for cleaving a neurotransmitter; determination of ligands for this type of receptor to modulate the action of an enzyme that cleaves a
neurotransmitter is useful in developing drugs that can be used in the treatment of disorders of neurotransmission.
c) Antibodies: For instance, the invention may be useful in investigating a receptor that comprises a ligand-binding site on an antibody molecule which combines with an epitope of an antigen of interest; determining a sequence that mimics an
antigenic epitope may lead to the development of vaccines in 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 autoimmune
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 that act as receptors for synthesized sequence.
e) Catalytic Polypeptides: Polymers, preferably antibodies, 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 and others are described in, for example, PCT
Publication No. WO 90/05746, WO 90/05749, and WO 90/05785, which are incorporated herein by reference for all purposes.
f) Hormone receptors: Determination of the ligands which bind with high affinity to a receptor such as the receptors for insulin and growth hormone 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 or a replacement for growth hormone. Other examples of hormone receptors include the vasoconstrictive hormone receptors; determination of ligands for these receptors 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.
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. In some embodiments, the substrate itself contains wells, trenches, flow through regions, etc. which form all or part of the synthesis regions. According to other
embodiments, small beads may be provided on the surface, and compounds synthesized thereon may be released upon completion of the synthesis.
Channel Block
A material having a plurality of grooves or recessed regions on a surface thereof. The grooves or recessed regions may take on a variety of geometric configurations, including but not limited to stripes, circles, serpentine paths, or the like.
Channel blocks may be prepared in a variety of manners, including etching silicon blocks, molding or pressing polymers, etc.
Predefined Region
A predefined region is a localized area on a substrate which is, was, or is intended to be used for formation of a selected polymer and is otherwise referred to herein in the alternative as "reaction" region, a "selected" region, or simply a
"region." The predefined region may have any convenient shape, e.g., circular, rectangular, elliptical, wedge-shaped, etc. In some embodiments, a predefined region and, therefore, the area upon which each distinct polymer sequence is synthesized is
smaller than about 1 cm.sup.2, more preferably less than 1 mm.sup.2, and still more preferably less than 0.5 mm.sup.2. In most preferred embodiments the regions have an area less than about 10,000 .mu.m.sup.2 or, more preferably, less than
100.mu.m.sup.2. Within these regions, the polymer synthesized therein is preferably synthesized in a substantially pure form. Additionally, multiple copies of the polymer will typically be synthesized within any preselected region. The number of
copies can be in the thousands to the millions.
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. Preferably the region is sufficiently pure such that the predominant species in the predefined region is
the desired sequence. According to preferred aspects of the invention, the polymer is at least 5% pure, more preferably more than 10% to 20% pure, more preferably more than 80% to 90% pure, and most preferably more than 95% pure, where purity for this
purpose refers to the ratio of the number of ligand molecules formed in a predefined region having a desired sequence to the total number of molecules formed in the predefined region.
II. General
The compounds, compositions and methods of the present invention can be used in a number of solid phase synthesis applications, including light-directed methods, flow channel and spotting methods, pin-based methods and bead-based methods.
Light-Directed Methods
"Light-directed" methods (which are one technique in a family of methods known as VLSIPS.TM. methods) are described in U.S. Pat. No. 5,143,854, previously incorporated by reference. The light directed methods discussed in the '854 patent
involve activating predefined regions of a substrate or solid support and then contacting the substrate with a preselected monomer solution. The predefined regions can be activated with a light source, typically shown through a mask (much in the manner
of photolithography techniques used in integrated circuit fabrication). Other regions of the substrate remain inactive because they are blocked by the mask from illumination and remain chemically protected. Thus, a light pattern defines which regions
of the substrate react with a given monomer. By repeatedly activating different sets of predefined regions and contacting different monomer solutions with the substrate, a diverse army of polymers is produced on the substrate. Of course, other steps
such as washing unreacted monomer solution from the substrate can be used as necessary.
Flow Channel or Spotting Methods
Additional methods applicable to library synthesis on a single substrate are described in co-pending application Ser. Nos. 07/980,523, filed Nov. 20, 1992, and 07/796,243, filed Nov. 22, 1991, incorporated herein by reference for all
purposes. In the methods disclosed in these applications, reagents are delivered to the substrate by either (1) flowing within a channel defined on predefined regions or (2) "spotting" on predefined regions. However, other approaches, as well as
combinations of spotting and flowing, may be employed. In each instance, certain activated regions of the substrate are mechanically separated from other regions when the monomer solutions are delivered to the various reaction sites.
A typical "flow channel" method applied to the compounds and libraries of the present invention can generally be described as follows. Diverse polymer sequences are synthesized at selected regions of a substrate or solid support by forming flow
channels on a surface of the substrate through which appropriate reagents flow or in which appropriate reagents are placed. For example, assume a monomer "A" is to be bound to the substrate in a first group of selected regions. If necessary, all or
part of the surface of the substrate in all or a part of the selected regions is activated for binding by, for example, flowing appropriate reagents through all or some of the channels, or by washing the entire substrate with appropriate reagents. After
placement of a channel block on the surface of the substrate, a reagent having the monomer A flows through or is placed in all or some of the channel(s). The channels provide fluid contact to the first selected regions, thereby binding the monomer A on
the substrate directly or indirectly (via a spacer) in the first selected regions.
Thereafter, a monomer B is coupled to second selected regions, some of which may be included among the first selected regions. The second selected regions will be in fluid contact with a second flow channel(s) through translation, rotation, or
replacement of the channel block on the surface of the substrate; through opening or closing a selected valve; or through deposition of a layer of chemical or photoresist. If necessary, a step is performed for activating at least the second regions.
Thereafter, the monomer B is flowed through or placed in the second flow channel(s), binding monomer B at the second selected locations. In this particular example, the resulting sequences bound to the substrate at this stage of processing will be, for
example, A, B, and AB. The process is repeated to form a vast array of sequences of desired length at known locations on the substrate.
After the substrate is activated, monomer A can be flowed through some of the channels, monomer B can be flowed through other channels, a monomer C can be flowed through still other channels, etc. In this manner, many or all of the reaction
regions are reacted with a monomer before the channel block must be moved or the substrate must be washed and/or reactivated. By making use of many or all of the available reaction regions simultaneously, the number of washing and activation steps can
be minimized.
One of skill in the art will recognize that there are alternative methods of forming channels or otherwise protecting a portion of the surface of the substrate. For example, according to some embodiments, a protective coating such as a
hydrophilic or hydrophobic coating (depending upon the nature of the solvent) is utilized over portions of the substrate to be protected, sometimes in combination with materials that facilitate wetting by the reactant solution in other regions. In this
manner, the flowing solutions are further prevented from passing outside of their designated flow paths.
The "spotting" methods of preparing compounds and libraries of the present invention can be implemented in much the same manner as the flow channel methods. For example, a monomer A can be delivered to and coupled with a first group of reaction
regions which have been appropriately activated. Thereafter, a monomer B can be delivered to and reacted with a second group of activated reaction regions. Unlike the flow channel embodiments described above, reactants are delivered by directly
depositing (rather than flowing) relatively small quantities of them in selected regions. In some steps, of course, the entire substrate surface can be sprayed or otherwise coated with a solution. In preferred embodiments, a dispenser moves from region
to region, depositing only as much monomer as necessary at each stop. Typical dispensers include a micropipette to deliver the monomer solution to the substrate and a robotic system to control the position of the micropipette with respect to the
substrate, or an ink-jet printer. In other embodiments, the dispenser includes a series of tubes, a manifold, an array of pipettes, or the like so that various reagents can be delivered to the reaction regions simultaneously.
Pin-Based Methods
Another method which is useful for the preparation of compounds and libraries of the present invention involves "pin based synthesis." This method is described in detail in U.S. Pat. No. 5,288,514, previously incorporated herein by reference.
The method utilizes a substrate having a plurality of pins or other extensions. The pins are each inserted simultaneously into individual reagent containers in a tray. In a common embodiment, an array of 96 pins/containers is utilized.
Each tray is filled with a particular reagent for coupling in a particular chemical reaction on an individual pin. Accordingly, the trays will often contain different reagents. Since the chemistry disclosed herein has been established such that
a relatively similar set of reaction conditions may be utilized to perform each of the reactions, it becomes possible to conduct multiple chemical coupling steps simultaneously. In the first step of the process the invention provides for the use of
substrate(s) on which the chemical coupling steps are conducted. The substrate is optionally provided with a spacer having active sites. In the particular case of oligonucleotides, for example, the spacer may be selected from a wide variety of
molecules which can be used in organic environments associated with synthesis as well as aqueous environments associated with binding studies. Examples of suitable spacers are polyethyleneglycols, dicarboxylic acids, polyamines and alkylenes,
substituted with, for example, methoxy and ethoxy groups. Additionally, the spacers will have an active site on the distal end. The active sites are optionally protected initially by protecting groups. Among a wide variety of protecting groups which
are useful are FMOC, BOC, t-butyl esters, t-butyl ethers, and the like. Various exemplary protecting groups are described in, for example, Atherton et al., Solid Phase Peptide Synthesis, IRL Press (1989), incorporated herein by reference. In some
embodiments, the spacer may provide for a cleavable function by way of, for example, exposure to acid or base.
Bead Based Methods
Yet another method which is useful for synthesis of polymers and small ligand molecules on a solid support "bead based synthesis." A general approach for bead based synthesis is described copending application Ser. Nos. 07/762,522 (filed Sep.
18, 1991); 07/946,239 (filed Sep. 16, 1992); 08/146,886 (filed Nov. 2, 1993 now abandoned); U.S. Pat. No. 5,541,061 and PCT/US93/04145 (filed Apr. 28, 1993), the disclosures of which are incorporated herein by reference.
For the synthesis of molecules such as oligonucleotides on beads, a large plurality of beads are suspended in a suitable carrier (such as water) in a container. The beads are provided with optional spacer molecules having an active site. The
active site is protected by an optional protecting group.
In a first step of the synthesis, the beads are divided for coupling into a plurality of containers. For the purposes of this brief description, the number of containers will be limited to three, and the monomers denoted as A, B, C, D, E, and F.
The protecting groups are then removed and a first portion of the molecule to be synthesized is added to each of the three containers (i.e., A is added to container 1, B is added to container 2 and C is added to container 3).
Thereafter, the various beads are appropriately washed of excess reagents, and remixed in one container. Again, it will be recognized that by virtue of the large number of beads utilized at the outset, there will similarly be a large number of
beads randomly dispersed in the container, each having a particular first portion of the monomer to be synthesized on a surface thereof.
Thereafter, the various beads are again divided for coupling in another group of three containers. The beads in the first container are deprotected and exposed to a second monomer (D) , while the beads in the second and third containers are
coupled to molecule portions E and F respectively. Accordingly, molecules AD, BD, and CD will be present in the first container, while AE, BE, and CE will be present in the second container, and molecules AF, BF, and CF will be present in the third
container. Each bead, however, will have only a single type of molecule on its surface. Thus, all of the possible molecules formed from the first portions A, B, C, and the second portions D, E, and F have been formed.
The beads are then recombined into one container and additional steps such as are conducted to complete the synthesis of the polymer molecules. In a preferred embodiment, the beads are tagged with an identifying tag which is unique to the
particular double-stranded oligonucleotide or probe which is present on each bead. A complete description of identifier tags for use in synthetic libraries is provided in co-pending application Ser. No. 08/146,886 (filed Nov. 2, 1993 now abandoned)
previously incorporated by reference for all purposes.
The advent of methods for the synthesis of diverse chemical compounds on solid supports has resulted in the genesis of a multitude of diagnostic applications for such chemical libraries. A number of these diagnostic applications involve
contacting a sample with a solid support, or chip, having multiple attached biological polymers such as peptides and oligonucleotides, or other small ligand molecules synthesized from building blocks in a stepwise fashion, in order to identify any
species which specifically binds to one or more of the attached polymers or small ligand molecules.
For example, patent application Ser. No. 08/082,937, filed Jun. 25, 1993, now abandoned describes methods for making arrays of oligonucleotide probes that can be used to provide the complete sequence of a target nucleic acid and to detect the
presence of a nucleic acid containing a specific oligonucleotide sequence. U.S. Pat. No. 5,556,752 describes methods of making arrays of unimolecular, double-stranded oligonucleotides which can be used in diagnostic applications involving protein/DNA
binding interactions such as those associated with the p53 protein and the genes contributing to a number of cancer conditions. Arrays of double-stranded oligonucleotides can also be used to screen for new drugs having particular binding affinities.
A number of factors contribute to the successful synthesis and use of oligomer arrays on solid supports. For example, issues of relevance to the use of derivatized glass substrates for carrying out VLSIPS synthesis of peptide arrays are the
spacing of the synthesis initiation sites, the wettability of the surface by organic solvents and aqueous solutions, and the extent to which non-specific binding of receptors, antibodies or other biological macromolecules occurs.
The spacing of the synthesis initiation sites (typically, primary amines) is of concern since very high site densities will affect binding events between tethered ligands and receptors. Additionally, increased yields in synthesis can be achieved
by control of phenomena such as free radical formation during photolytic reaction, solvent accessibility and surface electrostatic effects.
It will be apparent to those of skill in the art that certain aspects of the present invention will find application in all methods of solid phase polymer synthesis, whereas other aspects of the invention will find application in only particular
methods of solid phase synthesis. For example, compounds of the present invention which are useful as hydrophilic linking groups are useful in all of the above solid phase techniques. Other compounds such as the photolabile linking groups will find
application more preferably in methods involving "flow channel" and "spotting" techniques, "bead-based" synthesis and "pin-based" synthesis.
Other aspects of the present invention involving conditions and reagents for removing the NVOC protecting group will be directed primarily to the light-directed synthesis described in, for example, U.S. Pat. No. 5,143,854.
Still other methods for determining the fidelity of polymer synthesis will find application in all solid phase techniques.
III. Novel Linking Groups
The use of VLSIPS and ESL technologies, disclosed in U.S. Pat. No. 5,143,854, and U.S. patent application Ser. Nos. 07/946,239 and 08/146,886 now abandoned, and incorporated herein by reference for all purposes, often requires that the
support used to assemble the ligands also be used to display the ligands for biological binding experiments. As a result, any linking groups used in preparation of the ligands must perform well in the organic environment used in synthesizing the ligands
as well as the aqueous environment typically used in binding assays.
Thus, in one aspect, the present invention provides novel linking groups which can facilitate polymer synthesis on a solid support and which provide other advantageous properties for biological assays. Some of the linking groups are hydrophilic
and provide a "wettable" surface which aids in synthesis of the polymers as well as in screening of the polymers for activity. Still other linking groups are more hydrophobic.
One group of "hydrophilic" compounds which can be used as linking groups are represented by the formula:
in which P is a protecting group, X is a polymer chain having amine functional groups at the termini, and Y is a radical of formula:
in which Z is an alkyl radical having from 1 to 10 carbon atoms, and R is hydrogen or an activating group.
In one group of embodiments, P is a photocleavable protecting group, preferably an NVOC, MeNPOC, Dimethoxybenzoinyl, or DDZ. More preferably, P is a MeNPOC protecting group.
In another group of embodiments, P is FMOC or BOC.
In still another group of embodiments, X is a polyether chain having an amine functionality at the termini. Preferably, X is --NH--(CH.sub.2 CH.sub.2 O).sub.n CH.sub.2 CH.sub.2 NH--, --NH--CH.sub.2 (CH.sub.2 CH.sub.2 O).sub.n CH.sub.2 CH.sub.2
CH.sub.2 NH--, --NH--CH.sub.2 (CH.sub.2 CH.sub.2 CH.sub.2 O).sub.n CH.sub.2 CH.sub.2 CH.sub.2 NH-- or --NH--(CH.sub.2).sub.m O(CH.sub.2).sub.n O(CH.sub.2).sub.m NH--, in which n is an integer of from 1 to 10 and m is an integer of from 1 to 6.
In yet another group of embodiments, Z is an alkyl radical of formula --(CH.sub.2).sub.q -- in which q is an integer of from 1 to 6.
In a particularly preferred embodiment, P is MeNPOC or FMOC, X is --NH--(CH.sub.2 CH.sub.2 O).sub.2 CH.sub.2 CH.sub.2 NH-- and Y is --CO--(CH.sub.2).sub.3 --CO.sub.2 H.
The hydrophilic linking groups of the present invention can be synthesized by methods which are known to those of skill in the art. For example, commercially available hexaethylene glycol (Aldrich Chemical Company, Milwaukee, Wis. USA) can be
treated with p-toluenesulfonyl chloride and ammonia to produce a glycol derivative having amino groups at the chain termini. Treatment of the diamine with glutaric anhydride, followed by protection of the remaining amino group with, for example, NVOC-Cl
produces a protected form of the linking group referred to as 24-ATOM-PEG (see Table 1, Example 2). Other methods for the synthesis of hydrophilic linking groups begin with pentaethylene glycol and proceed with a one carbon homologation on each termini
via treatment of the diol with p-toluenesulfonyl chloride followed by cyanide ion. Reduction of the resultant dinitrile provides a diamine which can be treated with glutaric anhydride followed by protection of the remaining amine to provide a protected
form of the linking group referred to as 20-ATOM-PEG (see Table 1, Example 2). Still other diamines which are useful in the present linking groups are available from Fluka Chemical Co. (Ronkonkoma, N.Y., USA).
In another aspect, the present invention provides novel compounds which are useful as photochemically cleavable linking groups. Linking groups which are photochemically cleavable are useful in several applications. In one application, these
linking groups can be used for the photoinduced release of oligomers or small ligand molecules from a surface for characterization purposes following a bioassay. In another application, such linking groups are useful for the mild cleavage of oligomers
from a surface after various side chain protecting groups are removed. The oligomers can then be used in subsequent bioassays.
A number of reports of photochemically cleavable linking groups have appeared in the literature for use in peptide synthesis. A phenacyl based linking group (see 1 below) was first disclosed in Wang, J. Org. Chem. 41:3258 (1976). An
ortho-nitrobenzyl based linking group (see 2 below) was disclosed in Rich, et al., J. Am. Chem. Soc. 97:1575-1579 (1975). Both of these have been reported to give modest yields of peptides upon photolytic cleavage from a support, but only after
extended photolysis (e.g. 10 hours of photolysis in trifluoroethanol). ##STR1##
Compounds of the present invention which are useful as photochemically cleavable linking groups are based upon nitroveratryl (NVOC) and .alpha.-methylnitroveratryl (MeNVOC) groups and can be represented by the formula: ##STR2## in which R.sup.1
is C.sub.1 -C.sub.8 alkyl, aryl or aralkyl; R.sup.2, R.sup.3 and R.sup.4 are each independently hydrogen, C.sub.1 -C.sub.8 alkyl, or C.sub.1 -C.sub.8 alkoxy; X.sup.11 and Y.sub.11 are each independently halogen, --SH, --SP, --OH, --OP, --NH.sub.2 or
--NHP, in which P is a suitable protecting group; and q is an integer of from 1 to 10, preferably from 1 to 4.
In one group of embodiments, the compounds are represented by the formula: ##STR3## in which R.sup.1 is C.sub.1 -C.sub.8 alkyl; R.sup.2 and R.sup.4 are each independently hydrogen, C.sub.1 -C.sub.8 alkyl or C.sub.1 -C.sub.8 alkoxy; R.sup.3 is
C.sub.1 -C.sub.8 alkoxy; X.sup.11 and Y.sup.11 are each independently --Br, --Cl, --OH, --NH.sub.2, --OP, and --NHP, wherein P is a suitable protecting group; and q is an integer of from 1 to 4. In particularly preferred embodiments, R.sup.1 is methyl,
R.sup.2 and R.sup.4 are both hydrogen, R.sup.3 is methoxy, Y.sup.11 is --OH, q is 3, and X.sup.11 is --Br, --OH, --NH.sub.2, --ODMT, --NHBOC or --NHFMOC.
In another group of embodiments, the compounds are represented by the formula: ##STR4## in which the symbols R.sup.1, R.sup.2, R.sup.3, R.sup.4, X.sup.11, Y.sup.11 and q represent those groups described above for the first group of embodiments.
As above, embodiments which are particularly preferred are those in which R.sup.1 is methyl, R.sup.2 and R.sup.4 are both hydrogen, R.sup.3 is methoxy, Y.sup.11 is --OH, q is 3, and X.sup.11 is --Br, --OH, --NH.sub.2, --ODMT, --NHBOC or --NHFMOC.
Exemplary of the photochemically cleavable linking groups of the present invention are structures 3-6, below. ##STR5##
Compounds 3-6 were prepared in order to evaluate the effectiveness of each as a linking group. As a result, each compound was provided with an amide linkage (--CONHBn) on one terminus to represent the linking moiety on a support and an ester
(--OAc) or amide (--NHAc) linkage on the other terminus. One of skill in the art will appreciate that these photochemically cleavable linking groups can be prepared and stored in their unprotected forms as hydroxy-acids or amino-acids, or they can be
provided with protecting groups which are suitable for a variety of synthetic applications. These compounds which are suitable as photochemically cleavable linking groups can be prepared by standard synthetic methods known to those of skill in the art.
For example, linking group 3 can be prepared from commercially available vanillin (Aldrich Chemical Company, Milwaukee, Wis., USA). Alkylatlon the hydroxyl functionality of vanillin with t-butyl bromoacetate provides a phenoxyacetic ester derivative
which can be nitrated using nitric acid. The carboxylic acid functionality which is formed via ester cleavage during the nitration process can be convened to the benzamide using standard methods. Reduction of the aldehyde with sodium borohydride
followed by acylation of the hydroxyl group thus formed with acetic anhydride provides linking group 3.
Preparation of linking group 4 can be achieved in a similar sequence of steps beginning with acetovallinone (Aldrich Chemical Company). Preparation of linking group 5 can be achieved using methods similar | | |
