FIELD OF THE INVENTION The present invention relates to the field of photosensitive protecting groups. More specifically, the invention provides ortho-nitrobenzylic groups for protecting carboxylic, amino, thiol, hydroxyl, and other moieties.

BACKGROUND OF THE INVENTION Chemical protecting groups are used during synthesis reactions to temporarily protect certain functional groups on a compound against undesired reactions. When a reaction sequence is complete, and protection is no longer necessary, the protective group is removed to restore the protected functional group to its natural activity. Protective groups are removed by various procedures such as exposure to acidic or basic conditions. One class of protective groups, the photolabile or photosensitive protecting groups, are removed by exposure to electromagnetic radiation of a prescribed wavelength.

The properties and uses of some photolabile protecting compounds have been reviewed. See, McCray et aL. , Ann. Rev, of Biophys. and Biophys. Chem. (1989) 18:239-270 which is incorporated herein by reference. A particularly useful class of photoremovable protecting groups is the ortho-

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nitrobenzyl derivatives. When exposed to radiation (typically near ultra-violet) , these compounds undergo an internal rearrangement that results in deprotection of the protected compound. In the process, the protected compound splits off an ortho-nitroso aldehyde or ketone, leaving a free hydroxyl, amino, or other previously protected group. See Cameron and Frechet, J. Am. Chem. Soc. (1991) 113 :4303-4313. incorporated herein by reference for all purposes.

Examples of known ortho-nitrobenzyl photoremovable protecting groups are described in, for example, Patchornik,

J. Amer. Chem. Soc. (1970) j32_:6333, A it et al. , J■ Orα. Che . (1974) H:192, Pillai, Synthesis (Jan. 1980) 1-26, and E.P. Application 046 083, all of which are incorporated herein by reference. Known members of this class include ortho- nitrobenzyl and ortho-nitrobenzyloxycarbonyl protected compounds.

Patchornik and others have reported some phenyl-substituted ortho-nitrobenzyl groups for protecting amino acid functional groups. For example, the phenyl ring has been substituted methoxy groups at the 3 and 4 positions (nitroveratryl) . In other known compounds, one of the benzylic hydrogens has been replaced with a methyl or phenyl (substituted and unsubstituted) group.

Ortho-nitrobenzyl protected compounds have now found use in a variety of fields. For example, they have proven useful in Very Large Scale Immobilized Polymer Synthesis (VLSIPS™) which provides techniques of forming vast arrays of peptides and other polymer sequences using, for example, light-directed synthesis techniques. The details of VLSIPS ^" " are disclosed in Pirrung et al. , U.S. Patent No. 5,143,854

(see also PCT Application No. WO90/15070) , Fodor et al. , PCT Publication No. WO92/10092, and Fodor et al. , Science (1991)

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251:767-777. all incorporated herein by reference for all purposes.

Some preferred photosensitive groups for protecting amino acids and nucleosides during VLSIPS™ have been described in PCT Publication No. W092/10092. These include nitroveratryloxycarbonyl (NVOC) , nitropiperonyloxycarbonyl (NPOC) , alpha-methyl-nitroveratryloxycarbonyl (MeNVOC) , alpha- methyl-nitropiperonyl (MeNPOC) , and the benzylic form of each of these (i.e. NV, NP, MeNV, and MeNP) .

VOC MeNVOC

In addition to VLSIPS™, ortho-nitrobenzyl photolabile protecting groups have been proposed for use in photolithography for electronic device fabrication (see e.g. Reichmanis, et ail., J. Polymer Sc. Polymer Chem. Ed. (1985) 2.3:1-8, incorporated herein by reference for all purposes), and monitoring transport of various molecules and ions in biological milieus (see e.g. Kaplan et al. , PNAS USA (1987) 8 _. 5:6571-6575, incorporated herein by reference for all purposes) .

In VLSIPS™, a "basis set" of protected monomers is used to synthesize a diverse collection of polymers. The basis set is, for example, the twenty naturally occurring amino acids having protected alpha-amino groups. It is desirable to have each member of the basis set photolyze at

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approximately the same rate. Unfortunately, this is often not possible when a single protecting group (e.g. ortho- nitrobenzyloxycarbonyl) is used to protect each member of the basis set. For example, when two different amino acids protected with nitrobenzyloxycarbonyl groups are exposed to radiation of the same wavelength and intensity, they may be deprotected at substantially different rates. Thus, it is desirable to develop methods that "equalize" the photolysis rates of the basis set members. Although some ortho-nitrobenzyl photoremovable protecting groups have proven useful, additional photoremovable protecting groups having various structures and photolysis rates are desirable.

SUMMARY OF THE INVENTION

The present invention provides new ortho-nitrobenzyl photosensitive protecting groups. These groups can be coupled to amino, carboxyl, hydroxyl, thiol and other moieties of compounds protected during certain chemical reactions. For example, the photosensitive groups of this invention can be used to protect functional groups of nucleosides, amino acids, or saccharides from unwanted side reactions during polymer synthesis.

According to one aspect, the invention provides improved photoremovable protecting groups having the formula:

In this structure, n is 0 or 1; A is -C(0)-, - (CQ ₁ Q ₂ )-, or -C(S)-; R _: , Q _: , and Q ₂ are independently hydrogen, C _: -C ₃ alkyl, aryl, alkoxy, aryloxy, or carboxy; and R- is a functional group of a molecule such as a natural or unnatural amino acid or peptide, a nucleoside, nucleoside analog, or

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oligonucleotide. R-j- _c are selected independently from among the groups hydrogen, alkoxy, aryloxy, benzyloxy, acyloxy, nitro, alkylthio, arylthio, hydroxyl, halogen, or a group having the formula -NR'R" where R' and R" are selected independently from the group consisting of hydrogen, C-_-C ₈ alkyl, aryl, or benzyl. R ₃ -R ₆ may also be a cyclic bridge between adjacent substituents. Exemplary bridges include acetal, ketal, orthoester, thioester, fused aromatic, or ether groups. In preferred novel embodiments, when n is 0 or A is

-C(O)- (i) R ₄ and R ₅ are not both methoxy when R-_, R ₃ , and R _s are hydrogen; (ii) R _x is not hydrogen, methyl, phenyl, or 2- nitrophenyl when R3-R ₆ are hydrogen simultaneously; and (iii) R-, is not 2-nitrophenyl or 3,4-dimethoxy-6-nitrophenyl when R ₃ and R ₆ are hydrogen and R ₄ and R ₅ are both either hydrogen or methoxy.

The amino acid (and peptide) functional groups protected as described include side chain groups as well as "backbone" groups (i.e. the a ine and carboxyl groups that form the peptide bond) . Side chain functional groups found on natural amino acids include amines, carboxyl groups, thiols, hydroxy groups, imidazoles, amides, indole groups, etc. The nucleoside, nucleoside analog, and oligonucleotide functional groups include the 2 * , 3 * , and 5' ribose hydroxy groups. In addition, the purine and pyrimidine bases of nucleosides can be protected by the above protecting groups. For example, R ₂ in the above compound structure can represent base exocyclic amine groups in some embodiments.

A particularly preferred class of photoremovable protecting groups has the formula:

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Here, R-, is a molecule functional group that may be one of the following: an amine, a carboxyl group, a thiol, an i idazole, an amide, a hydroxy group, or the like. Otherwise, R-. Q-i , Q ₂₁ and R-.-R _s are the same as defined in the previous structure. In preferred embodiments, the functional group R ₂ is found on a natural or unnatural amino acid or peptide, a nucleoside, a nucleoside analog, or an oligonucleotide as described in connection with the above embodiment. Preferred reactive compounds employed to make protected compounds having the above structure include activated ortho- nitrobenzyloxymethyl groups such as ortho-nitrobenzyloxymethyl halides (e.g. ortho-nitrobenzyloxymethyl chlorides) . Other active groups that can replace the halo group include hydroxyl, tosyl, mesyl, trifluoromethyl, diazo, azido, and the like.

The invention also includes basis sets of protected natural or unnatural amino or nucleic acids in which a plurality of amino or nucleic acids are protected by photoremovable protecting groups of the invention such that all of the protected amino or nucleic acids are deprotected at substantially the same rate in a particular solvent.

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 various reaction paths that can be employed to produce alcohols of the ortho- nitrobenzyl protecting groups of this invention;

Fig. 2 schematically illustrates reaction paths to various protected compounds of this invention;

Fig. 3 schematically illustrates reaction paths to various benzyloxycarbonyl protected compounds of this invention;

Fig. 4 schematically illustrates reaction paths to various benzyl protected compounds of this invention; and

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Fig. 5 schematically illustrates reaction pat s tc various benzyloxymethyl protected compounds of this invention.

Fig. 6 presents various compounds for which photolysis data is provided in Table V.

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DESCRIPTION OF THE PREFERRED EMBODIMENTS

CONTENTS

I. Definitions

II. Function of Protecting Groups

III. Preferred Ortho-nitrobenzyl Protecting Groups

IV. Choice of Sidechain Protecting Groups

V. Synthesis of Protected Compounds 1. Example

2. Example

3. Example

4. Example

5. Example 6. ExaMple

7. Example

VI . Multiple-quantum Protecting Groups

VII. Photolysis of Protected Group Compounds

VIII.Basis Sets of Photoprotected Amino or Nucleic Acids

IX. Conclusion

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1. Definitions

Certain terms used herein are intended to have the following general definitions

A. Monomer: A member of the set of small molecules which are or can be joined together to form a polymer. Frequently, monomers have at least two different reaction sites (e.g. the amino and carboxyl groups of amino acids) . 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. 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 at- , β-, or ω-amino acids, heteropoly ers in which a known drug is covalently bound to any of the above, polynucleotides, polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, or other polymers which will be apparent to those of skill in the art. Methods of cyclization and polymer reversal of polymers are disclosed in copending application Serial No. 796,727 (Attorney Docket No. 11509-51) , filed on the same date as the present application, entitled "POLYMER REVERSAL ON SOLID SURFACES," incorporated herein by reference for all purposes.

B. Functional Group: A group or moiety on a chemical compound that in some environments imparts certain chemical properties to the compound. More specifically, "functional group" refers to groups that are capable of undergoing reaction such as oxidation or reduction under certain conditions. As used in the context of this invention,

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functional group most often refers to a group on a monomer or polymer that can be protected by a photolabile or other group. Typically, functional groups include the reactive sites on a monomer that participate in coupling reactions to produce a polymer. For example, amino acid functional groups include carboxyl and amino groups; nucleic acid functional groups include 2', 3', and 5' hydroxyl groups. Other common functional groups encountered in the context of this invention include side chain groups (thiol, i idazole, indole, etc.) on amino acids; purine/pyrimidine groups (e.g. exocyclic amines) on nucleic acids; amine or carboxyl groups on linkers; and hydroxyl and amine groups on carbohydrates or lipids.

C. Protecting group: A material which is chemically bound to a reactant functional group and which may be removed upon selective exposure to an activator such as electromagnetic radiation. A protecting group prevents the protected functional group from undergoing undesired side reactions. For instance, the amino group of glycine may be coupled to a protecting group to prevent the glycine amino group from reacting during a coupling reaction between the glycine carboxylic terminus and the amino terminus of a growing peptide. Examples of photosensitive protecting groups include nitropiperonyl, nitroveratryl, and nitrobenzyl groups. Other protective groups sensitive to acid or base include t- butyloxycarbonyl or fluorenylmethoxycarbonyl.

D. Basis Set: A set of protected reactants such as monomers used in, for example, a synthesis step in the formation of a polymer array. For example, the 20 naturally occurring amino acids form one basis set and the four naturally occurring deoxyribonucleic acids form another basis set. As a further example, the 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.

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E. Peptide: A polymer in which the constituent monomers are amino acids joined together through amide bonds. Peptides are alternatively referred to as polypeptides. In the context of this specification it should be appreciated that the peptide may include the L-optical or D-optical isomers of a- , β- , or ω-amino acids. In addition, the peptide may include amino acids having unnatural side chains or other deviations from the naturally occurring amino acids. Such "peptides" have been the subject of much study recently, and are discussed by Fauchere, J-L, in Advances in Drug Design. Vol. 15, Testa ed. , (1986), which is incorporated herein by reference for all purposes. Peptides are 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.

F. Radiation: Energy which may be selectively applied including energy having a wavelength of between 10 ^" - ⁴ and 10 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 material. The photosensitive protecting groups of this invention are typically photolyzed by electromagnetic

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radiation in the ultraviolet or visible regions of the spectrum.

Abbreviations: The following frequently used abbreviations are intended to have the following meanings:

BOC: t-butyloxycarbonyl.

BOP: benzotriazol-l-yloxytris-(dimethylamino) phosphoniu hexafluorophosphate.

DCC: dicyclohexylcarbodiimide. DCM: dichloromethane; methylene chloride.

DDZ: dimethoxydimethylbenzyloxy.

DIEA: N,N-diisopropylethylamine.

DMAP: 4-dimethylaminopyridine.

DMF: dimethyl formamide. DMT: dimethoxytrityl.

DTT: dithiothreitol

FMOC: fluorenylmethyloxycarbonyl.

HBTU: 2-(lH-benzotriazol-l-yl) -1, 1, 3 , 3-tetramethyluronium hexafluorophosphate. HOBT: 1-hydroxybenzotriazole.

NBOC: 2-nitrobenzyloxycarbonyl.

NMP: N-methylpyrrolidone.

NPOC: 6-nitropiperonyloxycarbony1.

NV: 6-nitroveratryl. NVOC: 6-nitroveratryloxycarbonyl.

TFA: trifluoracetic acid.

THF: tetrahydrofuran.

II. Function of Protecting Groups Protecting groups of the present invention can be used in conjunction with solid phase oligomer syntheses, such as peptide syntheses using natural or unnatural amino acids, nucleotide syntheses using deoxyribonucleic and ribonucleic acids, oligosaccharide syntheses, and the like. In a particularly preferred embodiment, the protecting groups of this invention are used to synthesize an array of polymers according to a VLSIPS™ process. Such processes employ a substrate containing oligomers or other materials protected

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with photosensitive groups. Selected regions of the substrate are irradiated to create regions having reactive moieties that are essentially free of side products resulting from the protecting group. The protecting groups serve two purposes: (1) protecting the substrate surface from unwanted reactions, and (2) blocking a reactive functional group of the monomer to prevent self-polymerization or other reaction. For instance, attachment of a protecting group to the amino terminus of an activated amino acid, such as an

N-hydroxysuccinimide-activated ester of the amino acid, prevents the amino terminus of one monomer from reacting with the activated ester portion of another during peptide synthesis. Alternatively, the protecting group may be attached to the carboxyl group of an amino acid to prevent reaction at this site. Most protecting groups can be attached to either the amino or the carboxyl group of an amino acid, and the nature of the chemical synthesis will dictate which reactive groups will require a protecting group. Analogously, attachment of a protecting group to the 5'-hydroxyl group of a nucleoside or nucleoside derivative during oligonucleotide synthesis using for example, phosphora idite coupling chemistry, prevents the 5'-hydroxyl of one nucleoside from reacting with the 3 '-activated phosphate-triester of another. In other embodiments, the photolabile protecting group may be attached to the 2'- or 3 '-hydroxyl group of a nucleoside. In alternative embodiments, the photosensitive protecting groups of this invention can be employed to protect side chain functionalities of amino acids during peptide syntheses employing conventional acid or base labile protecting groups such as BOC (t-butyloxycarbonyl) or FMOC (fluorenylmethoxycarbonyl) on the alpha backbone amine. In some preferred embodiments, the photolabile protecting groups protect any one or a combination of the following groups: the thiol group of cysteine; the basic residues (amine and i idazole groups) of lysine, arginine, and histidine; the acidic carboxy groups of aspartic and glutamic acids; the hydroxy groups of serine, threonine, and tyrosine; and the

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neterocyclic indole ring of tryptophan. Of course, otner amino acid side chains such as those occurring on non-natural amino acids can likewise be protected by the photosensitive groups of the present invention. Further, the purine and pyrimidine bases (and particularly the exocyclic amine groups) of nucleoside and nucleoside derivatives likewise can be protected with the photosensitive groups of this invention. Regardless of the specific type of compound being synthesized, protecting groups are employed to protect a moiety on a molecule from reacting with another reagent.

Typically, the reaction to be blocked will be the formation of a covalent bond such as a peptide bond between two amine acid molecules. However, the protecting groups of this invention can also block non-covalent reactions. For example, the photosensitive protecting groups of this invention can be used to "cage" (i.e. provide protection based upon steric hindrance) biotin or a biotin analog, thus preventing binding with avidin. Useful applications of this strategy are described in Barrett et al. , PCT Publication No. WO 91/07087, incorporated herein by reference for all purposes.

Protecting groups of the present invention preferably have the following general characteristics: they prevent selected reagents from modifying the group to which they are attached; they are stable (that is, they remain attached to the molecule) to the synthesis reaction conditions; they are removable under conditions that do not adversely affect the structure to which they are attached; and once removed, they do not react appreciably with the surface or surface-bound oligomer. In some embodiments, liberated byproducts of the photolysis reaction can be rendered unreactive toward the growing oligomer by adding a reagent that specifically reacts with the byproduct. The selection of a suitable protecting group will depend, of course, on the chemical nature of the monomer unit and oligomer, as well as the specific reagents they are to protect against.

The removal rate of the protecting groups depends on the wavelength and intensity of the incident radiation, as well as the physical and chemical properties of the protecting

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group itself. Preferred protecting groups are removed at a faster rate and with a lower intensity of radiation. For example, at a given set of conditions, MeNVOC and MeNPOC are photolytically removed from the N-terminus of a peptide chain faster than their counterparts, NVOC and NPOC. Therefore for most applications, MeNVOC and MeNPOC are preferred over NVOC and NPOC.

In many embodiments, the photosensitive protecting groups will be removable by radiation in the ultraviolet (UV) or visible portion of the electromagnetic spectrum. In preferred embodiments, the photosensitive protecting groups are sensitive to an electromagnetic spectral region that does not significantly overlap the absorption band of the monomers or polymers being protected. Higher energy radiation (i.e. low wavelength radiation) is generally more likely to damage monomers such as amino acids and nucleosides. For example, the four naturally occurring bases in deoxynucleosides absorb 250-280 nm light very strongly. In addition, short wave ultra-violet light is known to photo-destruct DNA formation of thymine di ers. Thus, in most preferred embodiments, the protecting groups will be removable by radiation in the near UV or visible portion of the spectrum. Of course, each collection of monomers (basis sets) will have its own characteristic absorption bands which can be avoided by choosing protecting groups that are sensitive to wavelengths well removed from the absorption band.

Ill. Preferred Ortho-nitrobenzyl Protecting Groups

Preferred photochemical protecting groups of the present invention have the general formula:

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In this structure, n is 0 or 1; A is -C(O)-, - (CQ ₁ Q ₂ ) - , or -C(S)-; R- , Q-, , and Q ₂ are independently hydrogen, C--C ₈ alkyl, aryl, alkoxy, aryloxy, or carboxy; and R ₂ is a functional group of a molecule such as a natural or unnatural amino acid or peptide, a nucleoside, nucleoside analog, or oligonucleotide. ₃ - ₆ ^are selected independently from among the groups hydrogen, alkoxy, aryloxy, benzyloxy, acyloxy (e.g., CH ₃ C(O)O-) nitro, alkylthio, arylthio, hydroxyl, halogen, or a group having the formula -NR'R" where R' and R" are selected independently from the group consisting of hydrogen, C±-C _Q alkyl, aryl, or benzyl. R ₃ -Rc may also be a cyclic bridge between adjacent substituents. Exemplary bridges include acetal, ketal, orthoester, thioester, fused aromatic (which together with the first benzene ring forms a derivative of naphthalene, quinoiine, anthracene, etc.) , or ether groups.

In preferred novel embodiments, when n is 0 or A is -C(O)- (i) R ₄ and R ₅ are not both methoxy when R _1# R ₃ , and R ₆ are hydrogen; and (ii) R _x is not hydrogen, methyl, phenyl, or 2-nitrophenyl when R ₃ -R _s are hydrogen simultaneously.

Further, compounds having hydroxyl substituents on the phenyl ring, and/or alkoxy or aryloxy substituents on the benzyl carbon may be too reactive for certain applications, and therefore are generally less preferred. The amino acid (and peptide) functional groups protected as described include side chain groups as well as "backbone" groups (i.e. the amine and carboxyl groups that form the peptide bond) . Those of skill in the art will recognize that a variety of other functional groups can be protected by the above photosensitive groups. Specific examples include hydroxy and amino groups on saccharides, hydroxy groups on steroids, and amino groups attached to linkers on a substrate surface (see PCT Publication No. WO92/10092, previously incorporated by reference) . In addition, certain molecules can be "caged" to provide steric protection against binding. For example, biotin and biotin analogs can be caged with the above photosensitive groups to

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prevent binding with avidin. The protecting group can be coupled through the biotin urea groups.

Although carboxyl, amino, and hydroxy groups of amino acids and nucleosides can, in principle, be protected with either benzyl or benzyloxycarbonyl protecting groups

(i.e. n=0 and 1, and ignoring the possibility that A is -CH2- or -C(S)-), certain couplings are preferred. Specifically, amino acid carboxyl groups preferably are protected with benzyl form photosensitive groups (n=0) , while amino acid amine groups and nucleoside hydroxy groups preferably are protected with benzyoxycarbonyl form of the photosensitive groups (n=l) .

When a primary amine group (e.g. those found on most natural amino acids) is protected by a benzylic group (n=0) of the above structure, the amine nitrogen must be further protected for some applications,. For example if the amine is not further protected during peptide synthesis using VLSIPS™, the free amine hydrogen may react with other amino acids activated with coupling reagents such as BOP. If, however, a second protecting is employed, no free hydrogen atoms are present on the amine to act as a reaction sites. Preferably, the additional protection is provided by C _: -C ₆ alkyl, aryl, acyl, or benzyl groups. More preferably, the additional protective group is methyl or acetyl. Such compounds are readily synthesized by methods well-known in the chemical arts. For example, following the coupling of the above- described photoprotective group to an amino acid (made, e.g., by using an activated benzyl derivative of the photoprotective group) , the photoprotected amino acid may be reacted with a reagent known to methylate amino acid nitrogen moieties, such as methyl iodide, to form the desired protected amino acid.

A particularly preferred class of protecting groups is the 6-nitrobenzyloxymethyl groups which have exhibited very rapid photolysis rates and have the general formula:

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Here, R ₂ is a molecule functional group that may be one of the following: an amine, a carboxyl group, a thiol, an imidazole, an amide, a hydroxy group, and the like.

Otherwise, R _α , Q _1; Q ₂ and R ₃ -R ₆ are the same as defined in the above structure. In preferred embodiments, the functional group R ₂ is found on a natural or unnatural amino acid or peptide, a nucleoside, a nucleoside analog, or an oligonucleotide. Both side chain and backbone groups may be protected. As in the above general embodiment, the photosensitive groups can also be used to protect functional groups found on a variety of compounds such as carbohydrates, lipids, biotin analogs, linker molecules, etc. Preferred reactive compounds employed to make protected compounds having the above structure include activated ortho-nitrobenzyloxymethyl compounds such as ortho- nitrobenzyloxymethyl halides (e.g. ortho-nitrobenzyloxymethyl chlorides) . Other active groups that can replace the halo group include hydroxyl, tosyl, mesyl, trifluoromethyl, diazo, azido, and the like.

Another class of preferred protecting groups includes compounds in which R ₅ and Ra are both alkyloxy groups or together form a cyclic bridge acetal or ketal. In particularly preferred embodiments R _: is hydrogen or methyl and R, and R ₄ are hydrogen. In alternative preferred embodiments, R ₄ and R ₅ are alkoxy groups or together form the cyclic acetal or ketal. Preferably, , and R _s will then be hydrogen. Such compounds are made by standard methods such as those described in Greene, Protective Groups in Organic Synthesis, Wiley 1981, which is incorporated herein by reference for all purposes. One example is where R ₄ and R ₌ or R- and R _c form a methylene acetal or an acetonide: (-0-CH--0-

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, or -0-C(CH ₃ ) ₂ -0-) . In the structures below, Y _η and Y ₂ are independently C-,-C ₈ alkyl groups, or fused to form a cyclic bridge acetal or ketal.

In addition to acetals and ketals, the photoprotecting groups of the invention may also include compounds where adjacent benzene ring substituents form a ring having the formula -O-CRR'-O- or -0-CRR'-CR"R ^/,/ -0-, where R, R', R", and R" can independently be hydrogen, C-^C _g alkyl, aryl, benzyl, alkoxy, aryloxy, carboxy, alkyl carboxylic acid ester (i.e. -C(O)O-alkyl) , or carbonyl. Specific examples include cyclic orthoesters (-O-CRR'-O-, where R and R' are alkoxy, aryloxy, or carboxy) or cyclic ethers (-O-CRR '-CR"R "'- O- where R, R', R", and R"" are selected from the group hydrogen, alkyl, aryl, benzyl, alkoxy, aryloxy, or carboxy) . In addition, R and R' together or R" and R'" together may be a carbonyl oxygen (e.g. forming the following bridges: -0- C(0)CH ₂ -O- or -0-CH ₂ C(0)-0-) . These compounds are readily synthesized by known methods (see, e.g., Greene). A preferred cyclic ether has R, R', R", and R" hydrogen (i.e., -0-CH ₂ -CH ₂ - 0-) . Preferred substitution patterns are those at the R, and R _c , and R ₅ and R ₆ positions:

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Still other preferred compounds are tnose where R ₌ and R _c are methoxy. In a specific preferred embodiment , is dimethylamino and R_ is hydrogen:

Yet still other preferred compounds are formed when R ₄ is methoxy and R ₅ is dimethylamino, or, conversely, where R ₄ is dimethylamino and R ₅ is methoxy:

As noted, the photoremovable protecting groups of the present invention can be attached to an activated ester of a natural or unnatural amino acid or peptide at the amino terminus. For use in VLSIPS™ or other peptide synthesis techniques, an amine protected amino acid may have the following structure:

where R is the side chain of a natural or unnatural amino acid, X is a photoremovable protecting group according to this invention, and Y is an activated carboxylic acid derivative. The activated ester, Y, is preferably a reactive derivative having a high coupling efficiency, such as an acyl halide, mixed anhydride, N-hydroxysuccinimide ester, perfluorophenyl ester, or urethane protected acid, and the like. Other activated esters and reaction conditions are well known (See

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Atherton et al. , "Solid Phase Peptide Synthesis" 1989, IRL Press, London, incorporated herein by reference for all purposes) .

As noted, the photoreactive protecting groups of the present invention are useful for protecting both natural and unnatural nucleosides or nucleotides. More specifically, the 2', 3', and/or 5' hydroxyl functions of such compounds. Such compounds can therefore have the general structure:

X may be oxygen or sulphur. R _η is a purine or pyrimidine base or analog thereof (for example, adenine, cytosine, thymine, guanine, uracil, 6-ketoadenine, or an analog thereof) . ₂ ~ ₄ may be one of the commonly used protecting groups (see below) , or a photoreactive group as described above. Typically, one position will be protected with a photoreactive protecting group while the other position(s) will be (a) protected or substituted with one of the commonly used protecting groups, or (b) activated with one of the commonly reactive groups. However, more than one position may be protected with a photoreactive protecting group of the invention.

With respect to R ₂ , R ₃ , and R ₄ , these may be hydrogen, hydroxy, an oligonucleotide or one of the commonly used protecting groups: alkoxy, tetrahydropyranyl, 8- benzoylpropionyl, acetyl, or silyl. They can also be -NRR ' , - OP(0) (OR") (OR") , -OP(0)0 ₂ ^"2 , -OP(0)0 ₂ H ^" , -OP(0) (OR") (NRR ') , or -OP(OR") (NRR* ) (i.e. phosphoramidite) where R and R' are independently selected from the group consisting of C- -C- alkyl, aryl, benzyl, or acetyl, and R" and R'" are selected independently from the group consisting of C-_-C ₃ alkyl, aryl, benzyl, or 0-, -C-^ cyanoalkyl. R ₂ or R- _> also together form a

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cyclic acetal, ketal, orthoester, cr ether. In some preferred embodiments, R ₄ can be triphenylmethyl, di-p-methoxytrityl, dimethylpropanoyl. These compounds and their methods of synthesis are well-known in the art (see, e.g., Fuhrhop and Penzlin, Organic Synthesis Concepts, Methods, and Starting Materials, Verlag Chemie 1983, or Gait, Oligonucleotide Synthesis a Practical Approach, IRL Press 1984, both of which are incorporated herein by reference for all purposes) .

An especially preferred photoreactive protecting group for use with nucleosides and nucleotides has the formula:

IV. Choice of Sidechain Protecting Groups It will be appreciated by those skilled in the art that functional groups other than those directly involved in coupling during polymer synthesis may have to be protected. It will also be appreciated that such protecting groups should not be removable under coupling conditions. Thus, for example, a desired polypeptide will not be contaminated as a result of cross reactions between sidechains which have inadvertently become unprotected and other monomers or the sidechains of other amino acids. Since the removal of the photoreactive protecting groups of the present invention typically creates an acidic environment, care must be taken to choose sidechain protecting groups which are moderately acid stable.

Herein, the term "acid stable" means that the sidechain protecting group in question is not removed in appreciable amounts under conditions where the effective pH is less than about 7. Determining stability will depend on the particular amino acid sidechain as well as the sidechain protecting group which is chosen. For lysine, it has been

SUBSTITUTE SHEET

found that €-amino moieties protected with BOC (t- butyloxycarbonyl) tend to degrade over time in 5mM sulf ric acid. Thus, a protecting group for the e-amino group of this amino acid should be less acid labile than BOC. Generally, trityl protecting groups are also too sensitive to protect e- amino groups. One preferred protecting group for the €-amino acid of lysine is dimethoxybenzyloxycarbonyl. Another preferred protecting group is FMOC (9- fluorenylmethyloxycarbonyl) . These groups are well-known in the art as are the techniques for forming the appropriately protected lysine amino groups.

Although trityl is not preferred with respect to lysine, trityl is stable enough to be used with cystine, asparagine, and glutamine. Generally, however, asparagine and glutamine do not require protection. With respect to histidine, protection of the imidazole nitrogen is needed during addition to the polymer in order to prevent self- condensation and racemization. However, once the histidine is bound to the substrate, protection is no longer required. FMOC and trityl are examples of suitable protecting groups for the imidazole nitrogen of histidine. It will be appreciated by those of skill in the art that various other protecting groups may be used depending upon the application.

V. Synthesis of Protected Compounds

The protected compounds of the present invention often can be prepared via a process employing a benzyl alcohol of the protecting group. Various synthetic routes to some useful benzyl alcohols are shown in Fig. 1. In the displayed benzyl alcohols, R, R', and R" represent hydrogen, methyl, C- to C _D alkyl, and other groups in accordance with the structures presented above. Each arrow shown in Fig. 1 depicts a different reaction pathway, employing different reagents. Typical reagents for suitable use in the various reaction paths are as follows: (a) RC(0)C1 and aluminum chloride; (b) DMF and P0C1,; (c) R'l or R"I or MeBr- or EtBr- and potassium carbonate; (d) concentrated nitric acid or other nitration agent; (e) NaBH^ ; and (f) RMgBr or RLi.

SUBSTITUTE SHEET

Fig. 2 displays reaction paths for making various protected compounds from benzyl alcohols. In the structures shown, X denotes substitution on the phenyl ring, R denotes substitution on the benzylic hydrogen, and Z denotes an oxygen or sulfur atom. In reaction path (a) , the alcohol is reacted with C1C(Z)0R' and triethylamine or C1C(Z)SR' and triethylamine to form the protected thiol or alcohol shown. The reactants (other than the benzyl alcohol of the protecting group) and the products for the various other reactions are as follows: (b) reaction with C1CH ₂ 0R and triethylamine produces a protected alcohol; (c) reaction with Z=C=NR' or C1C(Z)NR'R" produces a protected amine; (d) reaction with C1CH ₂ SR' and triethylamine produces a protected thiol; and (e) reaction with R'C(0)OH, DCC, and DMAP or with R'C(0)C1 and triethylamine produces a protected carboxylic acid.

Fig. 3 schematically'depicts some general reactions that can be employed to produce benzyloxycarbonyl (and thiocarbonyl) protected functional groups. A shown, a benzyl alcohol is reacted through a chloroformate intermediate to produce the desired compounds. In Fig. 3, X denotes substitution on the phenyl ring, R denotes substitution on the benzylic carbon, and Z denotes an oxygen or sulfur atom. Typical reagents employed in the noted routes are as follows: (a) C(0)C1 ₂ or C(S)C1 ₂ ; (b) R'OH and triethyl amine; (c) R'SH and triethyl amine; (d) R ^» R"NH; and (e) H ₂ NC(0)R'

In one specific embodiment, the appropriate benzyl alcohol can be reacted with phosgene or other agent to produce an activated benzyloxycarbonyl derivative of the protecting group. These compounds are then used to produce the above- described benzyloxycarbonyl-protected groups (e.g. n is 1 and A is -C(O)- in the above generic structures). These groups are preferably coupled to the amino nitrogen of a natural or unnatural amino acid; or the 2', 3', or 5' oxygen of a natural or unnatural nucleoside or nucleotide using standard methods, for example, reacting the amino group of an amino acid or a ribose hydroxyl moiety of a nucleoside with the desired activated benzyloxycarbonyl derivative. Examples of such activated protecting groups have the general formula:

SUBSTITUTE SHEET

where X is halogen, mixed anhydride, phenoxy, p-nitrophenoxy, N-hydroxysuccinimide, hydroxyl, tosyl, mesyl, trifluoromethyl, diazo, azido, and the like. R-_ is hydrogen, C^-C ₈ alkyl, aryl, alkoxy, aryloxy, or carboxy. R ₃ - C are selected independently from among the groups hydrogen, alkoxy, aryloxy, benzyloxy, nitro, alkylthio, arylthio, hydroxyl, halogen, or a group having the formula -NR'R" where R' and R" are selected independently from the group consisting of C _: -C ₈ alkyl, aryl, or benzyl. R ₃ -R _? -- may also be a'cyclic bridge between adjacent substituents. Exemplary bridges include acetal, ketal, orthoester, thioester, phenyl, or ether groups. Compounds falling within this formula are readily formed using known standard techniques such as those described in March, Advanced Organic Chemistry. 3 ^rd ed. , Wiley 1985, or Carey and Sundberg, Advanced Organic Chemistry Part B: Reactions and Synthesis. 2 ^n~ ed. , Plenum 1984, both of which are incorporated herein by reference for all purposes. It should be noted that the corresponding benzyloxythiocarbonyl (employing the -C(S)- group in place of -C(O)-) can be prepared by analogous methods.

Fig. 4 schematically depicts various reaction pathways from a benzyl alcohol starting material to benzyl- protected compounds via an activated benzyl intermediate. In the compounds shown, X denotes substitution on the phenyl ring and R denotes substitution on the benzylic carbon. Typical reagents used in the various reaction steps are as follows: (a) S0C1 ₂ ; (b) R'OH and triethylamine; (c) R'SH and triethylamine; (d) R"R'NH; and (e) H ₂ NC(0)R' and triethylamine; (f) R'C(0)OH and triethylamine.

In a specific embodiment, the carboxy terminus of an amino acid protected with a benzylic photoactivatable group

SUBSTITUTE SHEET

can be formed by esterifying the carboxy group with an activated benzyl derivative of the protecting group. Likewise, the amino terminus of an amino acid or a hydroxy group of a nucleoside (or nucleoside derivative) protected with a benzylic group can be formed by alkylating the amino or hydroxy group with an activated benzyl derivative of the protecting group. As noted above, benzyoxycarbonyl groups are more preferred for protecting amine groups of amino acids and hydroxy groups of nucleosides. Further, if a benzylic form of a photoreactive protecting group is used to protect the amino nitrogen of an amino acid, that nitrogen may have to be further protected with an alkyl, aryl, benzyl, or acyl group.

Examples of activated benzyl derivatives have the general formula:

where X is halogen, hydroxyl, tosyl, mesyl, trifluoromethyl, diazo, azido, and the like. R ₁ is hydrogen, C,-C ₈ alkyl, aryl, alkoxy, aryloxy, or carboxy. -J-R _C are selected independently from among the groups hydrogen, alkoxy, aryloxy, benzyloxy, nitro, alkylthio, arylthio, hydroxyl, halogen, or a group having the formula -NR'R" where R' and R" are selected independently from the group consisting of C _: -C ₈ alkyl, aryl, or benzyl. ₃ - C may also be a cyclic bridge between adjacent substituents. Exemplary bridges include acetal, ketal, orthoester, thioester, phenyl, or ether groups.

Another method for generating compounds protected with benzylic protecting groups is to react the benzylic alcohol derivative of the protecting group with an activated ester of the compound to be protected. For example, to protect the carboxyl terminus of an amino acid, an activated ester of the amino acid is reacted with the alcohol derivative of the protecting group. Examples of activated esters

SUBSTITUTE SHEET

suitable for such uses include halo-formate, mixed anhydride, imidazolyl formate, acyl halide, and also include formation of the activated ester m situ the use of common reagents such as DCC and the like. See Atherton et al. (previously incorporated by reference) for other examples of activated esters.

To protect the 5 '-hydroxyl group of a nucleic acid with a benzylic protecting group, a derivative having a 5'- activated carbon is reacted with the alcohol derivative of the protecting group. Examples of nucleotides having activating groups attached to the 5'-hydroxyl group have the general formula:

\s' B

OP where Y is a halogen atom, a tosyl, mesyl, trifluoromethyl, azido, or diazo group, and the like. Similarly, the 2'- and 3'- hydroxyl moieties of nucleotide, nucleosides and derivatives thereof may be protected.

Benzyloxymethyl-protected compounds can be synthesized using standard techniques known in the chemical arts. For example, 6-nitroveratryloxymethyl chloride (NV0MC1) is readily made from 6-nitroveratryl alcohol upon exposure to gaseous HCI and paraformaldehyde following the methods described in Organic Syntheses, Vol. 6, pp. 101-103 (1988), which is incorporated herein by reference. Reaction of NVOMC1 with the 5 '-hydroxyl group of a nucleoside to form the protected 5 '-NVOM nucleoside is readily performed using techniques such as those described in Greene, or by Corey, et al. , in Tetrahedron Letters, No. 11, pp. 809-812 (1976), which is incorporated herein by reference. For example, the hydroxy group to be protected is reacted with the activated form of the benzyloxymethyl protecting group in the presence of a tertiary amine to form the protected compound, a benzyloxy ethylether. Primary and secondary amines as well as carboxylic acids can protected in an analogous manner.

SUBSTITUTE SHEET

Examples of reactions to produce benzyloxymethyl- protected compounds via a chloromethyl ether intermediate are schematically presented in Fig. 5. As in Figs. 2 to 4 , X denotes substitution on the phenyl ring and R denotes substitution on the benzylic carbon. Typical reagents that can be used in the various reaction steps are as follows: (a) CH ₂ 0 and HCI; (b) R'OH and triethyl amine; (c) R'SH and triethylamine; (d) R"R'NH; (e) H ₂ NC(0)R' and triethylamine; and (f) R'C(0)0H and triethylamine.

If excess activated benzyloxymethyl compound is reacted with a primary amine to be protected, an amine having two protecting groups will be formed. Such protected compounds require two photons to be completely deprotected.

EX?ropJ,e j

A solution of 2,3-dihydroxy benzaldehyde (1.38 g, 0.01 mol), KF (2.90 g, 0.05 mol), and CH ₂ Br ₂ (1.91 g, 0.011 mol) in 30 mL of DMF was heated to 110-120°C for 2 hours. The reaction mixture was cooled to room temperature and partitioned between ether and water. The organic phase was washed (sat. NaCl) , dried (MgS0 ₄ ) , and evaporated to give 1.46 g of a light brown oil. Chromatography on silica gel with 10%

SUBSTITUTE SHEET

EtOAc in hexanes afforded 1.14 g of

2,3-methylenedioxybenzaldehyde as a light yellow solid.

The benzaldehyde compound was dissolved in 40 L of 70% HN0 ₃ cooled to 0°C. After stirring for 30 min., the reaction mixture was poured into cold water and extracted with EtOAc. The organic phase was washed (sat NaHC0 ₃ ) , dried (MgS0 ₄ ) , and evaporated to give 1.2 g of a yellow solid which was used without further purification. The solid was taken up in 25 mL of anhydrous THF and treated with NaBH ₄ (466 mg, 0.12 mol) at room temperature for 1 hour. The reaction mixture was partitioned between sat. NH ₄ C1 and EtOAc. The organic phase was dried (MgS0 ₄ ) and evaporated to afford 1.18 g of the benzyl alcohol as a light yellow solid, which was used without further purification. The alcohol was dissolved in 20 mL of CH ₂ C1 ₂ and treated with DMAP (0.26 g, 0.0018 mol) and Ac ₂ 0 (1.5 g, 0.15 mmol) for 18 hours. The reaction mixture was partitioned between CH ₂ C1 ₂ and 3 N HCI. The organic phase was washed (sat. NaHC0 ₃ ) , dried (MgS0 ₄ ) , and was evaporated to give 1.4 g of a mixture of the two regioisomers as a light yellow solid. Chromatography on silica gel with 10% hexanes in CH ₂ C1 ₂ afforded 0.15 g of the less polar compound 1 as a light yellow solid and 0.40 g of the more polar compound 2 as a light yellow solid and 0.69 g of a mixture of the two.

Example 2

A solution of veratrole (2.00 g; 14.5 mmol) and propionyl chloride (1.4 mL; 16.1 mmol) in 15 L of CS ₂ cooled to 0°C was treated with A1C1 ₃ (2.00 g; 15.0 mmol) . After stirring for 30 min. , the mixture was allowed to warm to room temperature and turned deep red in color. The reaction

SUB _S TITUTE SHEET

mixture was quenched after an additional 4.5 hours by cooling to 0°C and slowly adding water. The residue was partitioned between EtOAc and 1 N HCI, and the organic phase washed (sat. NaCl), dried (MgS0 ₄ ), and concentrated to yield 3.00 g of a yellow solid. The crude ketone was used without further purification.

The crude ketone was dissolved in 15 mL of glacial acetic acid and added to 100 mL of 70% HN0 ₃ cooled to 0°C. The reaction mixture darkened in color within 5 min. After stirring for 20 min. , the mixture was allowed to warm to room temperature for an additional 1 hour. The mixture was poured into cold water, and was extracted with EtOAc. The organic phase was washed (sat,. NaCl), dried (MgS0 ₄ ) , and evaporated to give a yellow solid. Recrystalization from MeOH/H ₂ 0 afforded 1.716 g (50% yield for two steps) of 3 as tan crystals.

Example 3

l,4-Benzodioxan-6-yl methyl ketone (1.00 g; 5.61 mmol) was dissolved in 25 mL of 70% HNO, cooled to 0°C. The reaction mixture darkened in color within 5 min. The solution was warmed to room temperature after 5 min and was quenched /after an additional 2.5 hours by pouring into cold water and was extracted with EtOAc. The organic phase was washed (sat. NaCl, sat NaHC0 ₃ ) , dried (MgS0 ₄ ) , and evaporated to give a tan solid which was used without further purification.

The crude nitrophenyl compound was dissolved in 25 mL of anhydrous THF and treated with NaBH^ (400 mg; 10.6 mmol) at room temperature. The solution darkened to dark brown after 2 min. After stirring for 30 mm. , 10 mL of EtOH was added and the stirring continued for an additional 30 min. The solution was partitioned between EtOAc and sat. NH ₄ C1, and

SUB _S TITUTESHEET

the organic phase was dried (MgS0 ₄ ) , and evaporated to afford a brown oil.

The crude benzyl alcohol was taken up in 20 mL of CH ₂ C1 ₂ and 6 L of pyridine and was treated with 2 mL of Ac ₂ 0 and 10 mg of DMAP for 14 hours. The reaction mixture was partitioned between EtOAc and 1 N HCI, and the organic phase was dried (MgS0 ₄ ) , and concentrated to give a brown oil. Chromatography on silica gel with 100% CH ₂ C1 ₂ gave 0.68 g of a yellow oil, which was shown to be a mixture of three compounds by NMR analysis. Chromatography of this mixture on silica gel with 25% EtOAc in hexanes afforded 255 mg of pure 5 as a light yellow solid, MP 82-85°C, and an additional 238 mg of slightly impure 5 as a light yellow solid.

Example 4

Piperonal (21.65 g; 129 mmol) was slowly added to 175 mL of 70% HN0 ₃ cooled to 10°C. The dark reaction mixture was allowed to warm to room temperature and stirred for 1 hour total. The resultant slurry was poured into cold water and filtered to give a light yellow solid. Recrystalization from MeOH/H ₂ 0 afforded 21.46 g of light yellow crystals having a melting point of 108-112 °C (80% yield).

To a solution of the nitroacetophenone (14.7 g; 70.4 mmol) in 500 L of MeOH and 100 mL of CHC1 ₃ was added NaBH^

(2.71 g; 71.6 mmol). After stirring for 5 hours, the solution was partitioned between CHC1, and sat. NH ₄ C1. The organic phase was dried (MgS0 ₄ ) , and evaporated to give a yellow solid. Recrystalization from MeOH/H ₂ 0 afforded 14.24 g of the alcohol as light yellow crystals (96% yield) .

A solution of the nitropiperonyl alcohol (1.00 g; 4.74 mmol) in 25 mL of THF was treated with phosgene (8 nl of

SUB _S TITUTESHEET

1.93 M in toluene; 15.4 mmol) at room temperature for 12 hours. The excess phosgene and solvent were removed under reduced pressure to give a tan solid, which was used without any further purification. A solution of the crude chloroformate in 10 mL of dioxane was added to a solution of L-phenylalanine (540 mg; 3.27 mmol) and NaHC0 ₃ (680 mg; 8.09 mmol) in 15 mL of H ₂ 0 and 5 mL of dioxane. The reaction mixture was a light brown, homogeneous solution. The solution was stirred at room temperature for 26 hours and then was partitioned between ether and 0.1 N NaOH. The ether _, phase was discarded and the aqueous phase was acidified to pH 2 with 3 N HCI and was extracted with CHC1 ₃ . The organic phase was dried (MgS0 ₄ ) and evaporated to give a white solid. Chromatography on silica gel with 15% MeOH in CHC1 ₃ afforded 1.11 g (84% yield) of

MeNPOC-L-Phe-OH as a white solid, MP 144-154 °C (decomposes).

Example 5

Alpha-methyl-6-nitropiperonyl alcohol was prepared as follows. All anhydrous reagents, methylene chloride, pyridine, triethylamine, and diisopropylethyl amine were obtained from Aldrich Chem Co. N-4-isobutyryl-2 '- deoxycytidine, N6-phenoxyacetyl-2 '-deoxyadenosine, N2- phenoxyacetyl-2 '-deoxyguanosine, 2 '-deoxythymidine, and 2- cyanoethyl-N,N-diisopropylchlorophosphoramidite were purchased from Sigma Chem Co. 4,5-methylenedioxy-2-nitroacetophenone was purchased from Lancaster Synthesis and 1.93M phosgene in toluene was from Fluke Chem Co. H NMR spectra were measured on a Varian Gemini 300 MHz Spectrometer and are reported in ppm. Mass Spectra were obtained from the U. C. Berkeley Mass Spectrometry Laboratory.

4,5-methylenedioxy-2-nitroacetophenone (30 g, 0.144 mol) was dissolved in 200ml ethanol and 10ml of tetrahydrofuran. Sodium borohydride (5.5 g, 0.144 mol) was

SUB _STI TUTESHEET

added to the stirred suspension over a 5 min. period. The suspension was stirred for an additional hour at room temperature, poured into 1.2L of ice water, and acidified to pH 2 with IN HCI. The yellow solid was collected by filtration, washed with water, and dried in vacuo. The solid was recrystallized from 1:1 toluene/hexane to yield 22.8 g (75%) of α-methyl-6-nitropiperonyl alcohol. ¹ H NMR (CDC1 ₃ ) : 1.55 (d, 3H) CH ₃ ; 5.45 (q, 1H) CH; 6.1 ( , 2H) 0-CH ₂ -0; 7.3 and 7.5 (s, 2H) aromatic. MS Alpha-methyl-6-nitropiperonyloxycarbonyl chloride was prepared as follows. Phosgene (500ml, 1.93M in toluene) was added to a rapidly stirring solution of (20 g, 0.095 mol) in 700ml anhydrous tetrahydrofuran . After stirring overnight at room temperature the mixture was evaporated to dryness in vacuo resulting in a thick brown oil. Tituration with hexane yielded 20 g (73%) of α-methyl-6-nitropiperonyloxycarbonyl chloride as a yellow-brown solid. ¹ H NMR (CDC1 ₃ ) : 1.7 (d, 3H) CH ₃ ; 6.15 (m, 2H) 0-CH ₂ -0; 6.5 (q, 1H) CH; 7.05 and 7.55 (s, 2H) aromatic. 5 '-0-(α-methy1-6-nitropiperonyloxycarbonyl) -2 '- deoxynucleosides was prepared as follows. N-protected deoxynucleoside (34 mmol) was evaporated twice from 100ml anhydrous pyridine and dried in vacuo. The resultant residue was dissolved in 150 ml anhydrous pyridine, cooled to 0°C in an ice water bath, and 37 mmol added. After stirring for 15 min. at 0°C the ice bath was removed and the reaction stirred for 4-5 hours at room temperature. The mixture was concentrated in vacuo to an oil and taken up in 100 ml of methylene chloride. The solution was washed successively with saturated sodium bicarbonate (25 ml) , water (25 ml) , and saturated sodium chloride (25 ml) . The organic phase was dried over anhydrous sodium sulfate and the solvent removed by rotary evaporation. The reaction product was purified by silica gel chromatography, eluting with 10% (v/v) methanol in methylene chloride. Fractions containing the major product were pooled and dried in vacuo.

5 •-O-(α-methyl-6-nitropiperonyloxycarbonyl) -2 '- deoxythymidine: yield 60%; R = 0.47 (silica, methylene

SUBSTITUTE SHEET

chloriαe/ ethanol 9:1); ^: H NMR (CDCl- : 1.7 (d, 3K) CH ₃ ; 1.9 (s, 3H) CH ₃ ; 2.0-2.4 (m, 2H) 2 ^• -CH ₂ ; 4.1-4.5 (m, 4H) 3'- H, 4'-H, 5'-CH ₂ ; 6.1 (m, 2H) 0-CH ₂ -0; 6.3 (m, 2H) l'-H, CH (benzyl); 7.0 and 7.5 (s and m, 2H) aromatic; 7.35 (s, 1H) 6H. MS

5 '-O-(ce-methyl-6-nitropiperonyloxycarbonyl) -N6- phenoxyacetyl-2 '-deoxyadenosine: yield 52%; R _f = 0.69 (silica, methylene chloride/methanol 9:1); ^λ H NMR (CDC1 ₃ ) : 1.65 (d, 3H) CH ₃ ; 2.6 and 2.9 (m, 2H) 2 '-CH ₂ ; 4.2-4.4 ( , 4H) 3'-H, 4'-H, 5 '-CH ₂ ; 4.9 (d, 2H) CO-CH ₂ -0; 6.25 (m, 1H) l'-H; 6.5 (q, 1H) CH (benzyl); 7.05 (m, 4H) aromatic (PAC) , aromatic (nitrobenzyl); 7.35 (m. 2H) aromatic (PAC); 7.45 (s, 1H) aromatic (nitrobenzyl); 8.25 and 8.75 (d, 2H) 2H, 8H; 9.4 (bs, 1H) NH. 5 '-0-(α-methyl-6-nitropiperonyloxycarbonyl) -N2- phenoxyacetyl-2 '-deoxyguanosine: yield 70%; R _f = 0.57 (silica, methylene chloride/methanol 6:1); 1H NMR (CDC1 ₃ /CD ₃ 0D) : 1.6 (dd, 3H) CH ₃ ; 2.4-2.8 (m, 2H) 2 '-CH ₂ ; 4.2-4.6 (m, 4H) 3'-H, 4'-H, 5'-CH ₂ ; 4.7 (s, 2H) CO-CH ₂ -0; 6.1-6.4 (m, 4H) 0-CH ₂ -0, l'-H, CH (nitrobenzyl); 6.9-7.15 (m, 4H) aromatic (PAC), aromatic (nitriobenzyl) ; 7.3-7.42 (m, 3H) aromatic (PAC), aromatic (nitrobenzyl); 8.1 (s, 1H) 8H. MS

5 '-0-(α-methyl-6-nitropiperonyloxycarbonyl) -N4- isobutyryl-2 '-deoxycytidine: yield 60%; R = 0.56 (silica, methylene chloride/methanol 9:1); 1H NMR (CDC1 ₃ ) : 1.2 (dd, 6H) 2CH ₃ (ibu); 1.65 (dd, 3H) CH ₃ , 2.1 (m, 1H) 2 ^» -H; 2.5- 2.8 (m, 2H) 2'-H, CH (ibu); 4.2-4.5 (m, 4H) 3'-H, 4'-H, 5'- CH ₂ ; 6.1 (m, 2H) 0-CH ₂ -0; 6.3 (m, 2H) l'-H, CH (benzyl) ; 7.0 and 7.49 (d, 2H) aromatic; 7.45 and 8.0 (dd, 2H) 5H, 6H; 8.35 (bs, 1H) NH. MS

5 '-O-(α-methy1-6-nitropiperonyloxycarbonyl) -2 '- deoxynucleoside 3 *-0-(2-cyanoethyl)-N,N'- diisopropylaminophosphites were prepared as follows. 5'- protected deoxynucleoside (10 mmol) was dissolved in 75 ml of methylene chloride. Diisopropylethylamine (30 mmol) was added and the solution cooled to 0°C in an ice water bath. 2- cyanoethyl-N,N'-diisopropylchlorophosphoramidite (25 mmol) was slowly added to the cooled mixture. The ice bath was removed

SUBSTITUTE SHEET

and the reaction stirred for 2 hours at room temperature. Anhydrous methanol (5 ml) was added and the reaction stirred for an additional 30 min. at room temperature. The mixture was diluted with methylene chloride (150 ml) and washed with saturated sodium bicarbonate (50 ml) followed by water (50 ml) . The organic phase was dried over anhydrous sodium sulfate and the solvent removed in vacuo. The product was purufied by silica gel chromatography, eluting with 45:45:10 ethyl acetate/methylene chloride/triethyla ine. Fractions containing the major product were pooled and dried in vacuo.

5'-0-(α-methyl-6-nitropiperonyloxycarbonyl)-2 *- deoxythymidine 3 '-O-(2-cyanoethy1)-N,N'- diisopropylphosphoramidite: yield 80%; R _f = 0.58 (silica, 45:45:10 ethyl acetate/methylene chloride/triethylamine) ; -"H NMR (CDC1 ₃ ): 1.1-1.3 (m, 12H) CH ₃ (isopropyl) ; 1.65 (d, 3H) CH ₃ (nitrobenzyl); 1.9 (s, 3H) .CH ₃ ; 2.1-2.3 (m, 1H) 2'-H; 2.4-2.7 (m, 3H) 2'-H, CH ₂ -CN; 3.5-3.9 (m, 4H) NH (isopropyl), P-0-P-CH ₂ ; 4.1-4.5 (m, 4H) 3'-H, 4'-H, 5'-CH ₂ ; 6.15 (s, 2H) 0-CH ₂ -0; 6.3 (m, 2H) l'-H, CH (nitrobenzyl); 7.0 and 7.5 (ε, 2H) aromatic; 7.3 (m, 1H) 6H; 8.4 (bs, 1H) NH (thymidine). MS

5 '-0-(α-methyl-6-nitropiperonyloxycarbonyl) -N4- isobutyryl-2 '-deoxycytidine 3 '-0-(2-cyanoethy1) -N,N'- diisopropylphosphoramidite: yield 85%; R _f = 0.52 (silica, 45:45:10 ethyl acetate/methylene chloride/triethylamine) ; ¹ H NMR (CDC1 ₃ ): 1.1-1.3 (m, 18H) CH ₃ (isopropyl), CH ₃ (ibu) ; 1.65 (m, 3H) CH ₃ (nitrobenzyl); 2.1 (m, 1H) 2'-H; 2.55-2.8 (m, 3H) 2'-H, CH ₂ -CN; 3.5-3.9 (m, 4H) NH (isopropyl), P-O- CH ₂ ; 4.2-4.5 (m, 4H) 3'-H, 4'-H, 5'-CH ₂ ; 6.1-6.35 (m, 4H) O- CH ₂ -0, l'-H, CH (nitrobenzyl); 7.0 and 7.5 (d, 2H) aromatic; 7.4 and 7.95 (m, 2H) 5-H, 6H; 8.25 (bs, 1H) NH (amide) . MS

5 '-0-(α-methyl-6-nitropiperonyloxycarbonyl) -N6- phenoxyacetyl-2 '-deoxyadenosine 3 '-o-(2-cyanoethy1) -N,N'- diisopropylphosphoramidite: yield 81%; R--= 0.50 (silica, 45:45:10 ethyl acetate/methylene chloride/triethylamine) ; ¹ H NMR (CDC1 ₃ ): 1.1-1.3 (m, 12H) CH-. (isopropyl); 1.65 (m, 3H) CH, (nitrobenzyl); 2.6-3.0 ( , 4H) 2'-H, CH _: -CN; 3.6-3.9 (m, 4H) NH (isopropyl), P-O-CH-; 4.3-4.6 (m, 4H) 3'-H, 4'-H, 5'-

SUB _S TITUTE SHEET

CH2 ' 4.7 (2s, 2H) CO-CH--0; 6.1 (r., 2H) 0-CH ₂ -0; 6.3 (-. , 1H) l'-H; 6.5 (m, 1H) CH (nitrobenzyl); 7.1 (m, 4H) aromaitc (nitrobenzyl), aromatic (PAC); 7.35 ( , 2H) aromatic (PAC) ; 7.45 (s, 1H) aromatic (nitrobenzyl); 8.2 and 8.8 (s, 2H) 2H, 8H; 9.4 (bs, 1H) NH (amide). MS

5 '-O-(α-methy1-6-nitropiperonyloxycarbonyl) -N2- phenoxyacetyl-2 '-deoxyguanosine 3 '-0-(2-cyanoethyl)-N,N'- diisopropylphosphoramidite: yield 75%; R = 0.55 (silica, 9:1 methylene chloride/methanol); ¹ H NMR (CDC1 ₃ ) : 1.3 (d, 12H) CH ₃ (isopropyl); 1.6 (dd, 3H) CH ₃ (nitrobenzyl); 2.5-2.9 (m, 4H) 2'-H, CH ₂ -CN; 3.55-3.95 (m, 4H) NH (isopropyl), P-0-CH ₂ ; 4.3-4.6 (m, 4H) 3 ^» -H, 4--H, 5 '-CH ₂ ; 4.7 (2s, 2H) CO-CH ₂ -0; 6.1-6.35 ( , 4H) O-CH ₂ -0, l'-H, CH (nitrobenzyl); 6.95-7.15 (m, 4H) aromaitc (nitrobenzyl), aromatic (PAC); 7.35-7.45 (m, 3H) aromatic (PAC), aromatic (nitrobenzyl); 7.9 (s, 1H) 8H. MS

2, 3-Dimethoxybezaldehyde (1.00 g; 6.02 mmol) was added to 10 L of 70% HN0 ₃ cooled to 0°C. The solution was warmed to room temperature after 5 min and stirring was continued for an additional 10 min. The reaction mixture was poured into cold water and filtered to give 1.16 g of an off-white solid precipitate. The crude 1:1 mixture of regioisomers was used without further purification.

SUBSTITUTE SHEET

The crude nitrobenzaldehyde was reduced Py treating a solution of the aldehyde (501 mg; 2.37 mmol) in 25 mL of anhydrous THF with NaBH (185 mg; 4.89 mmol) cooled to 15°C. After stirring the solution for 10 min. , 3 mL of MeOH was added to aid solubility. The reaction mixture was stirred an additional 30 min. and was then partitioned between EtOAc and sat. NH ₄ C1. The organic phase was washed (sat. NaCl) , dried (MgS0 ₄ ) , and evaporated to give 0.56 g of a 1:1 mixture of the two regioisomers as a white crystalline solid. The alcohols were used without further purification.

A solution of the crude alcohols (0.56 g) in 10 mL of CHC1 ₃ was treated with 5 mL of pyridine and 2 mL of Ac20 for 3 hours at room temperature. The reaction mixture was partitioned between CHC13 and 1 NHC1, and the organic phase was dried (MgS0 ₄ ) and evaporated to give .6 g of a colorless oil. Chromatography on silica gel with 25% EtOAc in hexanes afforded 314 mg of 6 as a white solid (52% yield for the two steps) and 261 mg of 7 as a white solid (43% yield for the two steps) .

Nitroveratryloxy ethylchloride (NV0M-C1) was prepared as follows. To a suspension/solution of 5g (0.0235 mol) of NV-OH in 50 ml of toluene was added l.Og (0.033 mol) dry powdered paraformaldehyde [(CH ₂ 0) _n ] with vigorous stirring. HCl(g) was bubbled into the solution (no cooling) until all of the solid had gone into solution (30 min) at which time the solution turned dark green with a black aqueous layer coating the walls of the flask. The mixture was then allowed to stand at room temperature for approximately 30 min and the green solution was then decanted off the black aqueous

SUB _ST ITUTE SHEET

layer. To the toluene solution was added 30 ml of toluene and 20 ml of hexane followed by 2.5 g of MgS0 ₄ (anhydrous) . The solution was stirred at approximately 10°C for 15 min, filtered, and the filtrate rotary evaporated (at approximately 30°C) to an amber oil. The oil was placed on a vacuum line whereupon yellow crystals began to come out of the oil. The semi-solid was then triturated 3 times with 40 ml of hexane each time decanting off the hexane. The yellow crystalline residue was then dried in vacuum at room temperature. The yield was 5.67 g on 92%.

An NVOM 3-estradiol-benzylether was prepared as follows. To a mixture of 400 mg (1.105 mmol) of β-estradiol- 3-benzyl ether and 434 mg (1.66 mmol) of NV0M-C1 in 5 ml of CH ₂ C1 ₂ was added 213 mg (1.65 mmol) of DIE. After stirring overnight at room temperature the mix was with H ₂ 0, 0. IN HCI, dried (Na ₂ S0 ₄ ) and solvent removed. The yield was 584 mg or 90%. NVOM protected 2-deoxyadenosine was prepared by an analogous method.

VI. Multiple-guantum Protecting Groups

In most of the above described compounds, only a single photon is required (in theory) to photolyze a single molecule of the compound. However, some photoreactive groups of this invention reguire two or more photons for photolysis. Such compounds may be useful during light-directed synthesis on substrates to prevent unwanted deprotections caused by the diffraction or light around the edges of the mask (see the discussion of binary synthesis strategies above) . Compounds useful for this type of "multiple quantum" deprotection include those in which a first photon alters the photoactive

SUBSTITUTE SHEET

isomerization, redox reaction, cyclization, or deprotection of a functional group on the protecting group itself. Upon exposure to the second photon, the altered protecting group is then removed. One example of such a deprotection is shown below:

OMe OMe

It will be appreciated by those skilled in the art that the compound shown in the example above is but one of many compounds which require two photons to become deprotected.

VII. Photolysis of Protected Group Compounds Removal of the protecting group is accomplished by irradiation to separate the reactive group and the degradation products derived from the protecting group. Not wishing to be bound by theory, it is believed that irradiation of NVOC- and MeNVOC-protected oligomers, for example, occurs by the following reaction schemes:

NVOC-AA -> 3 ,4-dimethoxy-6-nitrosobenzaldehyde + C0 ₂ + AA MeNV0C-AA-> 3 ,4-dimethoxy-6-nitrosoacetophenone + CO- +AA

where AA represents the N-terminus of an amino acid or oligomer.

Along with the unprotected amino acid, other

SUB _S TITUTE SHEET

3 , 4-dimethoxy-6-nitrosophenylcarbonyl compound, which can react with nucleophilic portions of the oligomer to form unwanted secondary reactions. In the case of an NVOC- protected amino acid, the degradation product is a nitrosobenzaldehyde, while the degradation product for MeNVOC is a nitrosophenyl ketone. It is believed that the product aldehyde from NVOC degradation reacts with free amines to form a Schiff base (imine) that affects the remaining polymer synthesis. Preferred photoremovable protecting groups react slowly or reversibly with the oligomer on the support.

Not wishing to be bound by theory, it is believed that the product ketone from irradiation of a MeNVOC-protected oligomer reacts at a slower rate with nucleophiles on the oligomer than the product aldehydes from irradiation of the same ΝVOC-protected oligomer. Although not unambiguously determined, it is believed that this difference in reaction rate arises from radical stabilization. However, it may also be due to the difference in general reactivity between aldehydes and ketones towards nucleophiles due to steric and electronic effects.

In some preferred embodiments, scavengers or other reagents are added to the reaction mixture to react with and render harmless photolysis byproducts that might otherwise react with a growing oligomer. Suitable scavengers (which are often nucleophiles) will be known to those of skill in the art. Specific examples include acids, bisulfites, hydroxy- containing compounds, amines, etc.

Because photolysis is a first order process, the protected compound must be illuminated for nine half-lives to effect 100% removal. Very fast deprotection rates are therefore desired because they lessen the overall exposure time, reducing the effects of stray light and the total time necessary to synthesize a polymer.

The photolysis of Ν-protected L-phenylalanine in solution having different photoremovable protecting groups was analyzed, and the results are presented in the following table:

SUBSTITUTE SHEET

Photolysis of Protected L-Phe-OH

t _{x 2} i- ^~ seconds

Solvent NBOC NVOC MeNVOC MeNPOC

Dioxane 1288 110 24 19

5mM H ₂ S0 ₄ /Dioxane 1575 98 33 22

The half life, t_ ₂ is the time in seconds required to remove 50% of the starting amount of protecting group. The photolysis was carried out in the indicated solvent with 362/364 nm-wavelength irradiation having an intensity of 10 mW/cm ² , and the concentration of each protected phenylalanine was 0.10 mM. The table shows that deprotection of NVOC-, MeNVOC-, and MeNPOC-protected phenylalanine proceeded faster than the deprotection of NBOC. Furthermore, it shows that the deprotection of the two derivatives that are substituted on the benzylic carbon, MeNVOC and MeNPOC, were photolyzed at the highest rates in both dioxane and acidified dioxane.

Another photolysis study was performed on various MeNPOC nucleosides. The t - ₂ rates for the disappearance of starting material are tabulated below and are normalized to a photolysis intensity of 10 mW/ciir power using the 350-450 nm dichroic reflector on a Hg(Xe) arc lamp. DTT was intended to act as scavenger for the nitrosoketone byproduct and was included in the study to see if it had any effect on the photolysis rates.

Table II

SUB _S T _I TUTE SHEET

In another photolysis study, a 0. ImM solution of each of the four 5 '-protected nucleosides prepared in Example 5, MeNPoc-dT, MeNPoc-dC , MeNPoc-dG , and MeNPoc-dA was prepared in dioxane. A 200 μL aliquot of one of the four deoxynucleoside solutions was irradiated with 350-450nm light at 14.5 mW/cm ² (Oriel) in a narrow path (2mm) quartz cuvette for a given period of time x_ . A fresh aliquot of the O.lmM solution was irradiated as before for a different time x ₂ . Four or five time points were collected for each MeNPoc- deoxynucleosides.

Each time point was analyzed for loss of starting material at 280 nm on a nucleosil 5-C ₈ HPLC column using a mobile phase of 60% (v/v) CH ₃ CN in water containing 0.1% (v/v) TFA. MeNPOC-dT required a mobile phase of 70% (v/v) methanol in water. The integrated peak area of the remaining 5 '- MeNPoc-2 '-deoxynucleoside was calculated. A plot of natural logarithm of area vs. time resulted in a straight line of slope k where t-_ ₂ = 0.639/k.

In other photolysis experiments, the deprotection of nitroveratryloxymethyl protected compounds was shown to compare favorably with certain NVOC and MeNPOC compounds.

. α . S - <

M OS DIOX NE 5mM 5 ^~ y.

SUBSTITUTE SHEET

λ ^* .r

365 131 125 102 315 4.214 C

365 79.: 97.1 = 3 . / 247 4,774:

365 35.3

365 10.4

365 13.1

Still other photolysis results are shown below. In the second solvent, 5mM H ₂ S0, is used and in the third solvent 5mM DTT is used. The photolysis radiation was provided at a wavelength of 365 nm and an intensity of 10 mW/cm". The

ITUTE SHEET

listed values in the chart are t- ₂ in seconds. The various compounds are shown in Fig. 6.

Table V

VIII. Basis Sets of Photoprotected Amino or Nucleic Acids The deprotection rate (or photolysis half life) of a protected compound is a function of the particular photosensitive protecting group as well as the amino or nucleic acid functional group being protected. It is also a function of the solvent in which photolysis is performed and

SHEET

neutralize photolysis decomposition products. As illustrated in the above Tables, the deprotection rate can vary substantially for the basis set of naturally occurring deoxynucleosides, each protected with the same photosensitive group. It has also been observed that the set of NVOC- protected amino acids (naturally occurring) have photolysis half lives ranging from 70-150 seconds.

In VLSIPS™, it is desirable that the protected compounds are deprotected at substantially the same rate to prevent unwanted side reactions between the byproducts described above and the unprotected amino acids. In other words, monomers that deprotect earliest are exposed to reactive photolysis byproducts until the substrate is washed. If all oligomers on the substrate deprotect at the same rate, the time of exposure will be minimized. In VLSIPS™, it is also desirable to minimize the exposure time so that stray light from an illuminated region does not deprotect compounds in adjacent regions. To ensure a minimal exposure time, it is preferred to match the various photoreactive protecting groups of the invention with different amino acids (or other monomer types) so that all of the amino acids will be deprotected at substantially the same rate in a particular solvent. Sets of such rate-matched photoprotected amino acids or nucleosides are hereinbelow referred to as "basis sets". It will be appreciated that basis sets can be created for any class or subclass of compounds, most notably the natural or unnatural amino acids and the natural or unnatural nucleic acids.

"At substantially the same rate" is defined herein to mean that the half-life for deprotection of the most rapidly deprotected compound in the basis set is within about 25% of the compound with the slowest deprotection half-life in a particular solvent. Preferably, the differences between half- lives is about 10% and most preferably about 5%.

One preferred class of compounds for forming a basis set is a plurality of the twenty naturally-occurring amino acids. Another preferred class is a subset of the D-isomers of the twenty naturally-occurring amino acids. Yet another

SUBSTITUTE SHEET

include 3 amino acids and amino acids whose side chains have been altered in order to adjust the stearic bulk, hydrophobicity, or electronic character of the corresponding natural amino acid. Such amino acid analogs have found increasing utility in drug research (see, e.g., Fauchere) .

Still another preferred class is formed by the common nucleosides adenosine, thymidine, guanosine, cytidine, and inosine in addition to their 2', or 3 '-deoxy analogs and their 2', 3', or 5 '-phosphates. "Phosphate" is meant herein to include the phosphotriesters (-OP(OR") (OR ^* ') ) , phosphoramidites (-OP(OR") (NRR ') , where R-R"" are described above), as well as phosphate anions (-OP(0)0 ₂ H ^~ , and -0P(0)0 ₂ ^" ^) .

Typically, the choice of protecting groups will depend on the character of the solvent 'Which, as described above, has an important effect on the rate of deprotection. One preferred solvent for both amino acids and nucleosides is dioxane. Another preferred solvent is acetonitrile.

IX. Conclusion The above description is illustrative and not restrictive. Many variations of the invention will become apparent to those of skill in the art upon review of this disclosure. Merely by way of example, while the invention is illustrated primarily with regard to peptide and nucleotide synthesis, the invention is not so limited. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents.

SUBSTITUTE SHEET