Synthesis of oligonucleotide arrays using photocleavable protecting groups

Novel compounds are provided which are useful as linking groups in chemical synthesis, preferably in the solid phase synthesis of oligonucleotides and polypeptides. These compounds are generally photolabile and comprise protecting groups which can be removed by photolysis to unmask a reactive group. The protecting group has the general formula Ar--C(R.sub.1)(R.sub.2)--O--C(O)-- wherein: PA1 Ar is an optionally substituted fused polycyclic aryl or heteroaromatic group or a vinylogous derivative thereof; PA1 R.sub.1 and R2 are independently H, optionally substituted alkyl, alkenyl or alkynyl, optionally substituted aryl or optionally substituted heteroaromatic, or a vinylogous derivative of the foregoing; and PA1 X is a leaving group, a chemical fragment linked to Ar--C(R.sub.1)(R.sub.2)--O--C(O)-- via a heteroatom, or a solid support; provided that when Ar is 1-pyrenyl and R.sub.1 =R.sub.2 =H, X is not linked to Ar--C(R.sub.1)(R.sub.2)--O--C(O)-- via a nitrogen atom. Preferred embodiments are those in which Ar is a fused polycyclic aromatic hydrocarbon and in which the substituents on Ar, R.sub.1 and R.sub.2 are electron donating groups. A particularly preferred protecting group is the "PYMOC" protecting group, pyrenylmethyloxycarbonyl, where Ar=pyrenyl and R.sub.1 =R.sub.2 =H. Also provided is a method of forming, from component molecules, a plurality of compounds on a support, each compound occupying a separate predefined region of the support, using the protected compounds described above.

BACKGROUND OF THE INVENTION 
The present invention relates to the area of chemical synthesis. More 
particularly, this invention relates to photolabile compounds, reagents 
for preparing the same and methods for their use as photocleavable linkers 
and protecting groups, particularly in the synthesis of high density 
molecular arrays on solid supports. 
The use of a photolabile molecule as a linker to couple molecules to solid 
supports and to facilitate the subsequent cleavage reaction has received 
considerable attention during the last two decades. Photolysis offers a 
mild method of cleavage which complements traditional acidic or basic 
cleavage techniques. See, e.g., Lloyd-Williams et al. (1993) Tetrahedron 
49:11065-11133. The rapidly growing field of combinatorial organic 
synthesis (see, e.g., Gallop et al. (1994) J. Med. Chem. 37:1233-1251; and 
Gordon et al. (1994) J. Med. Chem. 37:1385-1401) involving libraries of 
peptides and small molecules has markedly renewed interest in the use of 
photolabile linkers for the release of both ligands and tagging molecules. 
A variety of ortho-benzyl compounds as photolabile protecting groups have 
been used in the course of optimizing the photolithographic synthesis of 
both peptides (see Fodor et al. (1994) Science 251:767-773) and 
oligonucleotides (see Pease et al. Proc. Natl. Acad. Sci. USA 
91:5022-5026). See PCT patent publication Nos. WO 90/15070, WO 92/10092, 
and WO 94/10128; see also U.S. patent application Ser. No. 07/971,181, 
filed Nov. 2, 1992, (now abandoned) and Ser. No. 08/310,510, filed Sep. 
22, 1994 (now abandoned); Holmes et al. (1994) in Peptides: Chemistry, 
Structure and Biology (Proceedings of the 13th American Peptide 
Symposium); Hodges et al. Eds.; ESCOM: Leiden; pp. 110-12, each of these 
references is incorporated herein by reference for all purposes. Examples 
of these compounds included the 6-nitroveratryl derived protecting groups, 
which incorporate two additional alkoxy groups into the benzene ring. 
Introduction of an a-methyl onto the benzylic carbon facilitated the 
photolytic cleavage with &gt;350 nm UV light and resulted in the formation of 
a nitroso-ketone. 
Photocleavable protecting groups and linkers should be stable to a variety 
of reagents (e.g., piperidine, TFA, and the like); be rapidly cleaved 
under mild conditions; and not generate highly reactive byproducts. The 
present invention provides such protecting groups and methods for their 
use in synthesizing high density molecular arrays. 
SUMMARY OF THE INVENTION 
According to a first aspect of the invention, novel compounds are provided 
which are useful as linking groups in chemical synthesis, preferably in 
the solid phase synthesis of oligonucleotides and polypeptides. These 
compounds are generally photolabile and comprise protecting groups which 
can be removed by photolysis to unmask a reactive group. The protecting 
group has the general formula Ar--C(R.sub.1)(R.sub.2)--O--C(O)-- wherein: 
Ar is an optionally substituted fused polycyclic aryl or heteroaromatic 
group or a vinylogous derivative thereof; 
R.sub.1 and R.sub.2 are independently H, optionally substituted alkyl, 
alkenyl or alkynyl, optionally substituted aryl or optionally substituted 
heteroaromatic, or a vinylogous derivative of the foregoing; and 
X is a leaving group, a chemical fragment linked to 
Ar--C(R.sub.1)(R.sub.2)--O--C(O)-- via a heteroatom, or a solid support; 
provided that when Ar is 1-pyrenyl and R.sub.1 =R.sub.2 =H, X is not 
linked to Ar--C(R.sub.1)(R.sub.2)--O--C(O)-- via a nitrogen atom. 
Preferred embodiments are those in which Ar is a fused polycyclic aromatic 
hydrocarbon and in which the substituents on Ar, R.sub.1 and R.sub.2 are 
electron donating groups. Particularly preferred protecting groups are the 
"PYMOC" protecting group, pyrenylmethyloxycarbonyl, where Ar=1-pyrenyl and 
R.sub.1 =R.sub.2 =H, and the "ANMOC" protecting group, 
anthracenylmethyloxycarbonyl, where Ar=anthracenyl and R.sub.1 =R.sub.2 
=H. Methods are provided for preparing these compounds. 
This invention also provides reagents of the molecular formula 
Ar--C(R.sub.1)(R.sub.2)--O--C(O)--X, where Ar, R.sub.1, and R.sub.2 have 
the meanings ascribed above, for incorporating the protecting group into 
the molecule desired to be protected. 
Another aspect of this invention provides a method of attaching a molecule 
with a reactive site to a support comprising the steps of: 
(a) providing a support with a reactive site; 
(b) binding a molecule to the reactive site, the molecule comprising a 
masked reactive site attached to a photolabile protecting group of the 
formula Ar--C(R.sub.1)(R.sub.2)--O--C(O)-- ,wherein: 
Ar is an optionally substituted fused polycyclic aryl or heteroaromatic 
group or a vinylogously substituted derivative of the foregoing; 
R.sub.1 and R.sub.2 are independently H, optionally substituted alkyl, 
alkenyl or alkynyl, or optionally substituted aryl or heteroaromatic group 
or a vinylogously substituted derivative of the foregoing; 
to produce a derivatized support having immobilized thereon the molecule 
attached to the photolabile protecting group; and 
(c) removing the photolabile protecting group to provide a derivatized 
support comprising the molecule with an unmasked reactive site immobilized 
thereon. 
A related aspect of this invention provides a method of forming, from 
component molecules, a plurality of compounds on a support, each compound 
occupying a separate predefined region of the support, said method 
comprising the steps of: 
(a) activating a region of the support; 
(b) binding a molecule to the region, said molecule comprising a masked 
reactive site linked to a photolabile protecting group of the formula 
Ar--C(R.sub.1)(R.sub.2)--O--C(O)--, wherein: 
Ar is an optionally substituted fused polycyclic aryl or heteroaromatic 
group or a vinylogously substituted derivative of the foregoing; 
R.sub.1 and R.sub.2 are independently H, optionally substituted alkyl, 
alkenyl or alkynyl, or optionally substituted aryl or heteroaromatic group 
or a vinylogously substituted derivative of the foregoing; 
(c) repeating steps (a) and (b) on other regions of the support whereby 
each of said other regions has bound thereto another molecule comprising a 
masked reactive site linked to the photolabile protecting group, wherein 
said another molecule may be the same or different from that used in step 
(b); 
(d) removing the photolabile protecting group from one of the molecules 
bound to one of the regions of the support to provide a region bearing a 
molecule with an unmasked reactive site; 
(e) binding an additional molecule to the molecule with an unmasked 
reactive site; 
(f) repeating steps (d) and (e) on regions of the support until a desired 
plurality of compounds is formed from the component molecules, each 
compound occupying separate regions of the support. 
This method finds particular utility in synthesizing high density arrays of 
nucleic acids on solid supports.

DESCRIPTION OF THE PREFERRED EMBODIMENT 
The following definitions are set forth to illustrate and define the 
meaning and scope of the various terms used to describe the invention 
herein. 
The term "alkyl" refers to a branched or straight chain acyclic, monovalent 
saturated hydrocarbon radical of one to twenty carbon atoms. 
The term "alkenyl" refers to an unsaturated hydrocarbon radical which 
contains at least one carbon--carbon double bond and includes straight 
chain, branched chain and cyclic radicals. 
The term "alkynyl" refers to an unsaturated hydrocarbon radical which 
contains at least one carbon--carbon triple bond and includes straight 
chain, branched chain and cyclic radicals. 
The term "aryl" refers to an aromatic monovalent carbocyclic radical having 
a single ring (e.g., phenyl) or two condensed rings (e.g., naphthyl), 
which can optionally be mono-, di-, or tri-substituted, independently, 
with alkyl, lower-alkyl, cycloalkyl, hydroxylower-alkyl, aminolower-alkyl, 
hydroxyl, thiol, amino, halo, nitro, lower-alkylthio, lower-alkoxy, 
mono-lower-alkylamino, di-lower-alkylamino, acyl, hydroxycarbonyl, 
lower-alkoxycarbonyl, hydroxysulfonyl, lower-alkoxysulfonyl, 
lower-alkylsulfonyl, lower-alkylsulfinyl, trifluoromethyl, cyano, 
tetrazoyl, carbamoyl, lower-alkylcarbamoyl, and di-lower-alkylcarbamoyl. 
Alternatively, two adjacent positions of the aromatic ring may be 
substituted with a methylenedioxy or ethylenedioxy group. Typically, 
electron-donating substituents are preferred. 
The term "heteroaromatic" refers to an aromatic monovalent mono- or 
poly-cyclic radical having at least one heteroatom within the ring, e.g., 
nitrogen, oxygen or sulfur, wherein the aromatic ring can optionally be 
mono-, di- or tri-substituted, independently, with alkyl, lower-alkyl, 
cycloalkyl, hydroxylower-alkyl, aminolower-alkyl, hydroxyl, thiol, amino, 
halo, nitro, lower-alkylthio, lower-alkoxy, mono-lower-alkylamino, 
di-lower-alkylamino, acyl, hydroxycarbonyl, lower-alkoxycarbonyl, 
hydroxysulfonyl, lower-alkoxysulfonyl, lower-alkylsulfonyl, 
lower-alkylsulfinyl, trifluoromethyl, cyano, tetrazoyl, carbamoyl, 
lower-alkylcarbamoyl, and di-lower-alkylcarbamoyl. For example, typical 
heteroaryl groups with one or more nitrogen atoms are tetrazoyl, pyridyl 
(e.g., 4-pyridyl, 3-pyridyl, 2-pyridyl), pyrrolyl (e.g., 2-pyrrolyl, 
2-(N-alkyl)pyrrolyl), pyridazinyl, quinolyl (e.g. 2-quinolyl, 3-quinolyl 
etc.), imidazolyl, isoquinolyl, pyrazolyl, pyrazinyl, pyrimidinyl, 
pyridonyl or pyridazinonyl; typical oxygen heteroaryl radicals with an 
oxygen atom are 2-furyl, 3-furyl or benzofuranyl; typical sulfur 
heteroaryl radicals are thienyl, and benzothienyl; typical mixed 
heteroatom heteroaryl radicals are furazanyl and phenothiazinyl. Further 
the term also includes instances where a heteroatom within the ring has 
been oxidized, such as, for example, to form an N-oxide or sulfone. 
The term "optionally substituted" refers to the presence or lack thereof of 
a substituent on the group being defined. When substitution is present the 
group may be mono-, di- or tri-substituted, independently, with alkyl, 
lower-alkyl, cycloalkyl, hydroxylower-alkyl, aminolower-alkyl, hydroxyl, 
thiol, amino, halo, nitro, lower-alkylthio, lower-alkoxy, 
mono-lower-alkylamino, di-lower-alkylamino, acyl, hydroxycarbonyl, 
lower-alkoxycarbonyl, hydroxysulfonyl, lower-alkoxysulfonyl, 
lower-alkylsulfonyl, lower-alkylsulfinyl, trifluoromethyl, cyano, 
tetrazoyl, carbamoyl, lower-alkylcarbamoyl, and di-lower-alkylcarbamoyl. 
Typically, electron-donating substituents such as alkyl, lower-alkyl, 
cycloalkyl, hydroxylower-alkyl, aminolower-alkyl, hydroxyl, thiol, amino, 
halo, lower-alkylthio, lower-alkoxy, mono-lower-alkylamino and 
di-lower-alkylamino are preferred. 
The term "electron donating group" refers to a radical group that has a 
lesser affinity for electrons than a hydrogen atom would if it occupied 
the same position in the molecule. For example, typical electron donating 
groups are hydroxy, alkoxy (e.g. methoxy), amino, alkylamino and 
dialkylamino. 
The term "leaving group" means a group capable of being displaced by a 
nucleophile in a chemical reaction, for example halo, nitrophenoxy, 
pentafluorophenoxy, alkyl sulfonates (e.g., methanesulfonate), aryl 
sulfonates, phosphates, sulfonic acid, sulfonic acid salts, and the like. 
"Activating group" 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 can be 
used in the place of a hydroxyl group include --O(CO)Cl; --OCH.sub.2 Cl; 
--O(CO)OAr, where Ar is an aromatic group, preferably, a p-nitrophenyl 
group; --O(CO) (ONHS); and the like. 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 groups are known to those of skill in 
the art. 
"Chemical library" or "array" is an intentionally created collection of 
differing molecules which can be prepared either synthetically or 
biosynthetically and screened for biological activity in a variety of 
different formats (e.g., libraries of soluble molecules; and libraries of 
compounds tethered to resin beads, silica chips, or other solid supports). 
The term is also intended to refer to an intentionally created collection 
of stereoisomers. 
"Predefined region" refers to a localized area on a solid support which is, 
was, or is intended to be used for formation of a selected molecule and is 
otherwise referred to herein in the alternative as a "selected" region. 
The predefined region may have any convenient shape, e.g., circular, 
rectangular, elliptical, wedge-shaped, etc. For the sake of brevity 
herein, "predefined regions" are sometimes referred to simply as 
"regions." In some embodiments, a predefined region and, therefore, the 
area upon which each distinct compound is synthesized smaller than about 1 
cm.sup.2 or less than 1 mm.sup.2. Within these regions, the molecule 
synthesized therein is preferably synthesized in a substantially pure 
form. In additional embodiments, a predefined region can be achieved by 
physically separating the regions (i.e., beads, resins, gels, etc.) into 
wells, trays, etc. 
"Solid support", "support", and "substrate" refer to a material or group of 
materials having a rigid or semi-rigid surface or surfaces. In many 
embodiments, at least one surface of the solid support will be 
substantially flat, although in some embodiments it may be desirable to 
physically separate synthesis regions for different compounds with, for 
example, wells, raised regions, pins, etched trenches, or the like. 
According to other embodiments, the solid support(s) will take the form of 
beads, resins, gels, microspheres, or other geometric configurations. 
Isolation and purification of the compounds and intermediates described 
herein can be effected, if desired, by any suitable separation or 
purification procedure such as, for example, filtration, extraction, 
crystallization, column chromatography, thin-layer chromatography, 
thick-layer (preparative) chromatography, distillation, or a combination 
of these procedures. Specific illustrations of suitable separation and 
isolation procedures can be had by references to the examples hereinbelow. 
However, other equivalent separation or isolation procedures can, or 
course, also be used. 
A "channel block" is 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. 
This invention provides novel compounds which are useful as linking groups 
in chemical synthesis, preferably in the solid phase synthesis of 
oligonucleotides and polypeptides and high density arrays thereof. These 
compounds are generally photolabile and comprise protecting groups which 
can be removed by photolysis to unmask a reactive group. The protecting 
group has the general formula Ar--C(R.sub.1)(R.sub.2)--O--C(O)--, wherein: 
Ar is an optionally substituted fused polycyclic aryl or heteroaromatic 
group or a vinylogous derivative thereof; 
R.sub.1 and R.sub.2 are independently H, optionally substituted alkyl, 
alkenyl or alkynyl, optionally substituted aryl or optionally substituted 
heteroaromatic, or a vinylogous derivative of the foregoing; and 
X is a leaving group, a chemical fragment linked to 
Ar--C(R.sub.1)(R.sub.2)--O--C(O)-- via a heteroatom, or a solid support; 
provided that when Ar is 1-pyrenyl and R.sub.1 =R.sub.2 =H, X is not 
linked to Ar--C(R.sub.1)(R.sub.2)--O--C(O)-- via a nitrogen atom. 
Preferred embodiments are those in which Ar is a fused polycyclic aromatic 
hydrocarbon and in which the substituents on Ar, R.sub.1 and R.sub.2 are 
electron donating groups. Particularly preferred protecting groups are the 
"PYMOC" protecting group, 1-pyrenylmethyloxycarbonyl, where Ar=1-pyrenyl 
and R.sub.1 =R.sub.2 =H, and the "ANMOC" protecting group, 
anthracenylmethyloxycarbonyl, where Ar =anthracenyl (e.g. 9-anthracenyl) 
and R.sub.1 =R.sub.2 =H. 
Representative fused polycyclic aromatic hydrocarbons include naphthalene, 
phenanthrene, anthracene, benzoanthracene, dibenzoanthracene, heptalene, 
acenaphthalene, acephenanthrene, triphenylene, pyrene, fluorene, 
phenalene, naphthacene, picene, perylene, pentaphenylene, pyranthrene, 
fullerenes (including C.sub.60 and C.sub.70), and the like. A 
representative vinylogously substituted derivative of an aromatic 
hydrocarbon is styrene. 
The invention also provides reagents of the molecular formula 
Ar--C(R.sub.1)(R.sub.2)--O--C(O)--X, where Ar, R.sub.1, and R.sub.2 have 
the meanings ascribed above, for incorporating the protecting group into 
the molecule desired to be protected. X can be any suitable leaving group 
such as halo, oxycarbonyl, imidazolyl, pentafluorophenoxy and the like, 
which is capable of reacting with a nucleophilic group such as hydroxy, 
amino, alkylamino, thio and the like on the molecule being protected. 
Thus, the reagents comprising the protecting groups disclosed herein can 
be used in numerous applications where protection of a reactive 
nucleophilic group is required. Such applications include, but are not 
limited to polypeptide synthesis, both solid phase and solution phase, 
oligo--and polysaccharide synthesis, polynucleotide synthesis, protection 
of nucleophilic groups in organic syntheses of potential drugs, etc. 
The invention also provides compositions of the molecular formula 
Ar--C(R.sub.1)(R.sub.2)--O--C(O)--M, where Ar, R.sub.1 and R.sub.2 have 
the meaning outlined above and M is any other chemical fragment. 
Preferably, M will be a monomeric building block that can be used to make 
a macromolecule. Such building blocks include amino acids, nucleic acids, 
nucleotides, nucleosides, monosaccharides and the like. Preferred 
nucleosides are deoxyadenosine, deoxycytidine, thymidine and 
deoxyguanosine as well as oligonucleotides incorporating such nucleosides. 
Preferably, the building block is linked to the photolabile protecting 
group via a hydroxy or amine group. When nucleotide and oligonucleotide 
compositions are used, with the protecting groups of this invention, the 
protecting groups are preferably incorporated into the 3'--OH or the 
5'--OH of the nucleoside. Other preferred compounds are protected 
peptides, proteins, oligonucleotides and oligodeoxynucleotides. Small 
organic molecules, proteins, hormones, antibodies and other such species 
having nucleophilic reactive groups can be protected using the protecting 
groups disclosed herein. 
The use of nucleoside and nucleotide analogs is also contemplated by this 
invention to provide oligonucleotide or oligonucleoside analogs bearing 
the protecting groups disclosed herein. Thus the terms nucleoside, 
nucleotide, deoxynucleoside and deoxynucleotide generally include analogs 
such as those described herein. These analogs are those molecules having 
some structural features in common with a naturally occurring nucleoside 
or nucleotide such that when incorporated into an oligonucleotide or 
oligonucleoside sequence, they allow hybridization with a naturally 
occurring oligonucleotide sequence in solution. Typically, these analogs 
are derived from naturally occurring nucleosides and nucleotides by 
replacing and/or modifying the base, the ribose or the phosphodiester 
moiety. The changes can be tailor made to stabilize or destabilize hybrid 
formation or enhance the specificity of hybridization with a complementary 
nucleic acid sequence as desired. 
Analogs also include protected and/or modified monomers as are 
conventionally used in oligonucleotide synthesis. As one of skill in the 
art is well aware oligonucleotide synthesis uses a variety of 
base-protected deoxynucleoside derivatives in which one or more of the 
nitrogens of the purine and pyrimidine moiety are protected by groups such 
as dimethoxytrityl, benzyl, tert-butyl, isobutyl and the like. Specific 
monomeric building blocks which are encompassed by this invention include 
base protected deoxynucleoside H-phosphonates and deoxynucleoside 
phosphoramidites. 
For instance, structural groups are optionally added to the ribose or base 
of a nucleoside for incorporation into an oligonucleotide, such as a 
methyl, propyl or allyl group at the 2'--O position on the ribose, or a 
fluoro group which substitutes for the 2'--O group, or a bromo group on 
the ribonucleoside base. 2'--O--methyloligoribonucleotides (2'--O--MeORNs) 
have a higher afinity for complementary nucleic acids (especially RNA) 
than their unmodified counterparts. 2'--O--MeORNA phosphoramidite monomers 
are available commercially, e.g., from Chem Genes Corp. or Glen Research, 
Inc. Alternatively, deazapurines and deazapyrimidines in which one or more 
N atoms of the purine or pyrimidine heterocyclic ring are replaced by C 
atoms can also be used. 
The phosphodiester linkage, or "sugar-phosphate backbone" of the 
oligonucleotide analogue can also be substituted or modified, for instance 
with methyl phosphonates or O--methyl phosphates. Another example of an 
oligonucleotide analogue for purposes of this disclosure includes "peptide 
nucleic acids" in which a polyamide backbone is attached to 
oligonucleotide bases, or modified oligonucleotide bases. Peptide nucleic 
acids which comprise a polyamide backbone and the bases found in naturally 
occurring nucleosides are commercially available from, e.g., Biosearch, 
Inc. (Bedford, Mass.). 
Nucleotides with modified bases can also be used in this invention. Some 
examples of base modifications include 2-aminoadenine, 5-methylcytosine, 
5-(propyn-1-yl)cytosine, 5-(propyn-1-yl)uracil, 5-bromouracil, and 
5-bromocytosine which can be incorporated into oligonucleotides in order 
to increase binding affinity for complementary nucleic acids. Groups can 
also be linked to various positions on the nucleoside sugar ring or on the 
purine or pyrimidine rings which may stabilize the duplex by electrostatic 
interactions with the negatively charged phosphate backbone, or through 
hydrogen bonding interactions in the major and minor groves. For example, 
adenosine and guanosine nucleotides can be substituted at the N.sup.2 
position with an imidazolyl propyl group, increasing duplex stability. 
Universal base analogues such as 3-nitropyrrole and 5-nitroindole can also 
be included. 
Compounds of this invention can be prepared by carbonylating an aromatic 
carbinol of the general formula Ar--C(R.sub.1)(R.sub.2)--OH with a 
carbonylation reagent such as for example, phosgene (COCl.sub.2), 
carbonyldiimidazole or pentafluorophenoxy chloroformate and the like to 
provide Ar--C(R.sub.1)(R.sub.2)--O--C(O)--X where X is a leaving group 
derived from the carbonylating reagent (Cl, if phosgene was used, 
pentafluorophenoxy, if pentafluorophenoxy chloroformate was used, etc.). 
This intermediate, Ar--C(R.sub.1)(R.sub.2)--O--C(O)--X is then reacted 
with a molecule M carrying a nucleophilic group whose protection is 
desired to yield a protected building block 
Ar--C(R.sub.1)(R.sub.2)--O--C(O)--M. Representative aromatic carbinols are 
pyrenemethanol, naphthalenemethanol, anthracenemethanol, perylenemethanol 
and the like. Such aromatic carbinols are available from commercial 
suppliers such as Aldrich Chemical Co., Milwuakee, Wis. Alternatively, 
they may also be obtained from precursor aromatic hydrocarbons by 
acylation under Friedel-Crafts conditions with acid chlorides and 
anhydrides and subsequent reduction of the carbonyl group thus added to a 
carbinol. 
Alternatively, one may first carbonylate the group on the molecule being 
protected with a carbonylation reagent, such as one described above, and 
subsequently displace the leaving group X thus inserted with the hydroxyl 
group of the aromatic carbinol. In either procedure, one frequently uses a 
base such as triethylamine or diisopropylethylamine and the like to 
facilitate the displacement of the leaving group. 
One of skill in the art will recognize that the protecting groups disclosed 
herein can also be attached to species not traditionally considered as 
"molecules". Therefore, compositions such as solid surfaces (e.g., paper, 
nitrocellulose, glass, polystyrene, silicon, modified silicon, GaAs, 
silica and the like), gels (e.g., agarose, sepharose, polyacrylamide and 
the like to which the protecting groups disclosed herein are attached are 
also contemplated by this invention. 
The protecting groups of this invention are typically removed by 
photolysis, i.e. by irradiation, though in selected cases it may be 
advantageous to use acid or base catalyzed cleavage conditions. Generally 
irradiation is at wavelengths greater than about 350 nm, preferably at 
about 365 nm. The photolysis is usually conducted in the presence of 
hydroxylic solvents, such as aqueous, alcoholic or mixed aqueous-alcoholic 
or mixed aqueous-organic solvent mixtures. Alcoholic solvents frequently 
used include methanol and ethanol. The photolysis medium may also include 
nucleophilic scavengers such as hydrogen peroxide. Photolysis is 
frequently conducted at neutral or basic pH. 
This invention also provides a method of attaching a molecule with a 
reactive site to a support, comprising the steps of: 
(a) providing a support with a reactive site; 
(b) binding a molecule to the reactive site, said first molecule comprising 
a masked reactive site attached to a photolabile protecting group of the 
formula Ar--C(R.sub.1)(R.sub.2)--O--C(O)--, wherein: 
Ar is an optionally substituted fused polycyclic aryl or heteroaromatic 
group or a vinylogously substituted derivative of the foregoing; 
R.sub.1 and R.sub.2 are independently H, optionally substituted alkyl, 
alkenyl or alkynyl, or optionally substituted aryl or heteroaromatic group 
or a vinylogously substituted derivative of the foregoing; to produce a 
derivatized support having immobilized thereon the molecule attached to 
the photolabile protecting group; and 
(c) removing the photolabile protecting group to provide a derivatized 
support comprising the molecule with an unmasked reactive site immobilized 
thereon. 
As one of skill will recognize, the process can be repeated to generate a 
compound comprising a chain of component molecules attached to the solid 
support. In a "mix and match" approach, the photolabile protecting groups 
may be varied at different steps in the process depending on the ease of 
synthesis of the protected precursor molecule. Alternatively, photolabile 
protecting groups can be used in some steps of the synthesis and 
chemically labile (e.g. acid or base sensitive groups) can be used in 
other steps, depending for example on the availability of the component 
monomers, the sensitivity of the substrate and the like. This method can 
also be generalized to be used in preparing arrays of compounds, each 
compound being attached to a different and identifiable site on the 
support as is disclosed in U.S. Pat. Nos. 5,143,854, 5,384,261, 5,424,186 
5,445,934 and U.S. patent application, Ser. No. 08/376,963, filed Jan. 23, 
1995 (now abandoned), incorporated for reference for all purposes. 
Thus, a related aspect of this invention provides a method of forming, from 
component molecules, a plurality of compounds on a support, each compound 
occupying a separate predefined region of the support, said method 
comprising the steps of: 
(a) activating a region of the support; 
(b) binding a molecule to the region, said molecule comprising a masked 
reactive site linked to a photolabile protecting group of the formula 
Ar--C(R.sub.1)(R.sub.2)--O--C(O)--, wherein: 
Ar is an optionally substituted fused polycyclic aryl or heteroaromatic 
group or a vinylogously substituted derivative of the foregoing; 
R.sub.1 and R.sub.2 are independently H, optionally substituted alkyl, 
alkenyl or alkynyl, or optionally substituted aryl or heteroaromatic group 
or a vinylogously substituted derivative of the foregoing; 
(c) repeating steps (a) and (b) on other regions of the support whereby 
each of said other regions has bound thereto another molecule comprising a 
masked reactive site linked to the photolabile protecting group, wherein 
said another molecule may be the same or different from that used in step 
(b); 
(d) removing the photolabile protecting group from one of the molecules 
bound to one of the regions of the support to provide a region bearing a 
molecule with an unmasked reactive site; 
(e) binding an additional molecule to the molecule with an unmasked 
reactive site; 
(f) repeating steps (d) and (e) on regions of the support until a desired 
plurality of compounds is formed from the component molecules, each 
compound occupying separate regions of the support. 
A related method of forming a plurality of compounds on predefined regions 
of a support involves binding a molecule with a reactive site protected 
with a chemically labile protecting group to an activated region of the 
support and chemically removing the chemically labile protecting group to 
reveal the reactive site. The reactive site is then protected with a 
photolabile protecting group of this invention. This process is repeated 
for other regions of the support with other molecules as desired to 
provide a support having molecules with reactive sites protected by 
photolabile protecting groups on separate regions of the support. Reactive 
sites can be unmasked by removing the photolabile group from selected 
regions and coupled to additional molecules with photolabile protecting 
groups as described earlier to build up arrays of compounds on the 
support. Again, in a "mix and match" approach, monomers with chemically 
labile protecting groups can be attached to a reactive site on the 
substrate (i.e., on the support itself when the first layer of monomers is 
being assembled or subsequently onto an already attached monomer whose 
reactive site has been unmasked) and these chemically labile protecting 
groups can be replaced by a photolabile protecting groups of this 
invention. The replacement is accomplished by removing the chemically 
labile protecting group under conditions which do not affect any 
photolabile groups which may be on the support. This then reveals an 
unmasked reactive site on the monomer which had carried the chemically 
labile protecting group and this unmasked reactive site is reacted with a 
reagent of the formula Ar--C(R.sub.1)(R.sub.2)--O--C(O)--X, where X is a 
leaving group. Thereby, this region of the support is protected by a 
photolabile protecting group which can be selectively removed by light 
directed systems described in U.S. Pat. Nos. 5,143,854, 5,384,261, 
5,424,186 and 5,445,934 and further described below. This method is 
particularly useful when the monomers are more readily available carrying 
chemically labile protecting groups than the photolabile protecting groups 
described herein. It will be recognized that any method of forming a chain 
of compounds or an array of compounds on a support using in at least one 
step a protecting group/reagent or compound of this invention is within 
the scope of the methods this invention. 
Generally, these methods involve sequential addition of monomers to build 
up an array of polymeric species on a support by activating predefined 
regions of a substrate or solid support and then contacting the substrate 
with a protected monomer of this invention (e.g., a PYMOC protected 
nucleoside or amino acid). It will be recognized that the individual 
monomers can be varied from step to step. A common support is a glass or 
silica substrate as is used in semiconductor devices. 
The predefined regions can be activated with a light source, typically 
shown through a screen such as a photolithographic mask similar to the 
techniques used in integrated circuit fabrication. Other regions of the 
support remain inactive because they are blocked by the mask from 
illumination and remain chemically protected. Thus, a light pattern 
defines which regions of the support react with a given monomer. The 
protected monomer reacts with the activated regions and is immobilized 
therein. The protecting group is removed by photolysis and washed off with 
unreacted monomer. By repeatedly activating different sets of predefined 
regions and contacting different monomer solutions with the substrate, a 
diverse array of polymers of known composition at defined regions of the 
substrate can be prepared. Arrays of 10.sup.6, 10.sup.7, 10.sup.8, 
10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12 or more different polymers can 
be assembled on the substrate. The regions may be 1 mm.sup.2 or larger, 
typically 10 .mu.m.sup.2 and may be as small as 1 .mu.m.sup.2. 
The methods described herein may also employ component molecules comprising 
a masked reactive site attached to a photolabile protecting group of the 
formula Ar--C(R.sub.1)(R.sub.2)--, wherein Ar, R.sub.1, and R.sub.2 have 
the meanings ascribed earlier. In such cases, the protecting group is 
attached to a reactive site that is not an amine and is removed by 
photolysis. 
The solid substrate or solid support may be of any shape, although they 
preferably will be roughly spherical. The supports need not necessarily be 
homogenous in size, shape or composition, although the supports usually 
and preferably will be uniform. In some embodiments, supports that are 
very uniform in size may be particularly preferred. In another embodiment, 
two or more distinctly different populations of solid supports may be used 
for certain purposes. 
Solid supports may consist of many materials, limited primarily by capacity 
for derivatization to attach any of a number of chemically reactive groups 
and compatibility with the synthetic chemistry used to produce the array 
and, in some embodiments, the methods used for tag attachment and/or 
synthesis. Suitable support materials typically will be the type of 
material commonly used in peptide and polymer synthesis and include glass, 
latex, polyethylene glycol, heavily cross-linked polystyrene or similar 
polymers, gold or other colloidal metal particles, and other materials 
known to those skilled in the art. The chemically reactive groups with 
which such solid supports may be derivatized are those commonly used for 
solid phase synthesis of the polymer and thus will be well known to those 
skilled in the art, i.e., carboxyls, amines, and hydroxyls. 
To improve washing efficiencies, one can employ nonporous supports or other 
solid supports less porous than typical peptide synthesis supports; 
however, for certain applications of the invention, quite porous beads, 
resins, or other supports work well and are often preferable. One such 
support is a resin in the form of beads. In general, the bead size is in 
the range of 1 nm to 100 .mu.m, but a more massive solid support of up to 
1 mm in size may sometimes be used. Particularly preferred resins include 
Sasrin resin (a polystyrene resin available from Bachem Bioscience, 
Switzerland); and TentaGel S AC, TentaGel PHB, or TentaGel S NH.sub.2 
resin (polystyrene-polyethylene glycol copolymer resins available from 
Rappe Polymere, Tubingen, Germany). Other preferred supports are 
commercially available and described by Novabiochem, La Jolla, Calif. 
In other embodiments, the solid substrate is flat, or alternatively, may 
take on alternative surface configurations. For example, the solid 
substrate may contain raised or depressed regions on which synthesis takes 
place. In some embodiments, the solid substrate will be chosen to provide 
appropriate light-absorbing characteristics. For example, the substrate 
may be a polymerized Langmuir Blodgett film, functionalized glass, Si, Ge, 
GaAs, GaP, SiO.sub.2, SiN.sub.4, modified silicon, or any one of a variety 
of gels or polymers such as (poly)tetrafluorethylene, 
(poly)vinylidendifluoride, polystyrene, polycarbonate, or combinations 
thereof. Other suitable solid substrate material will be readily apparent 
to those of skill in the art. Preferably, the surface of the solid 
substrate will contain reactive groups, which could be carboxyl, amino, 
hydroxyl, thiol, or the like. More preferably, the surface will be 
optically transparent and will have surface Si--OH functionalities, such 
as are found on silica surfaces. 
The photolabile protecting groups and protected monomers disclosed herein 
can also be used in bead based methods of immobilization of arrays of 
molecules on solid supports. 
A general approach for bead based synthesis is described in application 
Ser. Nos. 07/762,522 (filed Sep. 18, 1991, abandoned in favor of 
continuation-in-part application Ser. No. 07/946,239, which was abandoned 
in favor of continuation-in-part application Ser. No. 08/146,886, which 
issued as U.S. Pat. No. 5,639,603); 07/946,239 (filed Sep. 16, 1992, 
abandoned in favor of continuation-in-part application Ser. No. 
08/146,886, which issued as U.S. Pat. No. 5,639,603); 08/146,886 (filed 
Nov. 2, 1993, now issued as U.S. Pat. No. 5,639,603); 07/876,792 (filed 
Apr. 29, 1992, now issued as U.S. Pat. No. 5,541,061) and PCT/US93/04145 
(filed Apr. 28, 1993), Lam et al. (1991) Nature 354:82-84; PCT application 
no. 92/00091 and Houghten et al, (1991) Nature 354:84-86, each of which is 
incorporated herein by reference for all purposes. 
Other methods of immobilization of arrays of molecules in which the 
photocleavable protecting groups of this invention can be used include pin 
based arrays and flow channel and spotting methods. 
Photocleavable arrays also can be prepared using the pin approach developed 
by Geysen et al. for combinatorial solid-phase peptide synthesis. A 
description of this method is offered by Geysen et al., J. Immunol. Meth. 
(1987) 102:259-274, incorporated herein by reference. 
Additional methods applicable to library synthesis on a single substrate 
are described in applications Ser. Nos. 07/980,523, filed Nov. 20, 1992 
(now issued as U.S. Pat. No. 5,677,195), and 07/796,243, filed Nov. 22, 
1991 (now issued as U.S. Pat. No. 5,384,261), 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. Photocleavable linkers are particularly suitable for this 
technology as this delivery method may otherwise result in poor synthesis 
fidelity due to spreading, reagent dilution, inaccurate delivery, and the 
like. By using a photocleavable linker, rather than a conventional 
acid-cleavable linker, the purest material can be selectively cleaved from 
the surface for subsequent assaying or other procedures. More 
specifically, masks can be used when cleaving the linker to ensure that 
only linker in the center of the delivery area (i.e., the area where 
reagent delivery is most consistent and reproducible) is cleaved. 
Accordingly, the material thus selectively cleaved will be of higher 
purity than if the material were taken from the entire surface. 
Typically, the molecules used in this method will be the monomeric 
components of complex macromolecules. These monomeric components can be 
small ligand molecules, amino acids, nucleic acids, nucleotides, 
nucleosides, monosaccharides and the like, thereby allowing one to 
synthesize arrays of complex macromolecules or polymeric sequences, such 
as polypeptides, nucleic acids and synthetic receptors, on the solid 
support. 
This invention discloses new nucleoside phosphoramidite monomers with 
1-pyrenylmethyloxy-carbonyl ("PYMOC") 5'-protecting groups. They are 
photolytically cleaved under irradiation at wavelengths greater than about 
350 nm, preferably at about 365 nm, in the presence of methanol, water, or 
water-solvent mixtures and/or with nucleophilic scavengers such as 
hydrogen peroxide at neutral or basic pH. The rate of photolysis is 
similar to that observed for the MeNPOC group. However, the yield of PYMOC 
photo-removal is much higher (.about.95%), so that the use of these 
monomers for photochemical synthesis of oligonucleotides leads to higher 
stepwise cycle yields and therefore higher-purity oligomers. 
The 1-pyrenylmethyloxycarbonyl group described here can be used for the 
protection of alcohols. The photolysis of PYMOC is faster than that of the 
1-pyrenylmethyl group, so it would also be a superior photo-removable 
protecting group for phosphates, carboxylates, amines, thiols, etc. 
Other "benzylic" oxycarbonyls may have similar or better efficiency than 
the PYMOC group. A general formula would be: 
##STR1## 
where Ar is an optionally substituted fused polycyclic aryl or an 
optionally substituted heteroaromatic group or a vinylogously substituted 
derivative of the foregoing; 
R.sub.1 and R.sub.2 are independently H, optionally substituted alkyl, 
alkenyl or alkynyl, optionally substituted aryl, optionally substituted 
heteroaromatic, or vinylogously substituted derivatives of the foregoing. 
Preferred embodiments are those in which Ar is a fused polycyclic aromatic 
hydrocarbon. FIG. 2 shows representative examples. Preferred substituents 
on the aromatic hydrocarbons would be electron-donating groups that 
stabilize an incipient excited state benzyl carbocation. 
Other embodiments of the PYMOC photogroup, for example, include at least 
one additional substituent at the a position, such as a methyl group or a 
methoxy-substituted phenyl. These substituents will increase 
photosolvolysis efficiency, and improve the selectivity for the 
5'-hydroxyl in the preparation of the monomer 5'-protected nucleoside. 
EXAMPLE 
SYNTHESIS OF 
5'--O--PYMOC-2'-DEOXYNUCLEOSIDE-3'--O--(N,N-DIISOPROPYL)CYANOETHYLPHOSPHOR 
AMIDITES 
All chemical reagents used were procured from commercial sources (Aldrich 
Chemical Co., Milwaukee, Wis. and Sigma Chemical Co., Milawaukee, Wis.). 
Intermediates and products were identified by mass spectrometry, .sup.1 
H-NMR, and .sup.31 P-NMR. 
Abbreviations: 
DIEA--Diethyl isopropylamine 
NHS--N-hydroxysuccinimide 
THF--Tetrahydrofuran 
MeNPOC--methylnitropiperonyloxycarbonyl 
TEA--Triethylamine 
DMAP--4-Dimethylaminopyridine 
Pentafluorophenyl Chloroformate 
Pentafluorophenol (30 g; 163 mmol) and triethylamine (20 g, 200 mmol) were 
combined in 200 ml dry THF, and then added dropwise to a stirring solution 
of phosgene (20 g; 200 mmol) in 100 ml of toluene at 0.degree. C. After 2 
hours, the solution was filtered and evaporated to give the crude product 
as an oil, which was recrystallized from hexane to obtain 30 g (75%) pure 
pentafluorophenyl chloroformate. 
5'--O--(1-Pyrenylmethyl)oxycarbonyl-2'-deoxynucleosides 
The following general procedure was used to prepare 5'-PYMOC-derivatives of 
thymidine, N-4-isobutyryl-2'-deoxycytidine, 
N-7-phenoxyacetyl-2'-deoxyadenosine, and N-4-isobutyryl-2'-deoxyguanosine: 
The base-protected nucleoside (20 mmol) was dried by co-evaporating 3 times 
with 50 ml dry pyridine, then dissolved in 20 ml CH.sub.2 Cl.sub.2 and 10 
ml dimethylsulfoxide (DMSO) containing 1.7 ml (21 mmol) pyridine. The 
resulting solution was cooled to -10.degree. C. under argon, and 5 g (20 
mmol) of pentafluorophenyl chloroformate was added all at once with 
stirring. After an additional 2-3 hours stirring at -10.degree. C., the 
reaction mixture was analyzed by TLC or HPLC to determine the extent of 
conversion. Additional quantities of pentafluorophenyl chloroformate and 
pyridine (.about.0.4-1.0 mmol each) were then added, as needed, until the 
nucleoside was completely converted to the 
5'--O--pentafluorophenoxycarbonyl derivative. Although isolable, at this 
point the intermediate was usually converted directly to the 
PYMOC-derivative, in situ, by the addition of 1-pyrenemethanol (6 g, 26 
mmol), followed by 10 ml of triethylamine and 0.25 g (2 mmol) of 
N,N-dimethyl-aminopyridine, and stirring overnight at room temperature. 
About 50-100 ml of CH.sub.2 Cl.sub.2 was then added, and in the case of 
thymidine, the pure PYMOC-derivative precipitated and could be collected 
by filtration. Otherwise, the solution was washed twice with 5% aqueous 
NaHCO.sub.3, once with saturated NaCl, dried with Na.sub.2 SO.sub.4, and 
evaporated to dryness. The crude material was finally purified by flash 
chromatography (silica gel, 2:8 ethyl acetate-CH.sub.2 Cl.sub.2 /1-6% 
methanol gradient) to obtain the pure 5'-PYMOC nucleoside products in 
.about.75% yield. 
5'--O--(1-Pyrenylmethyl)oxycarbonyl-2'-deoxynucleoside-3'--O--(N,N-diisopro 
pyl)cyanoethylphosphoramidites 
On a 12 mmol scale, the 5'-PYMOC nucleosides were first dried by 
co-evaporation with dry pyridine, and then dissolved or suspended in 50 ml 
of dry CH.sub.2 Cl.sub.2. Then 2-cyanoethyl-N,N,N.sup.1,N.sup.1 
-tetraisopropylphosphorodiamidite (4.4 g; 14.5 mmol) and 
N,N-diisopropylammonium tetrazolide (1 g; 6 mmol) were added, and the 
mixture was left stirring under argon overnight. The solution was washed 
twice with 10% aqueous NaHCO.sub.3, once with saturated NaCl, dried with 
Na.sub.2 SO.sub.4, and then evaporated to dryness. The crude products were 
purified by flash chromatography (silica gel, eluting with a 1-5% methanol 
gradient in 2:8 ethyl acetate-CH.sub.2 Cl.sub.2 containing 0.5% 
triethylamine) to obtain the pure phosphoramidites in .about.80% yield. 
Table 1 compares the efficiency of photolytic cleavage of PYMOC protected 
nucleosides to MeNPOC (methylnitropiperonyloxycarbonyl) protected 
nucleosides. 
TABLE 1 
______________________________________ 
Photolysis Rates: 
5'-Protecting 
Base Group Solvent Power 
.sub.1/2 
______________________________________ 
T MeNPOC dioxane 35 mW/cm.sup.2 
9 sec 
T PYMOC MeOH " 10 sec 
T PYMOC 1:1 dioxane-H.sub.2 O " 10 sec 
T PYMOC 9:1 dioxane-MeOH " 43 sec 
G.sup.ibu MeNPOC dioxane 27 mW/cm.sup.2 11 sec 
G.sup.ibu PYMOC MeOH " 13 sec 
______________________________________ 
Table 2 compares the coupling cycle efficiency (six cycles) of PYMOC 
protected and MeNPOC protected nucleosides to a phosphoramidite solid 
support using surface fluorescence analysis. 
TABLE 2 
______________________________________ 
Stepwise Coupling Cycle Efficiencies: 
1. Surface fluorescence analysis ("staircase" assay): 
5'-Protecting 
Yield (6 steps) 
Base Group Net Avg. Stepwise 
______________________________________ 
T MeNPOC 15 73 
T PYMOC 56 91 
dG.sup.ibu MeNPOC 29 81 
dG.sup.ibu PYMOC 61 92 
dC.sup.ibu MeNPOC 37 85 
dC.sup.ibu PYMOC 68 94 
______________________________________ 
Table 3 compares the coupling cycle efficiency (six cycles) of PYMOC 
protected and MeNPOC protected nucleosides to a phosphoramidite solid 
support using HPLC analysis. 
TABLE 3 
______________________________________ 
2. HPLC analysis (DOP#AF001; 3" ethenodeoxyadenosine tag): 
5'-Protecting 
Yield (3 steps) 
Base Group Net Avg. Stepwise 
______________________________________ 
T PYMOC 92 97.2 
T " 93 97.6 avg 97.4 
T MeNPOC 45 77 
T " 43 75 
T " 48 78 
T " 40 74 
T " 48 78 avg 76.4 
______________________________________ 
The foregoing invention has been described in some detail by way of 
illustration and example, for purposes of clarity and understanding. It 
will be obvious to one of skill in the art that changes and modifications 
may be practiced within the scope of the appended claims. Therefore, it is 
to be understood that the above description is intended to be illustrative 
and not restrictive. The scope of the invention should, therefore, be 
determined not with reference to the above description, but should instead 
be determined with reference to the following appended claims, along with 
the full scope of equivalents to which such claims are entitled. 
All patents, patent applications and publications cited in this application 
are hereby incorporated by reference in their entirety for all purposes to 
the same extent as if each individual patent, patent application or 
publication were so individually denoted.