Photocleavable protecting groups and methods for their use

Details Photocleavable protecting groups and methods for their use Photocleavable protecting groups and methods for their use

2000-03-14

2003-05-20

Wilson, James O. (Department: 1623)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S025310, C536S025320, C536S025330, C536S025340, C536S025600, C536S124000, C435S006120, C435S007920, C435S007940, C435S007950

Reexamination Certificate

active

06566515

ABSTRACT:

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; Holmes et al. (1994) in
Peptides: Chemistry, Structure and Biology
(
Proceedings of the
13
th American Peptide Symposium
); Hodges et al. Eds.; ESCOM: Leiden; pp. 110-12, each of these references is incorporated herein by reference. Examples of these compounds included the 6-nitroveratryl derived protecting groups, which incorporate two additional alkoxy groups into the benzene ring. Introduction of an &agr;-methyl onto the benzylic carbon facilitated the photolytic cleavage with >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 for providing protecting 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 compounds have the general formula Ar—C(R
1
)(R
2
)—O—C(O)—X, wherein:
Ar is an optionally substituted fused polycyclic aryl or heteroaromatic group or a vinylogous derivative thereof;
R
1
and R
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
1
)(R
2
)—O—C(O)— via a heteroatom, or a solid support; provided that when Ar is 1-pyrenyl and R
1
=R
2
=H, X is not linked to Ar—C(R
1
)(R
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
1
and R
2
are electron donating groups. Particularly preferred protecting groups are the “PYMOC” protecting group, pyrenylmethyloxycarbonyl, where Ar=1-pyrenyl and R
1
=R
2
=H, and the “ANMOC” protecting group, anthracenylmethyloxycarbonyl, where Ar=anthracenyl and R
1
=R
2
=H. Methods are provided for preparing these compounds.
This invention also provides reagents of the molecular formula Ar—C(R
1
)(R
2
)—O—C(O)—X, where Ar, R
1
, and R
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
1
)(R
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
1
and R
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
1
)(R
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
1
and R
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.
The present invention also provides methods of performing chemical reactions on a surface, by providing at least one chemical reactant on the surface, and applying a coating to the surface. The coating provides an environment that is favorable to reaction of the chemical reactant. In a more preferred aspect, the invention provides a method of activating a functional group on a surface that is protected with a protecting group of the invention. The method involves applying a nucleophilic coating to the surface, and exposing the surface to light to remove the protecting group.
The methods and compositions described herein find particular utility in the synthesis of high density arrays of compounds, and particularly nucleic acids, on solid supports.


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