Trityl-type compounds and their use

Details Trityl-type compounds and their use Trityl-type compounds and their use

2003-05-19

2004-10-26

Kumar, Shailendra (Department: 1621)

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

Organic compounds

Amino nitrogen containing

C530S300000, C530S350000, C536S022100

Reexamination Certificate

active

06809220

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to trityl-type compounds and to their use as fluorescent labels for solution and solid support applications.
BACKGROUND OF THE INVENTION
Triarylmethyl (trityl)-type cations are stabilised by the resonance effect of the phenyl rings, which makes their ethers acid-labile, and they are consequently a useful family of protective groups, especially in nucleoside chemistry (1). Conjugation to a positively charged (cationic) carbon atom dramatically changes the spectral properties of the fluorophore. Trityl groups generating cations of different colours have been used to protect different nucleotides in oligonucleotide synthesis (2).
A modified trityl group bearing a pyrenyl residue in place of one of the aryl groups has fluorescent properties similar to non-modified pyrene and has been used, in its non-cationic (non-charged) form, for more precise fluorescent detection (down to 10
−10
M) of detritylation (3). The modified trityl compound is attached to a nucleoside by linkage to the carbon atom. Triphenylmethyl-based structures bearing a side-chain have been used to reversibly label synthetic oligonucleotides with biotin, etc (4), to purify them by immobilisation on to a solid support after synthesis (5), and to controllably activate prodrug antibody conjugates (6). These trityl-based structures combine the useful properties of a trityl group (easy cationisation, and easy control of the rate of cationisation, by introducing more or less methoxy groups) with a ‘hook’ which keeps them in the right place even after ionization, unlike more conventional trityl-based protective groups.
Various (non-acidic) ways of removing the triarylmethyl protective group are known. These include treatment with anion radicals (7), ZnBr
2
(8) and irradiation of the starting material (photochemical ionization) (9).
Derivatives of trityl groups with different masses have been used as unique mass-tags in combinatorial synthesis (10). They benefit from the high desorption rate of triphenylmethyl cation-based tags under conditions of laser desorption/ionisation time of flight (TOF) mass-spectrometry. Again, the trityl cations could be released either by acidic treatment or directly by laser irradiation. In the latter case, it can be beneficial to tune the absorbance of the tag more finely, by making it closer to the wavelength of the laser used for ionization of the sample, in a way similar to that described for porous silica used as a matrix (11).
Both single fluorophore (12) and energy transfer (13-17)-based fluorescence detection methods find wide applications in the analysis of nucleic acids.
SUMMARY OF THE INVENTION
The present invention is based, firstly, on the discovery of new multi-purpose fluorescent tags derived from polycyclic aromatic hydrocarbons (PAHs).
According to a first aspect of the invention, a trityl-type compound comprises, bonded to the same atom, three aryl groups, of which at least one is a fluorophore and at least one has a substituent including a functional group, and wherein the compound can exist in a non-ionised state or in an ionised state conjugated with the aryl groups. Such compounds can be soluble, have controllable fluorescence, and can be used, in the form of phosphoramidites, for nucleotide synthesis, or in dendrimers.
Having the functional group located on one of the aryl groups, permits the compound to undergo ionisation while at the same time being joined to another molecule or solid support. This is in contrast to the prior art where the fluorescent trityl-type compounds are bound to other molecules through the central carbon atom.
In a second aspect of the present invention, a fluorescence-resonant compound is obtainable by linking two different trityl-type compounds each comprising three aryl groups of which at least one has a substituent including a functional group, and wherein each trityl-type compound can exist in a non-ionised state or in an ionised state conjugated with the respective aryl groups, the linking being via the respective functional groups. Such compounds are useful in FRET, eximer and other energy-transfer systems where the ability to modulate different fluorescent signals by altering the reaction conditions, can be beneficial.


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