Organic compounds -- part of the class 532-570 series – Organic compounds – Nitrogen attached directly or indirectly to the purine ring...
Reexamination Certificate
2001-11-27
2004-05-25
Desai, Rita (Department: 1626)
Organic compounds -- part of the class 532-570 series
Organic compounds
Nitrogen attached directly or indirectly to the purine ring...
Reexamination Certificate
active
06740755
ABSTRACT:
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from European Patent Application No. 00126019.9, filed in English on Nov. 28, 2000, the disclosure of which is incorporated by reference herein in its entirety.
FIELD AND BACKGROUND OF THE INVENTION
Indocyanine dyes conform to the generalised formula:
where R
11
, R
12
, R
13
, R
14
, R
21
, R
22
, R
23
and R
24
are either hydrogens or substituents; the substituent couples R
13
, R
14
and/or R
23
, R
24
can form a condensed benzene ring, in turn bearing substituents; n=1-3; L
n1
, L
n2
are either methines (C—H) or substituted methines (C—R).
Comprehensive reviews regarding indocyanine dyes have been written by Frances M. Hamer, “The Chemistry of Heterocyclic Compounds”, vol. 18, “The Cyanine Dyes and Related Compounds”, Weissberger Ed., Wiley Interscience, New York, (1964); D. M. Sturmer, “The Chemistry of Heterocyclic Compounds”, “Special Topics in Heterocyclic Chemistry”, chapter VIII, “Synthesis and Properties of Cyanine and Related Dyes”, Weissberger Ed., Wiley, N.Y., (1977); “The Kirk-Othmer Encyclopaedia of Chemical Technology” vol. 7, p. 782, “Cyanine Dyes”, Wiley, N.Y., (1993).
For many years, indocyanine dyes have been very useful as sensitisers in photography, especially in the red and near infrared regions of the spectrum. However, in more recent years, there has been an upsurge of new uses of these dyes in innovative technological areas, such as laser and electro-optic applications, optical recording media, medical, biological and diagnostic. These new applications of indocyanine dyes place high demands on the degree of purity required, and the reproducibility of synthetic methods and purification steps is very important. These requirements are especially stringent for dyes designed to improve detection of ribonucleic acid (RNA), deoxyribonucleic acid (DNA) and of antigens in immunoassays. In these fields, the trend toward an increasing miniaturisation is accompanied by an increasing demand on sensitivity of the reporter molecules or labels. One way to increase the sensitivity of conventional fluorescence method is to use laser sources for the excitation. However, traditional fluorescent labels based on fluoresceins or rhodamines required expensive and/or bulky lasers. Moreover, their fluorescence occurs in the blue-green to green regions of the visible spectrum, where interference from the sample matrix is more likely to occur. Indocyanine dyes do not suffer from these limitations. They can be efficiently excited by means of small, inexpensive solid state devices such as laser diodes or light emitting diodes, with extinction coefficients often several times higher than fluoresceins and rhodamines; they emit in the red and near-infrared regions of the spectrum, where non-specific fluorescence from the sample is low or lacking; another sources, Raman noise, becomes smaller with the inverse fourth power of wavelength.
To be useful as a label, a dye has to be provided with a suitable side chain containing a functional group. While the main part of the dye structure is generally known from previous applications, the introduction of a functional group into the structure for the purpose of conjugation, or binding to another molecule, represents the innovative step in the inventions concerning the use of the dye as a labelling reagent. In general, only one such functionalised side arm is preferable, in order to avoid cross-linking or purification problems. With a few exceptions, limited to heptamethine dyes, the standard approach in the design of indocyanine labelling reagents has been to attach the functionalised side arm to one of the heterocyclic nuclei of the dye:
HET
1
—HET
2
—Z
See, for instance: J. S. Lindsey, P. A. Brown, and D. A. Siesel, “Visible Light-Harvesting in Covalently-Linked Porphyrin-Cyanine Dyes”, Tetrahedron, 45, 4845, (1989); R. B. Mujumdar, L. A. Ernst, Swati R. Mujumdar, C. J. Lewis, and A. S. Waggoner, “Cyanine Dye Labelling Reagents: Sulfoindocyanine Succinimidyl Esters”, Bioconjugate Chemistry, 4, 105, (1993); G. Mank, H. T. C. van der Laan, H. Lingeman, Cees Goojer, U. A. Th. Brinkman, and N. H. Velthorst, “Visible Diode Laser-Induced Fluorescence Detection in Liquid Chromatography after Precolumn Derivatization of Amines”, Anal. Chem., 67, 1742, (1995).
The general synthetic strategy necessary to prepare these labelling reagents is as follows. First, a quaternised nitrogen heterocycle HET
1
is prepared. Then, this heterocyclic base is reacted with an electrophilic reagent such as PhNH—(CH═CH)
n
—CH═NHPh.HCl or RO—(CH═CH)
n
—CH(OR)
2
, where Ph is a phenyl ring and R a methyl or ethyl group, to obtain a so-called hemicyanine dye, HET
1
—(CH═CH)
n
NHPh/HET
1
—(CH═CH)
n
NAcPh, where Ac is the acetyl radical or HET
1
—(CH═CH)
n
—OR. These intermediates are then reacted with a different quaternary nitrogen heterocycle, HET
2
. The functionalised side arm can be attached either to the first or to the second quaternised nitrogen heterocycle. The final result is an asymmetric polymethine labelling reagent, HET
1
—(CH═CH)
n
—HET
2
—Z.
Unfortunately, the hemicyanine intermediates are notoriously difficult to obtain in good yields and/or in a pure form. For example, the condensation of N-methyl-2,3,3-trimethyl[3H]indolium iodide with malonaldehyde dianil monochloride in acetic anhydride is said (Piggott and Rodd, BP 355,693/1930) to give rise to a green intermediate, indicating a strong contamination of the desired, yellow hemicyanine intermediate (yellow) with symmetric, blue indocyanine dye, FIG.
2
. Moreover, when F. M. Hamer, in “Some Unsymmetrical Pentamethincyanine Dyes and their Tetramethin Intermediates” tried to prepare a pure sample of the same hemicyanine intermediate, obtained it in an 8% yield, after a lengthy and wasteful procedure based on multiple extractions and precipitations. More recently, R. B. Mujumdar, L. A. Ernst, Swati R. Mujumdar, C. J. Lewis, and A. S. Waggoner, in “Cyanine Dye Labelling Reagents: Sulfoindocyanine Succinimidyl Esters”, Bioconjugate Chemistry, 4, 105, (1993) described the synthesis of hemicyanine intermediates for the preparation of sulfoindocyanine dyes active esters, useful as labelling reagents. One intermediate was obtained by condensing 1-ethyl-2,3,3-trimethyl[3H]indolium-5-sulfonate with N,N′-diphenylformamidine in acetic acid for four hours. While the reported yield of the crude compound was 30%, the carboindocyanine dyes prepared from it were obtained only in 25% and 5% yields, after extensive purification by reverse phase HPLC chromatography. Similarly, the condensation of 1-ethyl-2,3,3-trimethyl[3H]indolium-5-sulfonate with malonaldehyde dianil hydrochloride in a mixture of acetic acid and acetic anhydride at reflux for four hours was said to produce the corresponding hemicyanine intermediate in an unreported yield. Again, the yields of the dicarboindocyanine dyes obtained from these intermediates were very low (5%). In fact, when Mank (Anal. Chem., 67, 1744) tried to synthesise the same dicarbocyanine label described in the previous reference he obtained a total yield of 18% of dicarbocyanines, from which the desired product was difficult to separate. He then devised an alternative approach based on 1,3,3-trimethoxypropene. Unfortunately, this chemical is no longer available commercially. Similar difficulties were encountered by us when trying to repeat the syntheses indicated above.
For these reasons it became necessary to investigate more carefully the technique employed to prepare the required hemicyanine intermediates and the properties of the latter. We thus discovered two main sources of trouble. The first was the formation of symmetrical indocyanine dye in variable and often erratic amounts in the condensation step. The other complication arose from the reversibility of this reaction. For example, when a pure sample of hemicyanine intermediate was exposed to the same conditions used for the formation of the asymmetric cyanine dye, name
Caputo Giuseppe
Ciana Leopoldo Della
Desai Rita
Myers Bigel & Sibley Sajovec, PA
Saeed Kamal
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