METHODS OF LABELING POLYNUCLEOTIDE WITH DIBENZORHODAMINE DYES

• US 20110124510A1
• Filed: 10/06/2010
• Published: 05/26/2011
• Est. Priority Date: 11/25/1997
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Lam, Joe Y. L., BENSON, Scott C., Menchen, Steven Michael
• Application Number:
  US12/899,508
• Publication Number:
  US 20110124510A1
• Time in Patent Office: Days
• US Class Current: 506/7
• CPC Class Codes: C07D 209/60 Naphtho [b] pyrroles; Hydrogenated naphtho [b] pyrroles
• CPC Class Codes: C07D 221/10 Aza-phenanthrenes
• CPC Class Codes: C07D 311/78 Ring systems having three or more relevant rings
• CPC Class Codes: C07D 491/14 Ortho-condensed systems
• CPC Class Codes: C07D 491/147 the condensed system containing one ring with oxygen as ring hetero atom and two rings with nitrogen as ring hetero atom
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical
• CPC Class Codes: C09B 11/02 derived from diarylmethanes
• CPC Class Codes: C09B 11/24 Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2563/107 fluorescence
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]

Abstract

Dibenzorhodamine compounds having the structure

are disclosed, including nitrogen- and aryl-substituted forms thereof. In addition, two intermediates useful for synthesizing such compounds are disclosed, a first intermediate having the structure

including nitrogen- and aryl-substituted forms thereof, and a second intermediate having the structure

including nitrogen- and aryl-substituted forms thereof, wherein substituents at positions C-14 to C18 taken separately are selected from the group consisting of hydrogen, chlorine, fluorine, lower alkyl, carboxylic acid, sulfonic acid, —CH₂OH, alkoxy, phenoxy, linking group, and substituted forms thereof. The invention further includes energy transfer dyes comprising the dibenzorhodamine compounds, nucleosides labeled with the dibenzorhodamine compounds, and nucleic acid analysis methods employing the dibenzorhodamine compounds.

Claims

1. -105. (canceled)

106. A method of identifying polynucleotide classes comprising the steps of:

providing multiple classes of polynucleotides distributed among locations in an array;

wherein a first class of polynucleotide comprises a dibenzorhodamine dye having a structure of the formula;

107. The method of claim 106 wherein the dibenzorhodamine dye comprises a first bridging group which when taken together with the C-12-bonded nitrogen and the C-12 and C-13 carbons forms a first ring structure having from 4 to 7 members;

and/or

a second bridging group which when taken together with the C-2-bonded-nitrogen and the C-1 and C-2 carbons forms a second ring structure having from 4 to 7 members.

108. The method of claim 107 wherein one or both of the first and second ring structures has five members.

109. The method of claim 108 wherein the five membered ring structure includes one gem disubstituted carbon.

110. The method of claim 109 wherein the gem substituents are lower alkyl.

111. The method of claim 110 wherein the gem substituents are methyl.

112. The method of claim 108 wherein the five membered ring is not aromatic.

113. The method of claim 107 wherein the first and second ring structures are the same.

114. The method of claim 107 wherein the first and second ring structures are different.

115. The method of claim 106 wherein the dibenzorhodamine dye comprising one or more nitrogen substituents selected from the group consisting of lower alkyl, lower alkene, lower alkyne, phenyl, aromatic, electron-rich heterocycle, polycyclic aromatic, water-solubilizing group, linking group, including substituted forms thereof.

116. The method of claim 115 wherein the nitrogen substituents are selected from the group consisting of lower alkyl, phenyl, and substituted forms thereof.

117. The method of claim 115 wherein the nitrogen substituents are selected from the group consisting of substituted lower alkyl and substituted phenyl, wherein the substituent is linking group or sulfonate.

118. The method of claim 115 wherein the linking group is isothiocyanate, sulfonyl chloride, 4, 6, dichlorotriazinyl, succinimidyl ester, maleimide, haloacetyl or iodoacetamide.

119. The method of claim 115 wherein the linking group is carboxylate.

120. The method of claim 115 wherein the nitrogen substituents are water-solubilizing group.

121. The method of claim 115 wherein the water-solubilizing group is selected from the group consisting of sulfonate, phosphate, quaternary amine, sulfate, polyhydroxyl, and water-soluble polymer.

122. The method of claim 106 wherein the dibenzorhodamine dye and the polynucleotide are connected by a linkage at a position on the polynucleotide selected from the 8-position of a purine nucleobase, the 7- or 8-position of a deazapurine nucleobase, the 5-position of a pyrimidine nucleus, the 5′

terminus, the 3′

terminus, and the phosphodiester backbone.

123. The method of claim 122 wherein the linkage is attached to the linking group substituent of the dibenzorhodamine dye.

124. The method of claim 122 wherein the linkage is an acetylenic amido or alkenic amido linkage.

125. The method of claim 124 wherein the linkage has a structure of one of the following formulae:

126. The method of claim 106 wherein the dibenzorhodamine dye has a structure of the following formula: