Asymmetric rhodamine dye and use thereof in biological assays

Abstract

To provide rhodamine dyes having spectral properties uniquely suited to the creation of multiplex dye sets.SOLUTION: The present disclosure relates to N-protected NH-rhodamine dyes and their use in nucleic acid detection. In particular, the disclosure relates to methods of making N-protected NH-rhodamine dyes, and methods of use of N-protected NH-rhodamine dyes. Certain dyes provided herein have unique spectral properties that complement those in existing dye sets and can be used to expand the number of reporter dyes that can be included for HID applications and other biological assays. For example, fluorescent compounds represented by the following formula are useful to label synthetic oligonucleotides.SELECTED DRAWING: None

Description

[Background technology]

[0001] The use of fluorescent rhodamine dyes as detection labels finds widespread use in molecular biology, cell biology, and molecular genetics. For example, the use of fluorescently labeled oligonucleotides is now widespread in a variety of different assays, such as polynucleotide sequencing, fluorescence in situ hybridization (FISH), hybridization assays on nucleic acid arrays, fluorescence polarization studies, and nucleic acid amplification assays, including polymerase chain amplification assays, performed using optical probes and / or primers.

[0002] Various multiplex assay systems utilizing fluorescent dyes have been described, for example, rhodamine dyes have been described for use in multiplex assay systems, as described in WO 2012 / 067901 for use in Human Identification Assays (HID). Unfortunately, the spectral properties of existing dye sets, including rhodamine dyes, limit the ability to develop robust and sensitive assay systems using more than six dye combinations. To enable such more highly multiplexed systems, there is a need for the development of new rhodamine dyes with spectral properties that are uniquely suitable for the creation of such alternative multiplex dye sets. Summary of the Invention [Means for solving the problem]

[0003] Fluorescent compounds that can be used to label synthetic oligonucleotides are described. In one embodiment, the compounds have the formula (I): [ka]

[0004] In the formula, R 1 , R 2 , R 3 , R6 , R 7 , R 8 , R 11 , R 12 , R 13 , and R 14 are each independently hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, -R b , or -(CH 2 ) n -R b or R 1 and R 2 and / or R 6 and R 7 together with the carbon atom to which they are attached form an optionally substituted benzo group,

[0005] R 4 is selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, or R 4 and R 2 or R 3 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group;

[0006] R 5 is H or a protecting group,

[0007] R 9 is selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, or R 7 and R 9 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group;

[0008] R 10 is H or a protecting group, or R 8 and R 10 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group;

[0009] R 7 and R 9 or R 8 and R 10 at least one of, taken together with the atom and bond to which they are attached, forms an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group, and optionally, R 4 and R 2 or R 3 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group, provided that the compound has the formula [ka] Not a compound of

[0010] Each R a is lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl, -CX 3 and 6- to 20-membered heteroarylalkyl;

[0011] Each R b are independently X, -OH, -OR a , -SH, -SR a -NH 2 , -NHR a , N.R. c R c , N + R c R c R c , perhalo lower alkyl, trihalomethyl, trifluoromethyl, -P(O)(OH)2 , P(O)(OR a ) 2 , P(O)(OH)(OR a ), -OP(O)(OH) 2 , OP(O)(OR a ) 2 , -OP(O)(OR a )(OH), -S(O) 2 OH, S(O) 2 R a , -C(O)H, C(O)R a , -C(S)X, -C(O)OR a , -C(O)OH, -C(O)NH 2 , -C(O)NHR a , C(O)NR c R c , C(S)NH 2 , C(O)NHR a , -C(O)NR c R c , -C(NH)NH 2 , -C(NH)NHR a , and C(NH)NR c R c is selected from

[0012] Each R c are independently a or two R attached to the same nitrogen atom c together with the nitrogen atom form a 5-8 membered saturated or unsaturated ring which may optionally contain one or more of the same or different ring heteroatoms typically selected from O, N, and S;

[0013] Each R d and R e When alone, each independently represents hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, -R b , or -(CH 2 ) n -R b is selected from

[0014] n is an integer ranging from 1 to 10.

[0015] In another aspect, the disclosure describes an oligonucleotide that includes a labeling moiety prepared by reacting an oligonucleotide attached to a solid support with a reagent having the structure: LM-L-PEP

[0016] where PEP is a phosphate precursor group, L is an optional linker linking a labeling moiety to the PEP group, and LM comprises an N-protected NH-rhodamine moiety of formula (I), [ka]

[0017] In the formula, R 1 , R 2 , R 3 , R 6 , R 7 , R 8 , R 11 , R 12 , R 13 , and R 14 are each independently hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, -R b , or -(CH2) n -R b or R 1 and R 2 and / or R 6 and R 7 together with the carbon atom to which they are attached form an optionally substituted benzo group, R 2 , R 3 , R 7 , R 8 , R 12 , or R 13 one of which contains a group of formula -Y-, where Y is -C(O)-, S(O) 2 -, -S-, and -NH-;

[0018] R 4is selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, or R 4 and R 2 or R 3 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group;

[0019] R 5 is H or a protecting group,

[0020] R 9 is selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, or R 7 and R 9 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group;

[0021] R 10 is H or a protecting group, or R 8 and R 10 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group;

[0022] R 7 and R 9 or R 8 and R 10 at least one of, together with the atom to which they are attached, forms an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group, and optionally, R 4 and R 2 or R 3together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group, provided that the compound has the formula [ka] Not a compound of

[0023] Each R a is lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl, -CX 3 and 6- to 20-membered heteroarylalkyl;

[0024] Each R b are independently X, -OH, -OR a , -SH, -SR a -NH 2 , -NHR a , N.R. c R c , N + R c R c R c , perhalo lower alkyl, trihalomethyl, trifluoromethyl, -P(O)(OH) 2 , P(O)(OR a ) 2 , P(O)(OH)(OR a ), -OP(O)(OH) 2 , OP(O)(OR a ) 2 , -OP(O)(OR a )(OH), -S(O) 2 OH, S(O) 2 R a , -C(O)H, C(O)R a , -C(S)X, -C(O)OR a , -C(O)OH, -C(O)NH 2 , -C(O)NHR a , C(O)NR c R c , C(S)NH 2 , C(O)NHR a , -C(O)NRc R c , -C(NH)NH 2 , -C(NH)NHR a , and C(NH)NR c R c is selected from

[0025] Each R c are independently a or two R attached to the same nitrogen atom c together with the nitrogen atom form a 5-8 membered saturated or unsaturated ring which may optionally contain one or more of the same or different ring heteroatoms typically selected from O, N, and S;

[0026] Each R d and R e When alone, each independently represents hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, -R b , or -(CH2) n -R b and

[0027] n is an integer ranging from 1 to 10.

[0028] In another embodiment, a reagent useful for labeling oligonucleotides is a compound of the following structural formula: LM-L-PEP

[0029] wherein LM represents a labeling moiety comprising an N-protected NH-rhodamine moiety, PEP is a phosphate precursor group comprising a phosphoramidite group or an H-phosphonate group, L is an optional linker that attaches the labeling moiety to the phosphate precursor group, and the N-protected NH-rhodamine moiety is of structure (I): [ka]

[0030] In the formula, R 1 , R 2 , R3 , R 6 , R 7 , R 8 , R 11 , R 12 , R 13 , and R 14 are each independently selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5- to 14-membered heteroaryl, and 6- to 20-membered heteroarylalkyl; -R b , or -(CH2) n -R b , R 5 is a protecting group, R 4 is R 9 , and R 10 は , それぞれ When used alone, each independently represents hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, -P(O)(OH) 2 , P(O)(OR a ) 2 , P(O)(OH)(OR a ), -OP(O)(OH) 2 , OP(O)(OR a ) 2 , -OP(O)(OR a )(OH), -S(O) 2 OH, S(O) 2 R a , -C(O)H, C(O)R a , -C(S)X, -C(O)OR a , -C(O)OH, -C(O)NH 2 , -C(O)NHR a , C(O)NR c R c , C(S)NH 2 , C(O)NHR a , -C(O)NR c R c , -C(NH)NH 2 , -C(NH)NHR a , and C(NH)NR c R c wherein X is halo; or R 1and R 2 and / or R 6 and R 7 together with the carbon atom to which they are attached form an optionally substituted benzo group, and / or R 7 and R 9 and / or R 8 and R 10 and / or R 4 and R 2 or R 3 together with the carbon atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group;

[0031] n is an integer ranging from 1 to 10;

[0032] In the formula, R b are independently -X, -OH, -OR a , -SH, -SR a -NH 2 , -NHR a , N.R. c R c , N + R c R c R c , perhalo lower alkyl, trihalomethyl, -P(O)(OH) 2 , P(O)(OR a ) 2 , P(O)(OH)(OR a ), -OP(O)(OH) 2 , OP(O)(OR a ) 2 , -OP(O)(OR a )(OH), -S(O) 2 OH, S(O) 2 R a , -C(O)H, C(O)R a , -C(S)X, -C(O)OR a , -C(O)OH, -C(O)NH 2 , -C(O)NHR a , C(O)NR c R c , C(S)NH 2, C(O)NHR a , -C(O)NR c R c , -C(NH)NH 2 , -C(NH)NHR a , and C(NH)NR c R c Each R a is, independently of the others, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl, -CX 3 and 6- to 20-membered heteroarylalkyl, each R c is independent of others, R a or two R attached to the same nitrogen atom c together with the nitrogen atom form a 5-8 membered saturated or unsaturated ring which may optionally contain one or more of the same or different ring heteroatoms typically selected from O, N and S;

[0033] However, R of the compound of formula LM-L-PEP 2 , R 3 , R 7 , R 8 , R 12 , or R 13 At least one of the groups contains a group of formula -Y-, where Y is -C(O)-, -S(O) 2 -, -S-, and -NH-.

[0034] In another aspect, the method comprises:

[0035] Co-amplifying the nucleic acid sample with multiple amplification primer pairs to form multiple amplification products, at least one of each primer pair comprising a labeled nucleotide having the structural formula LM-L-PEP, and each amplification product comprising a different locus. [Brief description of the drawings]

[0036] [Figure 1] FIG. 1 shows exemplary linkers that can be used to link together various different moieties, including the reagents described herein. [Diagram 2]FIG. 1 shows an exemplary embodiment of a non-nucleoside synthesis reagent that does not include a synthetic handle. [Diagram 3] FIG. 1 shows an exemplary embodiment of a nucleoside synthesis reagent that does not include a synthetic handle. [Figure 4] FIG. 1 shows exemplary embodiments of non-nucleoside synthesis reagents that include synthetic handles. [Diagram 5] FIG. 1 shows an exemplary embodiment of a nucleoside synthesis reagent comprising a synthetic handle. [Figure 6] FIG. 1 shows an exemplary embodiment of a non-nucleoside solid support reagent. [Figure 7] FIG. 1 shows an exemplary embodiment of a nucleoside solid support reagent. [Figure 8A] FIG. 1 illustrates the use of specific embodiments of synthesis reagents to synthesize oligonucleotides labeled with NH-rhodamine dyes at the 5′-hydroxyl. [Figure 8B] FIG. 1 shows a specific embodiment of a synthesis reagent for the use of linker phosphoramidites and for the in situ synthesis of oligonucleotides labeled with energy transfer dyes at the 5′-end. [Figure 9] FIG. 1 illustrates the use of specific embodiments of synthesis reagents to synthesize oligonucleotides labeled with energy transfer dyes at the 3-hydroxyl. [Figure 10] 1 shows the spectra of a dye set proposed for use in multiplex assays, where Cmp A is an asymmetric rhodamine as described in PCT / US2019 / 67925 and Cmp B is an asymmetric rhodamine shown in structure D.1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the compositions and methods described herein. In this disclosure, the use of "or" includes "and / or" unless expressly stated otherwise. Similarly, the terms "comprise," "comprises," "comprising," "include," "includes," and "including" are not intended to be limiting.

[0038] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. It should be further noted that the claims may be drafted to exclude any element. Thus, this statement is intended to serve as a predicate for use of exclusive terminology, such as "solely," "only," and the like, in connection with the recitation of claim elements or the use of a "negative" limitation.

[0039] As used herein, the terms "including," "containing," and "comprising" are used in their open, non-limiting sense.

[0040] In order to provide a more concise description, some of the quantitative expressions given herein are not modified with the term "about". It is understood that all amounts described herein, whether or not the term "about" is explicitly used, are meant to refer to the actual given value, and also to refer to an approximation to the given value, including equivalents and closes, as reasonably estimated based on a person of ordinary skill in the art, according to experimental and / or measurement conditions for such given value. Whenever a yield is given as a percentage, such a yield refers to the amount of the entity for which the yield is given, relative to the maximum amount of the same entity that can be obtained under specific stoichiometric conditions. Concentrations given as percentages refer to mass ratios, unless otherwise indicated.

[0041] 4.1 Definition As used in this document, the following terms and expressions are intended to have the following meanings:

[0042] "Alkyl" by itself or as part of another substituent refers to a saturated or unsaturated, branched, straight chain or cyclic monovalent hydrocarbon radical having the specified number of carbon atoms (i.e., C1-C6 means 1 to 6 carbon atoms) derived by the removal of one hydrogen atom from a single carbon atom of a parent alkane, alkene, or alkyne. Exemplary alkyl groups include, but are not limited to, methyl; ethyl, such as ethanyl, ethenyl, ethynyl; propyl, such as propan-1-yl, propan-2-yl, cyclopropan-1-yl, prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, cycloprop-1-en-1-yl; cycloprop-2-en-1-yl, prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butan-1-yl, butan-2-yl, 2-methyl-propan-1-yl, 2-methyl- butyl such as propan-2-yl, cyclobutan-1-yl, but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl; and the like. Where specific levels of saturation are intended, the nomenclature "alkanyl", "alkenyl" and / or "alkynyl", as defined below, is used. As used herein, "lower alkyl" means (C1-C8) alkyl.

[0043] "Alkanyl" by itself or as part of another substituent refers to a saturated, branched, straight chain or cyclic alkyl derived by removing one hydrogen atom from a single carbon atom of a parent alkane. Typical alkanyl groups include, but are not limited to, methanyl; ethanyl; propanyl, such as propan-1-yl, propan-2-yl (isopropyl), cyclopropan-1-yl; butanyl, such as butan-1-yl, butan-2-yl (sec-butyl), 2-methyl-propan-1-yl (isobutyl), 2-methyl-propan-2-yl (t-butyl), cyclobutan-1-yl; and the like. As used herein, "lower alkanyl" refers to (C1-C8) alkanyl.

[0044] "Alkenyl," by itself or as part of another substituent, refers to an unsaturated, branched, straight chain or cyclic alkyl having at least one carbon-carbon double bond derived by the removal of a hydrogen atom from a single carbon atom of a parent alkane. The group may be in either the cis or trans conformation about the double bond(s). Typical alkenyl groups include, but are not limited to, ethenyl; propenyl such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl, prop-2-en-2-yl, cycloprop-1-en-1; cycloprop-2-en-1-yl; butenyl such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobuta-1,3-dien-1-yl, and the like; and the like. As used herein, "lower alkenyl" refers to (C2-C8) alkenyl.

[0045] "Alkynyl" by itself or as part of another substituent refers to an unsaturated, branched, straight chain or cyclic alkyl having at least one carbon-carbon triple bond derived by removing one hydrogen atom from a single carbon atom of a parent alkyne. Typical alkynyl groups include, but are not limited to, ethynyl; propynyl such as prop-1-yn-1-yl, prop-2-yn-1-yl; butynyl such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, and the like; and the like. As used herein, "lower alkynyl" refers to (C2-C8) alkynyl.

[0046] "Alkyldiyl" by itself or as part of another substituent refers to a saturated or unsaturated, branched, straight-chain or cyclic divalent hydrocarbon radical having the specified number of carbon atoms (i.e., C1-C6 means 1 to 6 carbon atoms) derived by the removal of one hydrogen atom from each of two different carbon atoms of a parent alkane, alkene, or alkyne, or by the removal of two hydrogen atoms from a single carbon atom of a parent alkane, alkene, or alkyne. The two monovalent radical centers or each valency of the divalent radical center can form bonds with the same or different atoms.Typical alkyldiyl groups include, but are not limited to, methanediyl; ethyldiyl such as ethane-1,1-diyl, ethane-1,2-diyl, ethene-1,1-diyl, and ethene-1,2-diyl; propane-1,1-diyl, propane-1,2-diyl, propane-2,2-diyl, propane-1,3-diyl, cyclopropane-1,1-diyl, cyclopropane-1,2-diyl, prop-1-ene-1,1-diyl, and prop-1-ene-1,2-diyl. propyldiyls such as propane-2-ene-1,2-diyl, propane-1-ene-1,3-diyl, cyclopropane-1-ene-1,2-diyl, cyclopropane-2-ene-1,2-diyl, cyclopropane-2-ene-1,1-diyl, and propane-1-yn-1,3-diyl; butane-1-diyl, butane-1,2-diyl, butane-1,3-diyl, butane-1,4-diyl, butane-2,2-diyl, 2-methyl-propane-1,1-diyl, and 2-methyl-propane-2,2-diyl. butyldiyl such as cyclobutane-1,2-diyl, cyclobutane-1,1-diyl; cyclobutane-1,2-diyl, cyclobutane-1,3-diyl, but-1-ene-1,1-diyl, but-1-ene-1,2-diyl, but-1-ene-1,3-diyl, but-1-ene-1,4-diyl, 2-methyl-prop-1-ene-1,1-diyl, 2-methanylidene-propane-1,1-diyl, buta-1,3-diene-1,1-diyl, buta-1,3-diene-1,2-diyl yl, buta-1,3-diene-1,3-diyl, buta-1,3-diene-1,4-diyl, cyclobut-1-ene-1,2-diyl, cyclobut-1-ene-1,3-diyl, cyclobut-2-ene-1,2-diyl, cyclobuta-1,3-diene-1,2-diyl, cyclobuta-1,3-diene-1,3-diyl, but-1-yne-1,3-diyl, but-1-yne-1,4-diyl, buta-1,3-diyne-1,4-diyl, etc.; and the like. Where a specific level of saturation is intended, the nomenclature alkanyldiyl, alkenyldiyl, and / or alkynyldiyl is used. Where it is specifically intended that the two valencies are on the same carbon atom, the nomenclature "alkylidene" is used. In some embodiments, the alkyldiyl group is a (C1-C8) alkyldiyl.Particular embodiments include saturated acyclic alkanyldiyl groups in which the radical center is at the terminal carbon, such as methanediyl (methano), ethane-1,2-diyl (ethano), propane-1,3-diyl (propano), butane-1,4-diyl (butano), etc. (also referred to as alkyleno, as defined below). As used herein, "lower alkyldiyl" refers to (C1-C8) alkyldiyl.

[0047] "Alkylene," by itself or as part of another substituent, refers to a straight-chain, saturated or unsaturated alkyldiyl group having two terminal monovalent radical centers derived by the removal of one hydrogen atom from each of the two terminal carbon atoms of a straight-chain or branched parent alkane, alkene, or alkyne, or by the removal of one hydrogen atom from each of two different ring atoms of a parent cycloalkyl. If a double or triple bond locant is present in a particular alkylene, it is indicated by square brackets. Typical alkylene groups include, but are not limited to, ethylene, such as ethano, etheno, ethino; propylene, such as propano, prop[1]eno, prop[1,2]dieno, prop[1]yno; butylene, such as butano, but[1]eno, but[2]eno, but[1,3]dieno, but[1]yno, but[2]yno, but[1,3]diyno; and the like. Where a specific level of saturation is intended, the nomenclature alkano, alkeno and / or alkyno is used. In some embodiments, the alkylene group is (C1-C8) or (C1-C3) alkylene. Specific embodiments include straight chain saturated alkano groups, for example, methano, ethano, propano, butano, etc. As used herein, "lower alkylene" means (C1-C8) alkylene.

[0048] "Heteroalkyl", "heteroalkanyl", "heteroalkenyl", "heteroalkynyl", "heteroalkyldiyl" and "heteroalkylene", by themselves or as part of another substituent, refer to alkyl, alkanyl, alkenyl, alkynyl, alkyldiyl and alkylene groups, respectively, in which one or more carbon atoms are each independently replaced with the same or different heteroatom or heteroatomic group. Exemplary heteroatoms and / or heteroatomic groups which can replace carbon atoms include, but are not limited to, -O-, -S-, -SO-, -NR'-, -PH-, -S(O)-, -SO2-, -S(O)NR'-, -SON2NR'-, and the like, combinations thereof, where R' is hydrogen or a substituent such as (C1-C8)alkyl, (C6-C14)aryl or (C7-C20)arylalkyl.

[0049] "Cycloalkyl" and "heterocycloalkyl", by themselves or as part of another substituent, refer to cyclic versions of "alkyl" and "heteroalkyl" groups, respectively. In the case of a heteroalkyl group, a heteroatom can occupy the position of attachment to the remainder of the molecule. Typical cycloalkyl groups include, but are not limited to, cyclopropyl; cyclobutyls such as cyclobutanyl and cyclobutenyl; cyclopentyls such as cyclopentanyl and cyclopentenyl; cyclohexyls such as cyclohexanyl and cyclohexenyl; and the like. Typical heterocycloalkyl groups include, but are not limited to, tetrahydrofuranyl (e.g., tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, etc.), piperidinyl (e.g., piperidin-1-yl, piperidin-2-yl, etc.), morpholinyl (e.g., morpholin-3-yl, morpholin-4-yl, etc.), piperazinyl (e.g., piperazin-1-yl, piperazin-2-yl, etc.), and the like.

[0050] "Parent Aromatic Ring System" refers to an unsaturated cyclic or polycyclic ring system having a conjugated pi-electron system. Specifically included within the definition of "parent aromatic ring system" are fused ring systems in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as fluorene, indane, indene, phenalene, tetrahydronaphthalene, and the like. Exemplary parent aromatic ring systems include, but are not limited to, aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexylene, indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, tetrahydronaphthalene, triphenylene, trinaphthalene, and the like.

[0051] "Aryl," by itself or as part of another substituent, refers to a monovalent aromatic hydrocarbon radical having the specified number of carbon atoms (i.e., C6-C14 means 6 to 14 carbon atoms) derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Typical aryl groups include, but are not limited to, radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorene, hexacene, hexaphene, hexylene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene, octalene, ovalene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrene, picene, pleiadene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene, and the like, and their various hydro isomers. Specific exemplary aryls include phenyl and naphthyl.

[0052] "Arylalkyl" by itself or as part of another substituent refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, in some embodiments a terminal or sp3 carbon atom, is replaced with an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, 2-phenylethen-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, 2-naphthylethene-1-yl, naphthobenzyl, 2-naphthopheniethan-1-yl, and the like. When alkyl moieties having a specific degree of saturation are intended, the nomenclature arylalkanyl, arylalkenyl, and / or arylalkynyl is used. When a defined number of carbon atoms is recited, e.g., (C7-C20)arylalkyl, the number refers to the total number of carbon atoms that make up the arylalkyl group.

[0053] "Parent heteroaromatic ring system" refers to a parent aromatic ring system in which one or more carbon atoms are each independently replaced with the same or different heteroatom or heteroatom group. Exemplary heteroatoms or heteroatom groups replacing carbon atoms include, but are not limited to, N, NH, P, O, S, S(O), SO2, Si, and the like. Specifically included within the definition of "parent heteroaromatic ring system" are fused rings in which one or more of the rings are aromatic and one or more of the rings are saturated or unsaturated, such as benzodioxane, benzofuran, chromane, chromene, indole, indoline, xanthene, and the like. Also included within the definition of "parent heteroaromatic ring system" are recognized rings that contain common substituents, such as benzopyrone and 1-methyl-1,2,3,4-tetrazole. Exemplary parent heteroaromatic ring systems include, but are not limited to, acridine, benzimidazole, benzisoxazole, benzodioxane, benzodioxole, benzofuran, benzopyrone, benzothiadiazole, benzothiazole, benzotriazole, benzoxazine, benzoxazole, benzoxazoline, carbazole, β-carboline, chroman, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, Examples of such an alkyl group include isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenanthroline, phenanthroline, phenanthine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, and xanthene.

[0054] "Heteroaryl" by itself or as part of another substituent refers to a monovalent aromatic group having the specified number of ring atoms (e.g., "C5-C14 membered" means 5 to 14 carbon atoms) derived by the removal of one hydrogen atom from a single atom of a parent aromatic ring system. Exemplary heteroaryl groups include, but are not limited to, acridine, benzimidazole, benzisoxazole, benzodioxane, benzodiazole, benzofuran, benzopyrone, benzothiadiazole, benzothiazole, benzotriazole, benzoxazine, benzoxazole, benzoxazoline, carbazole, β-carboline, chromane, chromene, cinnoline, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isopropyl ... Examples of such groups include isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, perimidine, phenanthridine, phenanthroline, phenanthine, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolidine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and the like, and groups derived from various hydroisomers thereof.

[0055] "Heteroarylalkyl" by itself or as part of another substituent refers to an acyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, in some embodiments a terminal or sp3 carbon atom, is replaced with a heteroaryl group. Where alkyl moieties having a specific degree of saturation are intended, the nomenclature heteroarylalkanyl, heteroarylalkenyl and / or heteroarylalkynyl are used. When a defined number of atoms is recited, e.g., 6-20 membered heteroarylalkyl, that number refers to the total number of atoms that make up the arylalkyl group.

[0056] "Haloalkyl," by itself or as part of another substituent, refers to an alkyl group in which one or more of the hydrogen atoms are replaced with halogen. Thus, the term "haloalkyl" is meant to include monohaloalkyl, dihaloalkyl, trihaloalkyl, etc., up to perhaloalkyl. For example, the expression "(C1-C2)haloalkyl" includes fluoromethyl, difluoromethyl, trifluoromethyl, 1-fluoroethyl, 1,1-difluoroethyl, 1,2-difluoroethyl, 1,1,1-trifluoroethyl, perfluoroethyl, and the like.

[0057] The groups defined above can include prefixes and / or suffixes commonly used in the art to create additional well-recognized substituents. As non-limiting examples, "alkyloxy" and / or "alkoxy" refer to groups of the formula -OR", "alkylamine" refers to groups of the formula -NHR", and "dialkylamine" refers to groups of the formula -NR"R", where each R" is an alkyl.

[0058] As used herein, "DNA" refers to deoxyribonucleic acid in various forms as understood in the art, such as genomic DNA, cDNA, isolated nucleic acid molecules, vector DNA, and chromosomal DNA. "Nucleic acid" refers to any form of DNA or RNA (ribonucleic acid). As used herein, the term "isolated nucleic acid molecule"1 refers to a nucleic acid molecule (DNA or RNA) that has been removed from its original environment. Some examples of isolated nucleic acid molecules are recombinant DNA molecules contained in a recombinant vector,

[0059] "Isolated" nucleic acid molecules include recombinant DNA molecules maintained in heterologous host cells, partially or substantially purified nucleic acid molecules, and synthetic DNA molecules. An "isolated" nucleic acid may be free of sequences which naturally flank the nucleic acid in the genomic DNA of the organism from which the nucleic acid is derived (i.e., sequences located at the 5' and 3' ends of the nucleic acid). Moreover, an "isolated" nucleic acid molecule, such as a cDNA molecule, may be substantially free of other cellular material or culture medium when produced by recombinant techniques, or free of chemical precursors or other chemicals when chemically synthesized.

[0060] A "short tandem repeat" or "STR" locus refers to a region of genomic DNA that contains short repeated sequence elements. The repeated sequence elements are typically, but not limited to, 3-7 base pairs in length. Each sequence element is repeated at least once within a STR, and is referred to herein as a "repeat unit." The term STR encompasses regions of genomic DNA in which multiple repeat units are repeated in tandem or with intervening bases, provided that at least one of the sequences is repeated at least twice in tandem.

[0061] "Polymorphic short tandem repeat loci" refer to STR loci in which the number of repeated sequence elements (and therefore the net length of the sequence) in a particular region of genomic DNA varies from allele to allele and from individual to individual.

[0062] As used herein, "allelic ladder" refers to a standard size marker consisting of amplified alleles from a genetic locus. An "allele" is a genetic variation associated with a segment of DNA, i.e., one of two or more alternative forms of a DNA sequence occupying the same genetic locus.

[0063] "Biochemical nomenclature" refers to standard biochemical nomenclature used herein, where nucleotide bases are designated as adenine (A), thymine (T), guanine (G), and cytosine (C). The corresponding nucleotide is, for example, deoxyguanosine-5'-triphosphate (dGTP).

[0064] "DNA polymorphism" refers to the coexistence of two or more different nucleotide sequences in a DNA sequence within the same mating population.

[0065] "Locus" or "genetic locus" refers to a specific physical location on a chromosome. Alleles of a locus are located at the same site on homologous chromosomes.

[0066] A "locus-specific primer" refers to a primer that specifically hybridizes to a portion of a described locus, or its complementary strand, for at least one allele of that locus, and does not hybridize efficiently to other DNA sequences under the conditions used in the amplification method.

[0067] "Polymerase chain reaction" or "PCR" uses repeated cycles of denaturation, annealing with primers, and extension with a DNA polymerase enzyme to generate approximately 10 copies of a target DNA sequence. 6 PCR refers to a technique that amplifies nucleic acids by a factor of 2. The PCR process for amplifying nucleic acids is contained in U.S. Patent Nos. 4,683,195 and 4,683,202, which are incorporated herein by reference in their entireties for the description of the process. PCR reaction conditions include the chemical components of the reaction and their concentrations, the temperatures used in the reaction cycles, the number of cycles of the reaction, and the duration of the steps of the reaction cycles.

[0068] "Amplifying" as used herein refers to the process of enzymatically increasing the amount of a particular nucleotide sequence. This amplification is typically accomplished by, but not limited to, PCR. "Denaturation" as used herein refers to the separation of two complementary nucleotide strands from an annealed state. Denaturation can be caused by many factors, such as the ionic strength of the buffer, temperature, or chemicals that disrupt base pairing interactions. "Annealing" as used herein refers to the specific interaction between nucleotide strands, where each strand binds to each other substantially based on the complementarity between the strands, as determined by Watson-Crick base pairing. Complementarity does not need to be 100% for annealing to occur. "Extension" as used herein refers to the amplification cycle after the primer oligonucleotide and target nucleic acid are annealed, and the polymerase enzyme effects primer extension into appropriately sized fragments using the target nucleic acid as a replicative template.

[0069] "Primer" refers to a single stranded oligonucleotide or DNA fragment that hybridizes to a DNA strand of a locus such that the 3' end of the primer can act as a site for polymerization and extension using a DNA polymerase enzyme. "Primer pair" refers to two primers including primer 1 that hybridizes to a single strand at one end of a DNA sequence to be amplified and primer 2 that hybridizes to the other end of the complementary strand of the DNA sequence to be amplified. "Primer site" refers to the region of the target DNA to which a primer hybridizes.

[0070] A "genetic marker" is generally an allele of genomic DNA with a characteristic of interest for analysis, such as DNA typing, where individuals are differentiated based on DNA mutations. Most DNA typing methods are designed to detect and analyze differences in length and / or sequence of one or more regions of a DNA marker known to appear in at least two different forms or alleles in a population. Such mutations are called "polymorphisms" and any region of DNA where such mutations occur is called a "polymorphic locus." One possible method of performing DNA typing involves combining PCR amplification techniques (KB Mullis, U.S. Pat. No. 4,683,202) with the analysis of length mutation polymorphisms. Traditionally, PCR could only be used to reliably amplify relatively small DNA segments, i.e., DNA segments less than 3,000 bases in length (M. Ponce and L. Micol (1992), NAR 20(3):623; R. Decorte et al. (1990), DNA CELL BIOL 9(6):461 469). Short tandem repeats (STRs), minisatellites, and variable number of tandem repeats (VNTRs) are examples of length variation polymorphisms. DNA segments containing minisatellites or VNTRs are usually too long to be reliably amplified by PCR. In contrast, STRs, containing repeating units of about 3-7 nucleotides, are short enough to be useful as genetic markers in PCR applications because amplification protocols can be designed to generate products that are smaller than those possible from other variable length regions of DNA.

[0071] The term "kit" as used herein refers to any delivery system for delivering materials. In the context of a reaction assay, such delivery systems include systems that allow for the storage, transport or delivery of reaction reagents (e.g., oligonucleotides, enzymes, primer set(s) in suitable containers, etc.) and / or support materials (e.g., buffers, written instructions for performing the assay, etc.) from one location to another. For example, a kit can include one or more containers (e.g., boxes) that contain the relevant reaction reagents and / or support materials. The term "fragmentation kit" as used herein refers to a delivery system that includes two or more separate containers that contain each subportion of all the components of the kit. The containers are delivered together or separately to the intended recipient. For example, a first container contains the enzymes used in the assay and a second container contains the oligonucleotides. Indeed, any delivery system that includes two or more separate containers that contain each subportion of all the components of the kit is included in the term "fragmentation kit". In contrast, a "combined kit" refers to a delivery system that includes all the components of a reaction assay in a single container (e.g., a single box that contains each of the desired components). The term "kit" includes both fragmented and combined kits.

[0072] 4.2 Illustrative Embodiments

[0073] The present disclosure provides reagents that can be used to chemically synthesize oligonucleotides with labeling moieties that include rhodamine dyes. Previously, it was difficult to chemically synthesize rhodamine-labeled oligonucleotides, in part due to the lack of availability of rhodamine-containing synthesis reagents that are stable to the synthesis and / or deprotection conditions commonly used in the stepwise chemical synthesis of oligonucleotides. It has now been discovered that protecting the exocyclic amine group of NH-rhodamine dyes with base-labile protecting groups such as acetyl groups results in N-protected NH-rhodamine dyes that are stable to the chemical synthesis and deprotection conditions commonly used in solid-state synthesis of oligonucleotides. As a result, N-protected NH-rhodamine can be incorporated into reagents that can be used to synthesize oligonucleotides labeled with labeling moieties that include rhodamine dyes, thereby eliminating the need to attach the label after synthesis. Because the label is applied during synthesis, the resulting labeled oligonucleotides can be purified and used without the use of HPLC.

[0074] The reagents take advantage of various characteristics and chemistries of reagents well known in the stepwise solid-state synthesis of oligonucleotides and can take the form of synthesis reagents that are attached to hydroxyl groups during the stepwise solid-state synthesis of oligonucleotide chains, or solid support reagents to which nucleoside monomer reagents, e.g. nucleoside phosphoramidite reagents, and / or any other reagents are attached in a stepwise manner to obtain synthetic oligonucleotides.

[0075] The synthesis and solid support reagents can be nucleosidic in nature, in that they can include nucleoside moieties, or they can be non-nucleosidic in nature.

[0076] All of the reagents described herein that contain a labeling moiety include an N-protected NH-rhodamine dye or moiety. The N-protected NH-rhodamine dye can be the only dye that contains a labeling moiety, or it can be one of two or more dyes that comprise a larger dye network. The solid support reagent further includes a solid support and one or more synthetic handles to which additional groups can be attached. The synthetic reagent further includes a PEP group that is useful for attaching the reagent to a primary hydroxyl group, and may optionally include one or more synthetic handles. The various moieties and groups that comprise the reagent can be linked to each other in any manner and / or orientation that allows them to perform their respective functions. These moieties and groups can be linked to each other via a linking group contained in the moiety, or can be linked to each other with the aid of a linker.

[0077] The various moieties, groups, and linkers that comprise the reagents described herein are described in more detail below.

[0078] 4.3 Linkers and Linking Groups

[0079] The various groups and moieties comprising the reagents described herein are typically linked together by linkers. The identity of a particular linker will depend, in part, on the identity of the moieties that are linked together. In general, the linker includes a spacing moiety that can include virtually any combination of atoms or functional groups that are stable to the synthesis conditions used to synthesize labeled oligonucleotides, such as conditions commonly used to synthesize oligonucleotides by the phosphite triester method, and the linker can be linear, branched, or cyclic in structure, or can include a combination of linear, branched, and / or cyclic structures. The spacing moiety can be monomeric in nature, or can be or include a region that is polymeric in nature. The spacing moiety can be designed to have certain properties, such as the ability to be cleaved under certain conditions, or a certain degree of rigidity, flexibility, hydrophobicity, and / or hydrophilicity.

[0080] As described in more detail below, many embodiments of the reagents described herein are synthesized by condensing synthons together in a specific manner to obtain the desired reagent. Each synthon typically contains one or more linking groups suitable for forming the desired bond. Generally, the linking group is a functional group that can be linked to another functional group F. z or is activated and capable of reacting to give a covalent bond YZ. y wherein Y is F y and Z represents a linking moiety provided by F z Such a group F y and F z are referred to herein as "complementary functional groups."

[0081] Pairs of complementary functional groups capable of forming covalent bonds with one another are well known in the art. y or F z One of the groups contains a nucleophilic group, and F y or F z The other of the groups comprises an electrophilic group. Complementary nucleophilic and electrophilic groups useful for forming bonds (or precursors thereof that are suitably activated or can be activated to form bonds) that are stable to a variety of synthetic and other conditions are well known in the art. Examples of suitable complementary nucleophilic and electrophilic groups that can be used to effect bond formation in the various reagents described herein, and the resulting bonds formed therefrom, are provided in Table 1 below. [Table 1]

[0082] Thus, a linker synthon can be generally represented by the formula LG-Sp-LG, where each LG independently of the others represents a linking group and Sp represents a spacing moiety. In some embodiments, the linker synthon has the formula F z -Sp-F z where each zEach F independently represents a member of a pair of complementary nucleophilic or electrophilic groups, as described above. z are independently selected from the group listed in Table 1 above. Linker synthons of this type form linker moieties of the formula -Z-Sp-Z-, where each Z, independently of the others, represents a portion of the bond described above. Particular linkers suitable for linking together specific groups and moieties in the reagents described herein will be discussed in more detail in connection with exemplary embodiments of the reagents. Non-limiting exemplary embodiments of linkers that can be used to link together various groups and moieties comprising the reagents described herein are shown in FIG. 2. In FIG. 2, Z 1 and Z 2 each independently represents, as described above, a functional group F z and K is selected from -CH- and -N-. In some specific embodiments of the linker shown in FIG. 1 or Z 2 One of them is -NH-, and the other is -O-, C(O)-, and -S(O) 2 - is selected.

[0083] 4.4 Sign part

[0084] The reagents described herein can include labeling moieties that include NH-rhodamine dyes in which one of the exocyclic amine groups is protected with a protecting group having specific properties. In general, rhodamine dyes are characterized by four main features: (1) a parent xanthene ring, (2) an exocyclic amine substituent, (3) an exocyclic iminium substituent, and (4) a phenyl group substituted at the ortho position with a carboxyl group. In some embodiments, the NH-rhodamine dyes of the present disclosure can be generally described by formula (Ia). In some embodiments, the exocyclic amine and / or iminium groups are typically located at the C3' and C6' carbon atoms of the parent xanthene ring, although "extended" rhodamines are also known in which the parent xanthene ring contains benzo groups fused to the C3' and C4' and / or C5' and C6' carbons. In these extended rhodamines, the characteristic exocyclic amine and iminium groups are located at the corresponding positions of the extended xanthene ring.

[0085] The carboxyl-substituted phenyl group is attached to the C1 carbon of the parent xanthene ring. As a result of the orthocarboxyl substituent, the rhodamine dyes can exist in two different forms: (1) an open acid form, and (2) a closed lactone form. While not intending to be bound by any working theory, the NMR spectrum of the exemplary N-protected NH-rhodamine dyes described herein is consistent with the closed spirolactone form of the dye, and therefore, the N-protected NH-rhodamine dyes containing the labeling moieties of the reagents described herein are believed to be in the closed spirolactone form. Thus, the various rhodamines, and their unprotected counterparts, are shown herein in their closed spirolactone form. However, it should be noted that this is merely for convenience and is not intended to limit the various reagents described herein to the lactone form of the dye. In certain embodiments, the open acid form of the compound is fluorescent (or exhibits increased fluorescence) compared to the closed spirolactone form of the compound. The amine group of the compounds described herein can be protected in the form of closed spirolactone and used as phosphoramidite for high yield and purity of nucleic acid labeling.Therefore, fluorescently labeled nucleic acid probes and primers are also provided herein that include the compounds in the deprotected open lactone form.Representative examples of the open lactone form after deprotection of the amine group and cleavage of the nucleic acid probe from the solid support are shown in Figures 8 and 9.

[0086] In the closed spirolactone form, the A and C rings of the parent xanthene ring are aromatic and the C3' and C6' substituents are both amines. The exocyclic amine groups of the rhodamine dyes included in the labeling moieties described herein are either unsubstituted or monosubstituted such that these amine groups are primary or secondary amines. Such rhodamine dyes are referred to herein as "NH-rhodamines." Thus, as used herein, "NH-rhodamines" generally includes the parent NH-rhodamine ring structure: [ka]

[0087] In the formula, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 , R 10 , R 11 , R 12 , R 13 , and R 14 Each of the carbon atoms is as defined herein. In the parent NH-rhodamine ring shown above, the various carbon atoms are numbered using an arbitrary numbering convention adopted from the numbering convention commonly used for closed spirolactone forms of rhodamine dyes. This numbering system is used merely for convenience and is not intended to be limiting in any way.

[0088] In any of the embodiments described herein, exemplary labeling moieties are represented by the following formulas (II.1), (II.2), (II.3), (II.4): [ka] and the like.

[0089] Those skilled in the art will appreciate that this disclosure f and R g Any double bond between e and R f It will be readily understood that the present invention describes other labeling moieties in formula I, such as those of formulas (II.1), (II.2), and (II.3), which exist between R e and R f There is no double bond between R e and R f If the additional group above is H, then the skilled artisan can easily determine the additional R d The group is R e and R f Furthermore, it will be understood by those skilled in the art that R h and Ri Between or R i and R j It will be appreciated that further embodiments may be utilized in which an optional double bond between

[0090] In the NH-rhodamine ring of structural formula (Ia) and other formulae described herein, R 5 is hydrogen or R 5 represents a substituent that substitutes the exocyclic amine to which it is attached. In some embodiments, R 5 may be a substituted or unsubstituted alkylaryl or arylalkyl group. In some embodiments, R 5 can be a protecting group.

[0091] In some embodiments, R 4 , R 9 , and / or R 10 may contain a substituent bridged to adjacent carbon atoms such that the exemplified nitrogen atom is included in a ring containing 5- or 6-ring atoms. The ring may be saturated or unsaturated, and one or more of the ring atoms may be optionally substituted. When a ring atom(s) is / are substituted, the substituents are typically selected, independently of one another, from lower alkyl, C6-C14 aryl, and C7-C20 arylalkyl groups.

[0092] Alternatively, two adjacent ring atoms may be included in an aryl bridge, such as a benzo or naphtho group. 4 , R 9 , and / or R 10Non-limiting exemplary embodiments of rhodamine dyes comprising a parent NH-rhodamine ring of structural formula (Ia), wherein the group is hydrogen or a lower alkyl group, or is optionally included in the ring substituted at adjacent carbon atoms. One or more of the carbon atoms at the C1, C4, C5, C6, C7, C1', C2', C4', C5', C7', and C8' positions of the parent NH-rhodamine ring of structural formula (Ia) are, independently of one another, unsubstituted or substituted with a substituent as defined herein. Furthermore, groups useful for substituting rhodamine dyes at these positions are well known in the art, for example, U.S. Pat. No. 4,622,400. Nos. 5,750,409, 5,847,162, 6,017,712, 6,080,852, 6,184,379, and 6,248,884, the disclosures of which are incorporated herein by reference.

[0093] In some embodiments, the substituents are, independently of each other, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, R b and -(CH 2 ) x R b wherein x is an integer from 1 to 10; R b are independently -X, -OH, -OR a , -SH, -SR a -NH 2 , -NHR a , -NR c R c , N + R c R c R c , perhalo lower alkyl, trihalomethyl, trifluoromethyl, -P(O)(OH) 2 , P(O)(OR a ) 2 , P(O)(OH)(OR a ), -OP(O)(OH) 2 , OP(O)(OR a ) 2 , -OP(O)(OR a)(OH), -S(O) 2 OH, S(O) 2 R a , -C(O)H, C(O)R a , -C(S)X, -C(O)OR a , -C(O)OH, -C(O)NH 2 , -C(O)NHR a , C(O)NR c R c , C(S)NH 2 , C(O)NHR a , -C(O)NR c R c , -C(NH)NH 2 , -C(NH)NHR a , and C(NH)NR c R c wherein X is halo (preferably fluoro or chloro), and each R a are independently selected from lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5- to 14-membered heteroaryl, and 6- to 20-membered heteroarylalkyl; c is, independently of the others, R a or two R attached to the same nitrogen atom ctogether with its nitrogen atom is a 5-8 membered saturated or unsaturated ring that may optionally contain one or more of the same or different ring heteroatoms, typically selected from O, N and S. Alternatively, the C1' and C2' substituents, and / or the C7' and C8' substituents together form a substituted or unsubstituted aryl bridge. Such a benzo bridge is provided that the C1' and C2' substituents, and the C7' and C8' substituents are not simultaneously included in the aryl bridge. Although quenching substitutions may be desirable, in some embodiments, the groups used to substitute the C1, C4, C5, C6, C7, C1', C2', C4', C5, C7', and C8' carbons do not promote quenching of the rhodamine dye. Substituents capable of quenching the rhodamine dye include carbonyl, carboxylate, heavy metal, nitro, bromo, and iodo. The carbon atoms at positions C4, C5, C6 and C7 of the parent NH-rhodamine ring of structural formula (Ia) may also contain optional substituents, independently of one another. These substituents may be selected from the various substituents described above. In some embodiments, the carbon atoms at positions C4 and C7 are substituted with chloro groups, such that the parent NH-rhodamine dye is an NH-4,7-dichlororhodamine dye.A vast number of rhodamine dyes containing the parent NH rhodamine ring of structural formula (Ia) that can be included in the labeling moiety of the reagents described herein are known in the art and are described in, for example, U.S. Pat. No. 6,248,884; U.S. Pat. No. 6,111,116; U.S. Pat. No. 6,080,852; U.S. Pat. No. 6,051,719; U.S. Pat. No. 6,025,505; U.S. Pat. No. 6,017,712; U.S. Pat. No. 5,936,087; U.S. Pat. No. 5,847,162; U.S. Pat. No. 5,840,999; U.S. Pat. No. 5,750,409; U.S. Pat. No. 5,366,860; U.S. Pat. No. 5,231,191; U.S. Pat. No. 5,227,487; WO 97 / 36960; WO 99 / 27020; Lee et al., 1992; Nucl. Acids. Res. 20:2471-2483; Arden-Jacob, "New Lanwellige Xanthen Farbstoffe fur FluoreszenZSonden und Farbstoff Lauer, Springer-Verlag, Germany, 1993; Sauer et al., 1995, Fluorescence 5:247-261; Lee et al., 1997, Nucl. Acids Res. 25:2816 2822; and Rosenblum et al., 1997, Nucl. Acids Res. 25:4500 4504, the disclosures of which are incorporated herein by reference. Any of the dyes described in these references in which the exocyclic amine is a primary or secondary amine as described herein, or the 4,7-dichloro analogs of such NH rhodamine dyes, can be included in the labeling moiety of the reagents described herein.

[0094] The reagents described herein are used for the chemical synthesis of labeled oligonucleotides and therefore R 5 may be a protecting group that is stable to the organic synthesis conditions used in the synthesis of oligonucleotides. 5R can be a protecting agent that protects the amine in the amide form, e.g., a carboxamide, sulfonamide, or phosphoramidite, and can be selected to protect the exocyclic amine in this manner, "locking" the protected NH-rhodamine in the closed lactone form and contributing to the stability of the reagents described herein. Although not required, R can be selected from those that are labile under the conditions used to remove the groups protecting the exocyclic amines of the nucleobases of synthetic oligonucleotides. 5 It is sometimes convenient to employ a protecting group so that the protecting group can be removed in a single step.

[0095] Conditions used for the synthesis and deprotection of synthetic oligonucleotides are well known in the art and are described, for example, in Current Protocols in Nucleic Acid Chemistry, Vol. I, Beacage et al., Eds. John Wiley & Sons, 2002, the disclosure of which is incorporated herein by reference. Briefly, the synthesis method using phosphoramidite reagents involves multiple rounds of: (i) DMT deprotection to remove the free hydroxyl, which can be done by treatment with 2.5% or 3% di- or trichloroacetic acid in dichloromethane; (ii) coupling of nucleosides or other phosphoramidite reagents to the free hydroxyl, which can be done in acetonitrile with 0.45M or 0.5M tetrazole; (iii) I 2 The reaction involves oxidation, which can be accomplished by treatment with 6.5% acetic anhydride in tetrahydrofuran (THF) followed by 10% 1-methylimidazole (MI) in THF.

[0096] Other conditions for carrying out the various steps of the synthesis are known in the art. For example, phosphoramidite coupling can be carried out in acetonitrile containing 0.25 M 5-ethylthio-1H-tetrazole, 0.25 M 4,5-dicyanoimidazole (DCI) or 0.25 M 5-benzylthio-1H-tetrazole (BTT). Oxidation can be carried out in THF / H 20.1M, 0.05M, or 0.02M I in O / pyridine (7:2:1) 2 Capping can be accomplished by treatment with THF / lutidine / acetic anhydride followed by 16% NMI in THF, 6.5% DMAP in THF followed by 10% mellum in THF, or 10% mellum in THF followed by 16% mellum in THF.

[0097] Nucleoside phosphoramidite reagents are protected with groups that can be removed under mild conditions, such as by treatment with concentrated ammonium hydroxide at room temperature for 4-17 hours, or by treatment with 0.05 M potassium carbonate in methanol, or by treatment with 25% t-butylamine in HO / EtOH, but those skilled in the art will also know that removal and cleavage of protecting groups from synthetic reagents can typically be accomplished by treatment with concentrated ammonium hydroxide at 60° C. for 1-12 hours. Those skilled in the art will be able to readily select protecting groups with suitable properties for use under particular synthetic and deprotection and / or cleavage conditions. For example, a wide variety of amine protecting groups are taught in Greene & Wuts' "Protective Groups In Organic Chemistry" 3d Edition, John Wiley & Sons, 1999 (hereinafter "Green & Wuts"), e.g., pages 309-405. Those skilled in the art will be able to readily select protecting groups R with suitable properties from those taught in Green & Wuts. 5 or R 10 In some embodiments, the protecting group R 5 or R 10 is the formula -C(O)R 15 where R 15 -CX is hydrogen, lower alkyl, methyl, 3 , -CHX 2 , -CH 2 X, -CH, O d , and lower alkyl, methyl, X, OR d, cyano, or nitro; R″ is selected from lower alkyl, phenyl, and pyridyl; and each X is a halo group, typically fluoro or chloro. In some embodiments, R 15 is methyl. In some embodiments, R 15 is trifluoromethyl. -C(O)R 15 Acyl protecting groups, such as those defined by, can be removed under a variety of basic conditions, as is well known in the art, including the mild conditions used to remove protecting groups from oligos synthesized with "base-labile" phosphoramidite reagents. In some embodiments, R 5 HA-C(O)R 15 where R 15 is hydrogen, lower alkyl, -CX 3 , -CHX 2 , -CH 2 X, -CH 2 -OR d and optionally lower alkyl, -X, -OR d , phenyl monosubstituted with a cyano or nitro group, d is selected from the group consisting of lower alkyl, phenyl and pyridyl, and each X is a halo group. Examples of conditions that can be used are specified above. As described in more detail in a later section, the N-protected NH-rhodamine moiety containing the labeling moiety can be attached to other groups or moieties. For example, the N-protected NH-rhodamine can be attached to another dye containing the labeling moiety, a PEP group, a linker, a synthetic handle, a quenching moiety, a moiety that functions to stabilize base pairing interactions (e.g., an intercalating dye or a minor groove binding molecule, etc.), or other moieties. Such attachment is typically made via a linking group LG (described above in connection with linkers) attached to the N-protected NH-rhodamine synthon used in the synthesis of the reagent.

[0098] The linking group LG can be attached to any available carbon atom of the N-protected NH-rhodamine synthon, or to a substituent attached to one of these carbon atoms. The location of the linking group can depend, in part, on the group or moiety to which the N-protected NH-rhodamine synthon is attached. In some embodiments, the linking group is attached at the C1', C2', C4', C5', C7', C8', C5, C6, or C7 position of the N-protected NH-rhodamine synthon. In certain embodiments, the linking group is attached at the C4', C5', C5, or C6 position.

[0099] The N-protected NH-rhodamine synthon may contain a single linking group LG or may contain multiple linking groups LG. In embodiments using multiple linking groups, the linking groups may be the same or different. The N-protected NH-rhodamine synthon containing multiple linking groups LG that are different from each other may have different groups or moieties attached to different positions of the parent NH-rhodamine ring using orthogonal chemistry. The identity of the linking group depends, in some cases, on the position of the linking group on the parent NH-rhodamine ring. In some embodiments where the linking group LG is attached to the C4'- or C5'-position of the parent NH-rhodamine ring, the linking group LG has the formula -(CH) n -F y where n is an integer ranging from 0 to 10; F y is as described herein. In some embodiments, n is 1 and F y is -NH.

[0100] In some embodiments where the linking group LG is attached to the 5-position or 6-position of the parent NH-rhodamine ring, the linking group LG has the formula (CH 2 ) n , -C(O)OR f where R f is selected from hydrogen and a good leaving group, and n is as previously defined. In some particular embodiments, the linking group LG comprises an NHS ester. In some particular embodiments, n is 0 and R f is the NHS.

[0101] As discussed above, the labeling moiety may include one or more additional dyes, such as N-protected NH-rhodamine, which, once deprotected, becomes a member of a larger energy transfer dye network. Such energy transfer dye networks are well known in the art and include combinations of fluorescent dyes with matched spectral characteristics and / or adjusted relative distances from one another, such that when one fluorescent dye in the network is excited by incident radiation of a suitable wavelength, it transfers its excitation energy to another fluorescent dye in the network, which then transfers its excitation energy to yet another fluorescent dye in the network, resulting in the final fluorescence by the acceptor dye in the network. The dye network provides a labeling moiety with a long Stokes shift. In such a network, a fluorophore that transfers or donates excitation energy to another fluorophore in the network is called a "donor." A fluorophore that receives or accepts excitation energy from another fluorophore is called an "acceptor." In a dye network that includes only two fluorescent dyes, one functions as a donor and the other as an acceptor. In dye networks containing three or more fluorescent dyes, at least one dye acts as both a donor and an acceptor. The principles of how dye networks function, as well as the criteria for selecting and combining suitable individual dyes to create such networks, are well known and are described, for example, in Hung et al., 1997, Anal. Biochem. 252:78-88.

[0102] In the labeling moieties described herein that include dye networks, the N-protected NH-rhodamine dyes, once deprotected, can function as donors or acceptors, or both, depending on the identity of the other dyes that comprise the network and the desired incidence and fluorescence wavelength. Many dyes suitable for use as donors and / or acceptors for NH-rhodamine dyes are known in the art, including, by way of example and not limitation, xanthene dyes (e.g., fluorescein, rhodamine and rhodol dyes), pyrene dyes, coumarin dyes (e.g., hydroxy and amino coumarins), cyanine dyes, phthalocyanine dyes and lanthanide complexes. Specific, non-limiting examples of these dyes in the context of energy transfer dye networks are described in Hung et al., 1996, Anal. Biochem. 238:165-170; Medintz et al., 2004, Proc. Nat'l Acad. Sci. USA 101(26):9612-9617; U.S. Patent No. 5,800,996; Sudhaker et al. 2003, Nucleosides, Nucleotides & Nucleic Acids 22:1443-1445; U.S. Patent No. 6,358,684; Majumdar et al., 2005, J. Mol. Biol. 351:1123-1145; Dietrich et al., 2002, Reviews Mol Biotechnology. 82(3):211-231; Tsuji et al., 2001, Biophysical J. 81(1):501-515; Dickson et al., 1995, J. Photochemistry & Photobiology 27(1):3-19; and Kumar et al., 2004, Developments in Nucl Acid Res. 1:251-274, the disclosures of which are incorporated herein by reference. Any of these dyes that can be suitably protected according to the principles described herein can be used as donor and acceptor dyes in labeling moieties that include dye networks.In some embodiments, one or more of the donor and / or acceptor dyes that comprise the network can be N-protected NH-rhodamine dyes as described herein.Specific positions for attaching donor and / or acceptor dyes to rhodamine dyes to form dye networks, as well as specific bonds and linkers that are useful for attaching such dyes, are well known in the art.Specific examples are described in, for example, U.S. Patent No. 6,811,979, U.S. Patent No. 6,008,379, U.S. Patent No. 5,945,526, U.S. Patent No. 5,863,727, and U.S. Patent No. 5,800,996, the disclosures of which are incorporated herein by reference.

[0103] In some embodiments, the linker connecting the donor dye and the acceptor dye is an anionic linker as described in U.S. Pat. No. 6,811,979, the disclosure of which is incorporated herein by reference (see, e.g., column 17, line 25 to column 18, line 37 and the disclosure in Figures 1-17).

[0104] In some embodiments of the reagents described herein, the label moiety comprises a donor dye for an NH-rhodamine dye. In some embodiments, the donor dye is a fluorescein or rhodamine dye, such as one of the NH-rhodamine dyes described herein. In certain embodiments, the donor dye is a fluorescein dye. Fluorescein dyes are structurally similar to rhodamine dyes, except that the 3- and 6-positions of the parent xanthene ring (corresponding to the 3'- and 6'-positions of the NH-rhodamine ring of structural formula (Ia)) are substituted with hydroxyl groups. Like rhodamine, fluoresceins also have extended ring structures in which the carbon atoms at the C3' and C4' and / or C5' and C6' positions of the parent xanthene ring are included in aryl bridges, such as benzo groups. Thus, fluoresceins generally include compounds of structural formulas (IVa), (IVb), and (IVc): [ka]

[0105] Like NH-rhodamine, the carbons at positions C1', C2', C2'', C4', C4'', C5', C5'', C7', C7'', C8', C4, C5, C6 and C7 of the fluorescein ring of structural formulas (IVa), (IVb) and (IVc) can be substituted with a variety of different substituents, such as those described above for NH-rhodamine.

[0106] When included in the labeling moiety described herein, the hydroxyls at the C3' and C6' positions should be protected with protecting groups having the same general properties as those protecting the exocyclic amine of NH-rhodamine described above.Thus, in certain embodiments, the protecting groups are stable to the conditions used to synthesize oligonucleotides, such as those used to synthesize and oxidize oligonucleotides via the phosphite triester method, but are unstable under conditions typically used to deprotect and / or cleave synthetic oligonucleotides from synthesis resins, such as incubation in concentrated ammonium hydroxide at room temperature or 55°C.

[0107] Fluoresceins where the C3' and C6' exocyclic hydroxyls contain protecting groups are referred to herein as "O-protected fluoresceins." The O-protected fluoresceins corresponding to the fluoresceins of structural formulas (IVa), (IVb) and (IVc), respectively, are shown below as structural formulas (Va), (Vb) and (Vc): [ka]

[0108] In the formula, R 5 represents a protecting group.

[0109] A variety of different fluorescein dyes are known in the art that can be suitably protected and incorporated into labeling moieties for use as donors of NH-rhodamine moieties.Certain exemplary fluorescein dyes are described, for example, in U.S. Pat. No. 6,221,604, U.S. Pat. No. 6,008,379, U.S. Pat. No. 5,840,999, U.S. Pat. No. 5,750,409, U.S. Pat. No. 5,654,441, U.S. Pat. No. 5,188,934, U.S. Pat. No. 5,066,580, U.S. Pat. No. 4,481,136, U.S. Pat. No. 4,439,356, WO 99 / 16832, and EP 0050684, the disclosures of which are incorporated herein by reference.Those skilled in the art can select fluoresceins that have suitable spectral properties for use as donors of specific NH-rhodamines.

[0110] The donor and the N-protected NH-rhodamine acceptor can be attached to each other in various orientations, either directly or with the aid of a linker. In some embodiments where the donor is an O-protected fluorescein or an N-protected NH-rhodamine, the donor is attached via the C2'-, C2"-, C4'-, C5'-, C7'-, C7"-, C5- or C6-position to the C2'-, C4'-, C5'-, C7'-, C5- or C6-position of the N-protected NH-rhodamine acceptor.

[0111] Specific exemplary bond orientations are shown in Table 2 below. [Table 2]

[0112] Labeling moieties that include dye networks, such as the donor-acceptor dye networks of Table 2, can be attached to any available position of the remainder of the reagent. In some embodiments, labeling moieties that include head-to-head linked acceptor / donor pairs are attached to the remainder of the reagent via the C5 or C6 position of the donor or acceptor moiety. In some embodiments, labeling moieties that include head-to-tail linked acceptor / donor pairs are attached to the remainder of the reagent via an available C4'-, C5'-, C5- or C6-position of the donor or acceptor moiety. In some embodiments, labeling moieties that include tail-to-tail linked acceptor / donor pairs are attached to the remainder of the reagent via the C4'- or C5'-position of the donor or acceptor moiety. In some embodiments, labeling moieties that include side-to-side linked acceptor / donor pairs are attached to the remainder of the reagent via the C4'-, C5'-, C5- or C6-position of the donor or acceptor. In some embodiments, a labeling moiety that comprises a side-to-head linked acceptor / donor pair is attached to the remainder of the reagent via an available C4'-, C5'-, C5-, or C6-position of the donor or acceptor. In some embodiments, a labeling moiety that comprises a side-to-tail linked acceptor / donor pair is attached to the remainder of the reagent via an available C4'-, C5'-, C5-, or C6-position of the donor or acceptor.

[0113] Regardless of these orientations, O-protected fluorescein or N-protected NH-rhodamine donors and N-protected NH-rhodamine acceptors are typically linked to one another via a linker. It has previously been discovered that such donor and acceptor dyes can be advantageously linked to one another via a linker that is essentially rigid and / or relatively long, for example, in the range of about 12-20 angstroms in length (as used herein, the "length" of a linker refers to the distance between the linking moieties, as determined by calculating the sum of the lengths of the chemical bonds that represent the shortest continuous path between the moieties). Without intending to be bound by any working theory, it is believed that linkers that tend to hold the donor and acceptor in close proximity to one another without the chromophores touching one another favorably result in efficient energy transfer. In this regard, the rigidity and length of the linker are binding parameters. In general, the shorter the linker (e.g., a linker having a length of about 5-12 angstroms), the greater the degree of rigidity required. Longer linkers (e.g., linkers having lengths in the range of about 15-30 angstroms) may be less rigid or non-rigid. Short, non-rigid (floppy) linkers should be avoided.

[0114] Rigidity can be achieved by using groups with restricted angles of rotation around their bonds, for example through the use of arylene or heteroarylene moieties and / or alkylene moieties containing double and / or triple bonds. A variety of linkers useful for linking rhodamine and fluorescein dyes to one another in the context of energy transfer dyes are known in the art and are described, for example, in U.S. Pat. No. 5,800,996, the disclosure of which is incorporated herein by reference. Specific examples of linkers useful for linking an O-protected fluorescein or N-protected NH-rhodamine donor to an N-protected NH-rhodamine acceptor of the labeling moieties described herein include, but are not limited to, linkers of the formula: (L.1)-Z-(CH 2 ) a -[(Ar) b-(CH 2 ) a ] c -Z-; (L.2)-Z-(CH 2 ) a -[C≡C-(CH 2 ) a ] c -Z-; (L.3)-Z-(CH 2 ) a -[C≡C-(Ar) b ] c -(CH 2 ) a -Z-; (L.4)-Z-(CH 2 ) d -NH-C(O)-[(CH 2 ) a -(Ar)-(CH 2 ) a -C(O)-NH] c -(CH 2 ) d -Z-; and (L.5)-Z-[CH 2 (CH 2 ) e O] f -CH 2 (CH 2 ) e O- is an example. wherein each Z is, independently of the other, a linking group F, as defined above. zeach a, independently of the others, represents an integer in the range of 0 to 4, each b, independently of the others, represents an integer in the range of 1 to 2, each c, independently of the others, represents an integer in the range of 1 to 5, each d, independently of the others, represents an integer in the range of 1 to 10, each e, independently of the others, represents an integer in the range of 1 to 4, each f, independently of the others, represents an integer in the range of 1 to 10, and each Ar, independently of the others, represents an optionally substituted monocyclic or polycyclic cycloalkylene, cycloheteroalkynene, arylene, or heteroarylene group. Non-limiting exemplary embodiments of Ar include groups derived from lower cycloalkanes, lower cycloheteroalkanes, parent aromatic ring systems, and parent heteroaromatic ring systems, as described above. Specific non-limiting exemplary embodiments of Ar include cyclohexane, piperazine, benzene, naphthalene, phenol, furan, pyridine, piperidine, imidazole, pyrrolidine, and oxadizole. Specific non-limiting exemplary embodiments of linkers are shown in Figure 1. In Figure 1, Z 1 and Z 2 each independently represents, as described above, a functional group F z and K is selected from -CH- and -N-. In some particular embodiments of the linker shown in FIG. 1 or Z 2の一方 is -NH-, and the others are -O-, -C(O)- and S(O) 2 - is selected.

[0115] In some embodiments, the linker connecting the donor dye and the acceptor dye is an anionic linker as described in U.S. Patent No. 6,811,979, the disclosure of which is incorporated herein by reference (see, e.g., column 17, line 25 to column 18, line 37 and the disclosure in Figures 1-17). Specific non-limiting exemplary embodiments of suitable anionic linkers include linkers of formulas (L.1)-(L.4) above, in which one or more of the Ar groups are substituted with one or more substituents that have a negative charge under the conditions of use, such as, for example, a pH in the range of about pH 7 to about pH 9. Specific non-limiting examples of suitable substituents include phosphate ester, sulfate ester, sulfonate, and carboxylate groups.

[0116] In some embodiments, the linker connecting the donor and acceptor dyes is an anionic linker as described in U.S. Patent No. 6,811,979, the disclosure of which is incorporated herein by reference (see, e.g., column 17, line 25 to column 18, line 37 and the disclosure in Figures 1-17). Specific non-limiting exemplary embodiments of suitable anionic linkers include linkers of formulas (L.1)-(L.4) above, in which one or more of the Ar groups are substituted with one or more substituents that have a negative charge under the conditions of use, such as, for example, a pH in the range of about pH 7 to about pH 9. Specific non-limiting examples of suitable substituents include phosphate ester, sulfate ester, sulfonate, and carboxylate groups.

[0117] In some embodiments, the labeling moiety is of formula (VI): A-Z 1 -Sp-Z 2 -D(VI) In the formula, A represents an N-protected NH-rhodamine acceptor, D represents a donor, such as an N-protected NH-rhodamine or an O-protected fluorescein donor, and Z 1 and Z 2 As mentioned above, functional group F zand Sp represents a portion of the bond provided by a linking moiety comprising the moiety: and Sp represents a spacing moiety, as described above. In some particular embodiments, A is an N-protected NH-rhodamine moiety as described herein, and D is selected from the group consisting of moieties having the structural formulas D.1, D.2, D.3, D.4, D.5, D.6, D.7, D.8, D.9, D.10, D.11, and D.12; [ka] JPEG2025060648000013.jpg23388 JPEG2025060648000014.jpg22190 JPEG2025060648000015.jpg21390 JPEG2025060648000016.jpg7788

[0118] In each of the formulas D.1 to D.12,

[0119] Each R 1’ , R 2’ , R 2’’ , R 4’ , R 4’’ , R 5’ , R 5’’ , R 7’ , R 7’’ , and R 8’ When alone, each independently represents hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, R b and -(CH 2 ) x -R b wherein x is an integer having a value of 1 to 10; b -X, -OH, -OR a -SH, -SR a -NH 2 , -NHR a -NR c R c , N + R c R c Rc , perhalo lower alkyl, trihalomethyl, trifluoromethyl, -P(O)(OH) 2 , P(O)(OR a ) 2 , P(O)(OH)(OR a ), -OP(O)(OH) 2 , OP(O)(OR a ) 2 , -OP(O)(OR a )(OH), -S(O) 2 OH, S(O) 2 R a , -C(O)H, C(O)R a , -C(S)X, -C(O)OR a , -C(O)OH, -C(O)NH 2 , -C(O)NHR a , C(O)NR c R c , C(S)NH 2 , C(O)NHR a , -C(O)NR c R c , -C(NH)NH 2 , -C(NH)NHR a , and -C(NH)NR c R c wherein X is halo and each R a is selected from the group consisting of lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, and 6-20 membered heteroarylalkyl, each R c is independently R a or two R attached to the same nitrogen atom c together with the nitrogen atom form a 5-8 membered saturated or unsaturated ring which may optionally contain one or more of the same or different ring heteroatoms selected from the group consisting of O, N, and S; or

[0120] Or, R 1’ and R 2’ or R 7’ and R 8’together with the carbon atoms to which they are attached form an optionally substituted (C6-C14) aryl bridge, and / or R 4’ and R 4’’ and / or R 5’ and R 5’’ together with the carbon atom to which they are attached form a benzo group,

[0121] Each R 4 , R 5 , R 6 , and R 7 are each independently hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 6- to 14-membered heteroaryl, 7- to 20-membered heteroarylalkyl, -R b and -(CH 2 ) x -R b is selected from

[0122] E 1 -NHR 9 , -NR 9 R 10 -OR 9b is selected from the group consisting of

[0123] E 2 -NHR 9 , -NR 9 R 10 -OR 9b is selected from the group consisting of

[0124] R 9 and R 10 is as described herein;

[0125] R 9b is R 9 and

[0126] Each Y 1a , Y 1b , Y 2a , Y 2b , Y 3a and Y 3b are independently -O-, -S-, -NH-, -C(O-) and -S(O)2 is selected from the group consisting of

[0127] However, each E 1 and E 2 -OR 9b If R 1’ and R 2’ and / or R 7’ and R 8’ may form an optionally substituted (C6-C14)aryl bridge only with the carbon atom to which they are attached. As used herein, an "asymmetric rhodamine" is a compound in which E1 and E2 are independently -NHR9 or -NR9R10, and E1 is not the same as E2.

[0128] In some particular embodiments of the labeling moiety of structural formula (VI), Y 1a , Y 2a and Y 3a is -NH- and Y 1b , Y 2b and Y 3b is -C(O)- and -S(O) 2 -, Z1 is selected from -C(O)- and -S(O) 2 - selected from Z 2 is -NH-, and Sp is the following group: -(CH 2 ) a -[(Ar) b -(CH 2 ) a ] c -;(Sp.1) -(CH 2 ) a -[C≡C-(CH 2 ) a ] c -;(Sp.2) -(CH 2 ) a -[C≡C-(Ar) b ] c -(CH 2 ) a -;(Sp.3) -(CH 2 ) d -NH-C(O)-[(CH 2 )a -(Ar)-(CH 2 ) a -C(O)-NH] c -(CH 2 ) d -(Sp.4); and -[CH 2 (CH 2 ) e O] f -CH 2 (CH 2 )-(Sp.5), wherein a, b, c, d, e, f, and Ar are as previously defined.

[0129] In some particular embodiments of the labeling moiety of structural formula (VI), R9 is -C(O)CH 3 and C(O)CF 3 Selected from R 9a is -C(O)C(CH 3 ) 3 It is.

[0130] 4.5 PEP group

[0131] Many embodiments of the reagents described herein include a PEP group ("PEP"). When used in a stepwise synthesis to synthesize a labeled oligonucleotide, the PEP group binds to an available hydroxyl group, which may be the 5'-hydroxyl group, of the nascent synthetic oligonucleotide, and ultimately, after any necessary oxidation and / or deprotection steps, contributes to a bond that attaches the label moiety to the synthetic oligonucleotide. The bond formed may be a phosphoester bond or a modified phosphoester bond as known in the art.

[0132] A variety of different groups suitable for attaching reagents to primary hydroxyl groups to give phosphate or modified phosphate linkages are well known in the art. Specific examples include, by way of example and not limitation, phosphoramidite groups (e.g., Letsinger et al., 1969, J. Am. Chem. Soc. 91:3350-3355; Letsinger et al., 1975 J. Am. Chem. Soc. 97:3278; Matteucci & Caruthers, 1981, J. Am. Chem. Soc. 103:3185; Beaucage & Caruthers, 1981, Tetrahedron Lett. 22:1859; the disclosures of which are incorporated herein by reference), 2-chlorophenyl- or 2,5-dichlorophenyl-phosphate groups (e.g., Sproat & Gait, ``Solid Phase Synthesis, A Practical Approach, Gait, Ed., 1984, IRL Press, pages 161-162, the disclosures of which are incorporated herein by reference), and 2-chlorophenyl- or 2,5-dichlorophenyl-phosphate groups (e.g., Sproat & Gait, ``Solid Phase Synthesis, A Practical Approach, Gait, Ed., 1984, IRL Press, pages 161-162, the disclosures of which are incorporated herein by reference). 83-115, the disclosures of which are incorporated herein by reference), and H-phosphonate groups (e.g., Garegg et al., 1985, Chem. Scr. 25:280-282; Garegg et al., 1986, Tet. Lett. 27:4055-4058; Garegg et al., 1986, Chem. Scr. 26:59-62; Froehler & Matteucci, 1986, Tet. Lett. 27:469-472; Froehler et al., 1986, Nucl. Acid Res. 14:5399-5407, the disclosures of which are incorporated herein by reference). In certain embodiments, the PEP group is a phosphoramidite group of formula (P.1): [ka]

[0133] In the formula, R 20 is a linear, branched or cyclic, saturated or unsaturated alkyl containing 1 to 10 carbon atoms, 2-cyanoethyl, an aryl containing 6 to 10 ring carbon atoms, and an arylalkyl containing 6 to 10 ring carbon atoms and 1 to 10 alkylene carbon atoms;

[0134] Each R 21 and R 22 are, independently of each other, linear, branched or cyclic, saturated or unsaturated alkyl containing 1 to 10 carbon atoms, aryl containing 6 to 10 ring carbon atoms, and arylalkyl containing 6 to 10 ring carbon atoms and 1 to 10 alkylene carbon atoms; or alternatively, R 21 and R 22 together with the nitrogen atom to which they are attached form a saturated or unsaturated ring containing 5 to 6 ring atoms, one or two of which, in addition to the exemplified nitrogen atom, can be a heteroatom selected from O, N and S.

[0135] In certain embodiments, R 20 is 2-cyanoethyl, R 21 and R 22 are each isopropyl.

[0136] 4.6 Composite Handles

[0137] Many of the embodiments of the reagents described herein include one or more synthetic handles that, after suitable deprotection, provide sites that can be used for the attachment of additional groups or moieties to the synthetic labeled oligonucleotide, if desired. These groups can be attached to the synthetic handle during the course of synthesis of the labeled oligonucleotide, or the synthetic handle can be deprotected after synthesis to reveal functional groups to which additional groups or moieties can be attached. For example, the synthetic handle can include a primary amine group protected with a protecting group that is stable to the conditions used to carry out the synthesis of the labeled oligonucleotide. Removal of the protecting group following synthesis, either simultaneously with or separately from the removal of various other protecting groups on the synthetic oligonucleotide, provides a primary amino group to which additional groups and / or moieties can be attached.

[0138] A variety of different types of reactive groups protected with protecting groups suitable for use in oligonucleotide synthesis are known in the art, including, by way of non-limiting example, amino groups (e.g., protected with trifluoroacetyl or 4-monomethoxytrityl groups), hydroxyl groups (e.g., protected with 4,4'-dimethoxytrityl groups), thiol groups (e.g., protected with trityl or alkylthiol groups), and aldehyde groups (e.g., protected with acetal protecting groups). All of these protected reactive groups can comprise the synthetic handles of the reagents described herein.

[0139] In some embodiments, the synthetic handle has the formula -OR k wherein R k represents an acid-labile protecting group that can be selectively removed during the synthesis of an oligonucleotide. For oligonucleotide synthesis, suitable acid-labile protecting groups for protecting primary hydroxyl groups are known in the art and include, by way of example and not limitation, triphenylmethyl (trityl), 4-monomethoxytrityl, 4,4'-dimethoxytrityl, 4,4',4''-trimethoxytrityl, bis(p-anisyl)phenylmethyl, naphthyldiphenylmethyl, p-(p'-bromophenacyloxy)phenyldiphenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, and 9-(9-phenyl-10)-oxo)anthryl. All of these groups can be removed by treatment with a weak acid, such as treatment with 2.5% or 3% dichloroacid or trichloroacid and dichloromethane. Methods for protecting primary hydroxyl groups with the acid-labile protecting groups listed above are well known.

[0140] 4.7 Solid support

[0141] Many embodiments of the reagents described herein include a solid support to which other moieties and / or groups are attached. The solid support is typically activated with a functional group, such as an amino or hydroxyl group, to which a linker is attached having a suitable attachment group for attachment of other moieties.

[0142] A variety of materials that can be activated with functional groups that are suitable for attachment to various moieties and linkers, and methods for activating materials to contain functional groups, are known in the art, examples of which include controlled pore glass, polystyrene, and graft copolymers. Any of these materials may be used as solid supports in the reagents described herein.

[0143] 4.8 Useful synthetic reagents for labeling terminal hydroxyl groups

[0144] Some embodiments of the synthetic reagents described herein are described by structural formula (VII): LM-L-PEP(VII)

[0145] In the formula, LM represents a label moiety as described herein, L represents any linker as described herein, and PEP represents a PEP group as described herein. The reagent can include additional groups or moieties, such as a synthesis handle. In some embodiments, the synthesis reagent includes a label moiety and a PEP group, but does not include additional moieties or groups. Such synthesis reagents can be attached to hydroxyl groups during the stepwise synthesis of oligonucleotides, and are particularly useful for attaching a label moiety to the terminal hydroxyl group, usually the 5'-hydroxyl, of synthetic oligonucleotides.

[0146] In some embodiments, the labeling moiety can be of the formula: [ka]

[0147] In the formula, R 1 ~R 14 , R a ~R j , and Y are as defined herein.

[0148] The PEP group can be directly attached to the labeling moiety or can be attached to the labeling moiety with the aid of a linker. Since the PEP group is generally attached to the molecule by attaching a suitable reagent to the primary hydroxyl group, in embodiments where the PEP group is directly attached to the labeling moiety, the labeling moiety should include a substituent that includes a primary hydroxyl group. In embodiments where the PEP group is attached to the labeling moiety with the aid of a linker, the linker synthon should include a linking group suitable for forming a bond with the linking group on the labeling moiety synthon and a primary hydroxyl group suitable for attachment to the PEP group. Suitable linker synthons include, but are not limited to, those of formula F z -Sp-OH synthon, z is a functional group complementary to the functional group on the labeling moiety synthon, and Sp represents a spacing moiety. The spacing moiety can include any combination of atoms and / or functional groups that are stable to the conditions used in the synthesis and deprotection of the labeled synthetic oligonucleotide. Non-limiting exemplary linkers are shown in FIG. 1, where Z 2 is O. In some embodiments, Sp is an optionally substituted alkylene chain containing 1 to 10 chain atoms. In certain embodiments, Sp is an unsubstituted alkylene chain containing 1 to 9 carbon chain atoms.

[0149] In some embodiments, the synthesis reagent is a compound of structural formula (VII): wherein LM is one of the embodiments of the labeling moiety specifically exemplified above; L is -Z-(CH 2 ) 3-6 -O-, -Z-(CH 2 ) a -[(Ar) b -(CH 2 ) a ] c -O-, -Z-(CH 2 ) a -[C≡C-(CH 2 ) a ] c -O-, -Z-(CH 2 ) a -[C≡C-(Ar)b ] c -(CH 2 ) a -O-, -Z-(CH 2 ) d -NH-C(O)-[(CH 2 ) a -(Ar)-(CH 2 ) a -C(O)-NH] c -(CH 2 ) d -O-, -Z-[CH 2 (CH 2 ) e O] f -CH 2 (CH 2 ) e O- and Z 2 is selected from one of the linkers shown in FIG. PEP is a phosphoramidite group, such as, for example, the phosphoramidite group of formula P.1 above. In some particular embodiments, Z of the linker L is -NH-.

[0150] In some embodiments, the linker in the synthesis reagent of structural formula (VII) comprises a nucleoside such that the synthesis reagent is a nucleoside. In some embodiments, the nucleoside synthesis reagent is a compound of structural formula (VII.1): [ka] wherein PEP represents a phosphate precursor group, B represents a nucleobase, LM represents a labeling moiety, and L2 represents a linker that connects the nucleobase B to the linker LM. The characteristics and properties of the nucleobase B and the linker L are described in more detail below. A non-limiting exemplary nucleoside synthesis reagent of structural formula (VII.1) is shown in FIG.

[0151] An exemplary scheme for synthesizing examples of synthetic reagents in which the PEP group is attached to the labeling moiety via an optional linker is provided in Scheme (I) below, where various R, F y , F z, Y, Z and SP groups are as previously defined. [ka]

[0152] In scheme (I), the functional group F y A parent NH-rhodamine synthon 100, containing a linking group containing is acetylated with anhydride 101 to generate the N-acetyl-protected NH-rhodamine synthon 102. Synthon 102 is then coupled to linker synthon 103 to generate compound 104. y Depending on the identity of F, synthon 102 may require activation prior to conjugation. For example, F y If is a carboxyl group, it can be activated as an ester, such as an NHS ester, prior to conjugation. In compound 104, -YZ- is a complementary functional group F, as previously described. y and F z and Y represents a bond formed by y represents the portion contributed by F z Compound 104 then reacts with a PEP synthon 105, which in the particular embodiment illustrated is a phosphine, to produce a phosphoramidite synthesis reagent 106.

[0153] 4.9 Synthesis Reagents Useful for Internal or 3'-Terminal Labeling

[0154] The synthesis reagents described herein may optionally include one or more synthesis handles useful for attaching additional groups and / or moieties. k The synthesis reagents containing a synthetic handle of the formula R krepresents an acid labile protecting group as described above, providing a primary hydroxyl group to which additional nucleotides can be attached. As a result, synthesis reagents containing such synthesis handles can be used to label synthetic oligonucleotides at the 5'-hydroxyl, the 3'-hydroxyl, or at one or more internal positions. These can also be combined with each other or with other phosphoramidite labeling reagents, allowing the synthesis of oligonucleotides containing multiple labeling moieties.

[0155] Labeling Moiety, PEP Group, and Synthetic Handle-OR k may comprise a synthesis reagent and may be joined together in any manner and / or orientation that allows them to perform their respective functions. As a specific example, the PEP group and the synthesis handle may each be attached to a labeling moiety, optionally via a linker. In some embodiments, such a synthesis reagent is a compound of structural formula (VIII): R k OL-LM-L-PEP(VIII)

[0156] wherein each L, independently of the others, represents an optional linker, LM represents a labeling moiety, and PEP represents a PEP group. k Non-limiting examples of linkers L, labeling moieties LM and PEP groups include those specifically exemplified above.

[0157] Another embodiment is a combination of a PEP group and a synthetic handle -OR k can be attached to a branched linker that is attached to a labeling moiety. In some embodiments, such a synthesis reagent is a compound of structural formula (IX): [ka] In the formula, L represents a linker, LM represents a labeling moiety, and PEP represents a PEP group.

[0158] In certain embodiments, the synthesis reagent of structural formula (IX) is a compound of structural formula (IX.1): [ka] In the formula, LM represents a labeling moiety, -Z- represents a functional group F on the linker. z represents the fraction of bonds contributed by 1 , Sp 2 and Sp 3 are the same or different and each represents a spacing moiety, G is CH, N, or a group including arylene, phenylene, heteroarylene, lower cycloalkylene, cyclohexylene, and / or lower cycloheteroalkylene, and PEP represents a PEP group. In some embodiments, LM is one of the embodiments of the labeling moieties specifically exemplified above, Sp 1 , Sp 2 and Sp 3 are each independently an alkylene chain containing 1 to 9 carbon atoms, Sp.1, Sp.2, Sp.3, Sp.4 and Sp.5 (as defined above), and / or PEP is a phosphoramidite group of formula P.1 above. Non-limiting specific embodiments of exemplary synthesis reagents of formula (IX.1) are shown in Figures 2 and 3.

[0159] In some embodiments, the synthetic handle -OR k is provided by a nucleoside such that the synthesis reagent is a nucleoside. In such nucleoside synthesis reagents, the labeling moiety is typically attached to the nucleobase of the nucleoside via a linker, and exocyclic functional groups on the nucleobase that are reactive under the conditions used to synthesize the labeled oligonucleotide, such as exocyclic amines, are protected. An example is shown in FIG.

[0160] The nucleoside can be any nucleoside that can be suitably protected for use in synthesizing oligonucleotides, including a 2'-deoxyribose sugar moiety, a 3'-deoxyribose sugar moiety (which is useful for synthesizing labeled oligonucleotides containing 2'-5' internucleotide linkages), a suitably protected ribose moiety, substituted versions of any of these ribose moieties, or even non-ribose sugar moieties.

[0161] In some embodiments, such nucleoside synthesis reagents are compounds of structural formulas (IX.2), (IX.3), (IX.4), and (IX.5): [ka]

[0162] where LM represents a labeling moiety, B represents a suitably protected nucleobase, and L 2 represents a linker that attaches the labeling moiety to the nucleobase, and R k represents an acid labile protecting group, PEP represents a PEP group, O represents an oxygen atom, and in structural formula (IX.4), R 16 represents a 2'-hydroxyl protecting group.

[0163] In the synthesis reagents of structural formulas (VII.1), (IX.2), (IX.3), (IX.4) and (IX.5), the nucleobase B can be virtually any heterocycle useful for incorporation into an oligonucleotide. For example, the nucleobase can be one of the genetically encoded purines (adenine or guanine), one of the genetically encoded pyrimidines (cytosine, uracil or thymine), a genetically encoded analog and / or derivative of a purine and / or pyrimidine (e.g., 7-deazadenine, 7-deazaguanine, 5-methylcytosine), a non-genetically encoded purine and / or pyrimidine (e.g., inosine, xanthene and hypoxanthene), or another type of heterocycle. A wide variety of heterocycles useful for incorporation into oligonucleotides are known in the art and are described, for example, in Practical Handbook of Biochemistry and Molecular Biology, Fasman, Ed., 1989, CRC Press (e.g., pages 385-393, and references cited therein), the disclosures of which are incorporated herein by reference. All of these various heterocycles, and any subsequently discovered heterocycles, can be included in the nucleoside synthesis reagents described herein.

[0164] In the synthetic reagents of structural formulas (VII.1), (IX.2), (IX.3), (IX.4) and (IX.5), when B is a purine, the illustrated sugar moiety is typically attached to the N9 position of the purine, and when B is a pyrimidine, the illustrated sugar moiety is typically attached to the NI position of the pyrimidine. Attachment sites for other nucleobases will be apparent to those skilled in the art.

[0165] The exocyclic amine or other reactive group(s) on the nucleobase are protected with a protecting group that is stable to the synthesis conditions used to synthesize the labeled oligonucleotide. For oligonucleotide synthesis, a variety of groups suitable for protecting the exocyclic amine groups of nucleoside nucleobases are well known in the art, as are methods for preparing such protected nucleosides.

[0166] For example, groups that have been used to protect the exocyclic amine of adenine include benzyl (Bz), phenoxyacetyl (Pac), and isobutyryl (iBu). Groups that have been used to protect the exocyclic amine of cytosine include acetyl (Ac) and Bz. Groups that have been used to protect the exocyclic amine of guanine include iBu, dimethylformamide (Dmf), and 4-isopropyl-phenoxyacetyl (iPr-Pac). All of these protecting groups can be removed by treatment with ammonium hydroxide at 55-65°C for 2-3 hours. However, some of these protecting groups can be removed under milder conditions. For example, A iBU , A Pac , C Ac and G iPr-Pac Cleavage of the protecting group from can be accomplished with ammonium hydroxide or 0.05M potassium carbonate in methanol at room temperature for 4-17 hours, or with H 2 This can be accomplished by treatment with 25% t-butylamine in 0 / EtOH. Nucleoside reagents that utilize protecting groups that can be removed under these milder deprotection conditions are preferred, since some NH-rhodamine and / or other dyes, including those described herein, may not be stable to the harsher deprotection conditions required by other protecting groups.

[0167] A linker L that connects the labeling moiety LM to the nucleobase B 2 can be attached to any position of the nucleobase. In some embodiments, when B is a purine, the linker is attached to the 8-position of the purine, when B is a 7-deazapurine, the linker is attached to the 7-position of the 7-deazapurine, and when B is a pyrimidine, the linker is attached to the 5-position of the pyrimidine.

[0168] In some embodiments, a linker L useful for attaching LM to a nucleobase is 2 is an acetylene or alkene amino bond, e.g., -C≡C-CH 2 -NH-, -C≡CC(O)-, -CH=CH-NH-, -CH=CH-C(O)-, -C≡C-CH 2 -NH-C(O)-(CH 2 ) 1-6 -NH-, and -CH=CH-C(O)-NH-(CH 2 ) 1-6 -NH-C(O)-, for example a bond of the formula C-CH-CH 2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 Propargyl-1-ethoxyamino bonds such as -NH- or robust bonds such as -C≡CC≡C-CH 2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 -NH-, -C≡C-(Ar) 1-2 -C≡C-CH 2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 -NH-, -C≡C-(Ar) 1-2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 -NH- and -C≡C-(Ar)1-2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 -NH-, and the like, wherein Ar is as previously defined.

[0169] In some embodiments, a linker L is useful for attaching LM to a purine nucleobase. 2 is, for example, a compound of the formula -NH-(CH 2 ) 1-6 Includes alkylamines such as -NH- bonds.

[0170] In some embodiments, a linker L is useful for attaching LM to a purine or pyrimidine nucleobose. 2 is an anionic linker as described in U.S. Pat. No. 6,811,979, the disclosure of which is incorporated herein by reference (see, e.g., column 17, line 25 to column 18, line 37 and the disclosure in Figures 1-17).

[0171] Methods for synthesizing such linker-derivatized nucleosides suitable for incorporation into the reagents described herein are described, for example, in Hobbs et al., 1989, J. Org. Chem. 54:3420, U.S. Patent No. 5,151,507 (Hobbs et al), U.S. Patent No. 5,948,648 (Khan et al), and U.S. Patent No. 5,821,356 (Khan et al), the disclosures of which are incorporated herein by reference. As described in more detail below, the derivatized nucleosides can be used as synthons to synthesize nucleoside synthesis reagents.

[0172] Certain exemplary embodiments of linker-derivatized nucleobases that may include the nucleoside reagents described herein are shown below. [ka]

[0173] Nucleoside synthesis reagents can be prepared from linker-derivatized nucleoside synthons as shown in Scheme (II) below. [ka]

[0174] In Scheme (II), the linker-derivatized nucleoside synthon 110 is protected at the 5′-hydroxyl with an acid-labile protecting group, and is depicted in the scheme as an exemplary chloride reagent R k Cl is used, R k is as defined above. Treatment with base to remove the trifluoroacetyl protecting group gives synthon 112. Reaction of synthon 112 with labeling moiety synthon 102 (see scheme (I) above), followed by treatment with PEP synthon 105 (in this particular example, treated with a phosphine (see scheme (I) above)) gives nucleoside synthesis reagent 114. The specific conditions for carrying out the various synthetic steps shown above are well known. As shown in FIG. 4, or of formula -OR k Non-nucleoside synthesis reagents containing the synthetic handle of the formula (II) can be prepared by routine application of Scheme (II).

[0175] 4.10 Solid-supported reagents

[0176] Many embodiments of the reagents described herein include a solid support. Such reagents generally include a solid support, a labeling moiety as described herein, and a synthetic handle, and may include additional groups or moieties, such as additional labeling moieties, quenching moieties, synthetic handles and / or groups useful for stabilizing oligonucleotide duplexes, such as agents that intercalate between base pairs (intercalating agents) and agents that bind to the minor groove of the duplex (minor groove binding, or MGB, agents). The solid support, labeling moiety, synthetic handle, and any additional moieties may be attached to each other in any manner or orientation that allows them to perform their respective functions.

[0177] In some embodiments, the solid support is attached to the remainder of the reagent via a linker. The linker that attaches the solid support to the remainder of the reagent typically comprises a bond that can be selectively cleaved under certain conditions, allowing the synthesized labeled oligonucleotide to be released from the solid support after synthesis. In some embodiments, the bond is labile to the conditions used to deprotect the synthesized labeled oligonucleotide, such that the oligonucleotide is deprotected and cleaved from the solid support in a single step. Such linkers typically comprise ester bonds, but may also comprise other bonds, such as carbonate esters, diisopropylsiloxy ethers, modified phosphate esters, etc.

[0178] Numerous selectively cleavable linkers useful in the context of oligonucleotide synthesis are known in the art, as are methods for derivatizing solid supports with such linkers.All of these various linkers can be adapted for use in the solid support reagents described herein.A non-limiting example of a solid support reagent that includes an exemplary linker that can be cleaved under basic conditions used to deprotect synthetic oligonucleotides is shown in Figure 6.

[0179] Like the synthesis reagents, the solid supported reagents can be non-nucleoside or nucleoside in nature. Exemplary embodiments of non-nucleoside solid supported reagents include reagents of structural formula (X): [ka] where LM represents a labeling moiety, L represents an optional selectively cleavable linker, -OR k represents a synthetic handle, where R k is an acid labile protecting group as described above.

[0180] In some embodiments, the solid support synthesis reagent of structural formula (X) is a non-nucleoside reagent of structural formula (X.1): [ka]

[0181] In the formula, Z, LM, G, Sp 1 , Sp 2 and R k is as defined above in relation to structural formula (IX.1), and Sp 4 represents a selectively cleavable spacing moiety. In some particular embodiments, the selectively cleavable spacing moiety Sp 4 contains an ester bond.

[0182] In some embodiments, the solid support synthesis reagent of formula (X) is a nucleoside reagent of formula (X.2), (X.3), (X.4) or (X.5): [ka] In the formula, LM, R k , B, and L 2 is as defined above for structural formulas (X.2), (X.3), (X.4) and / or (X.5); R 16 is as defined above for structural formula (IX.4), and Sp 4 represents a selectively cleavable spacing moiety that, in some embodiments, comprises an ester bond, as described above. Specific examples of (X.2) are shown in FIG.

[0183] 4.11 Additional Exemplary Embodiments

[0184] It should be understood that the specific embodiments of the various moieties, groups, and linkers described throughout this disclosure may be included in all of the reagents described herein. Moreover, the various specific embodiments may be combined with each other in any combination, as if the specific combinations were specifically exemplified. As a specific example, any one of the specific embodiments of the labeling moiety LM described herein may be included in any of the specifically exemplified embodiments of the non-nucleoside and nucleoside solid supports and synthesis reagents described herein. As another specific example, any one of the specific embodiments of the PEP group PEP, such as the phosphoramidite group of structural formula (P.1) above, may be included in any of the synthesis reagents described herein.

[0185] 4.12 Use of Reagents

[0186] The various reagents described herein can be used for the stepwise synthesis of oligonucleotides to synthesize oligonucleotides labeled with rhodamine dyes directly on synthesis resin.The various reagents thus enable the ability to synthetically label oligonucleotides with a myriad of different rhodamines, eliminating the need for laborious post-synthetic modification.The synthesis of oligonucleotides labeled with NH rhodamine dyes using exemplary synthesis reagents is shown in Figure 8A.

[0187] As will be appreciated by those skilled in the art, the availability of phosphoramidite reagents that can act as NH-rhodamine dye donors, acceptors, or quenchers allows the reagents described herein to synthesize oligonucleotides that are labeled with energy transfer dyes and / or NH-rhodamine-quencher dye pairs that are synthesized in situ. An exemplary synthesis of an oligonucleotide labeled with an NH-rhodamine-fluorescein energy transfer dye pair is shown in FIG. 8B and FIG. 9, which illustrates the versatility offered by the reagents described herein. Because the reagents described herein allow virtually any NH-rhodamine dye to be included in the solid support and / or synthesis reagent, oligonucleotides labeled with energy transfer dye pairs with spectral properties tailored for specific applications can be conveniently synthesized in situ without the need for post-synthetic modification. Furthermore, oligonucleotides labeled with a myriad of different energy transfer dye pair combinations can be synthesized from individual monomer reagents, eliminating the need to create synthesis reagents that contain specific dye pairs. Each part of the dye pair can be attached to the nascent oligonucleotide in a stepwise manner, with or without the addition of an intervening linking moiety.

[0188] Referring to Figure 8A, a support-bound synthetic oligonucleotide is treated with acid to remove the DMT group protecting its 5'-hydroxyl, generating a 5'-deprotected support-bound oligonucleotide. Coupling of an N-protected NH-rhodamine phosphoramidite reagent followed by oxidation yields the support-bound NH-rhodamine labeled oligonucleotide in the open lactone form. Treatment with concentrated ammonium hydroxide removes the protecting groups and cleaves the synthetic oligonucleotide from the solid support (resin) to yield an oligonucleotide labeled with an NH-rhodamine dye.

[0189] Referring to Figure 8B, a nascent support-bound oligonucleotide can be labeled with an in situ synthesized NH-rhodamine-fluorescein dye pair by coupling an N-protected NH-rhodamine phosphoramidite synthesis reagent to the 5'-hydroxyl of the oligonucleotide, yielding a rhodamine-labeled oligonucleotide after oxidation. Removal of the DMT group and subsequent coupling with an O-protected phosphoramidite (FAM-phosphoramidite in the specific example shown) yields the labeled support-bound oligonucleotide. Cleavage and deprotection yields the oligonucleotide, which is labeled with the NH-rhodamine-FAM energy transfer dye pair.

[0190] 9, a solid support reagent containing a protected NH-rhodamine-fluorescein energy transfer dye pair as the labeling moiety can undergo three cycles of synthesis to produce a labeled, support-bound oligonucleotide. Cleavage from the solid support yields the deprotected, labeled oligonucleotide.

[0191] The length and nature of the bond connecting the donor and acceptor dyes can also be manipulated using phosphoramidite linker reagents. By coupling with a FAM-phosphoramidite followed by oxidation, deprotection, and cleavage, oligonucleotides are generated and labeled with the NH-rhodamine-FAM energy transfer dye pair. In the linker phosphoramidite, "Sp" is a spacer as defined above. For example, "Sp" can be a spacer as defined above, e.g., (Sp 1 ), (Sp 2 ), (Sp 3 ), (Sp 4 ) or (Sp 5 ) can be represented as

[0192] The length and properties of the linker connecting the NH-rhodamine and FAM dyes can be adjusted by coupling additional linker phosphoramidites prior to coupling with the FAM-phosphoramidite. The linker phosphoramidites can be the same or different. In this way, oligonucleotides labeled with energy transfer dye pairs can be easily synthesized in situ, with tailored donor and acceptor dyes and linkers connecting the donor and acceptor dyes.

[0193] Those skilled in the art will understand that any N-protected NH-rhodamine reagent that functions as an acceptor for FAM can be used. In addition, other O-protected fluoresceins can be used, as well as other types of phosphoramidite dyes. Since the dyes are added as monomers, the number of available energy transfer dye labels is greater than the number of phosphoramidite reagents required for their synthesis. For example, oligonucleotides labeled with nine different energy transfer dye pairs can be synthesized from three N-protected NH-rhodamine phosphoramidite reagents (reagents A, B, and C) and three different O-protected fluorescein phosphoramidite reagents (reagents 1, 2, and 3), oligo-A1, oligo-A2, oligo-A3, oligo-B1, oligo-B2, oligo-B3, oligo-C1, oligo-C2, and oligo-C3.

[0194] Current analysis of cell and tissue functionality often requires extracting as much information as possible from often limited material. For example, samples such as tumor biopsies are difficult to obtain and typically yield only small amounts of usable nucleic acid. PCR detection and measurement of a single target analyte, referred to as a singleplex assay, is the gold standard for analyzing clinical research samples at the nucleic acid level and has been invaluable in expanding the boundaries of biological knowledge for over a quarter century.

[0195] However, the limited amounts of nucleic acid available from clinical research specimens often force choices about how best to utilize these precious samples. Furthermore, when samples are limited, the number of loci that can be analyzed is also limited, reducing the amount of information that can be extracted from the sample. Finally, the additional time and materials required to set up multiple single-assay reactions can significantly increase the cost of complex projects.

[0196] Multiplex PCR analysis of nucleic acids, a strategy to amplify and quantify multiple targets from a single sample aliquot, is an attractive solution to these problems. In multiplex PCR, a sample aliquot is queried with multiple probes containing fluorescent dyes in a single PCR reaction. This increases the amount of information that can be extracted from that sample. Multiplex PCR can achieve significant savings in sample and material. To increase the utility of this method, multiplex PCR was developed to amplify and measure multiple target sequences simultaneously using several pairs of gene-specific primers and probes. Multiplex PCR offers the following advantages: 1) Efficiency: Multiplex PCR helps to conserve sample material and avoid well-to-well variability by combining several PCR assays into a single reaction. Multiplexing makes more efficient use of limited samples, such as samples containing rare targets that cannot be split into multiple aliquots without compromising sensitivity. 2) Economy: Even though targets are amplified in unison, each is detected independently by using gene-specific probes with unique reporter dyes to distinguish amplification based on fluorescent signal. Once optimized, a multiplex assay is more cost-effective than the same assays amplified independently.

[0197] However, currently there is a limit to the number of targets that can be analyzed in a single multiplex PCR assay. Experimental design for multiplex PCR is more complex than for single reactions. The probes used to detect each individual target must contain unique reporter dyes with distinct spectra. The excitation and emission filter settings of real-time detection systems vary from manufacturer to manufacturer. Therefore, the instrument must be calibrated for each dye as part of the experimental optimization process. Thus, one limitation in the development of multiplex PCR assays is the number of fluorophores, and therefore probes, that can be effectively measured in a single reaction. For example, in multiplex PCR, signal crosstalk between different fluorescent reporters can impair quantification or cause false positives. Therefore, it is essential to select fluorophores with minimal spectral overlap. Additionally, the fluorophores, especially their emission and excitation spectra, must also be compatible with the PCR instrument used, especially the bandpass specifications of each filter set.

[0198] In a further aspect, a method is provided for performing singleplex or multiplex PCR, such as qPCR, end-point PCR, using the described probes. End-point PCR is an analysis after all cycles of PCR are completed. Unlike qPCR (exponential phase), which allows quantification once the template has doubled, end-point analysis is based on the plateau phase of amplification.

[0199] In particular, a method for amplifying and detecting multiple target DNA sequences includes providing a composition or reaction mixture containing the described probes, subjecting the reaction mixture to a thermal cycling protocol such that amplification of the multiple target sequences can occur, and monitoring the amplification by detecting fluorescence of the described probes at least once during multiple amplification cycles.

[0200] The nucleic acid target(s) of the described method can be any nucleic acid target known to those of skill in the art. Furthermore, the target can be a region of low mutation or a region of high mutation. For example, one particularly valuable use of the methods disclosed herein includes targeting highly mutated nucleic acids, such as RNA virus genes, or regions of high genetic variability, such as single nucleotide polymorphisms (SNPs). In some embodiments, targets, such as material from forensic samples and / or fixed tissues, can be fragmented or degraded. Targets can be of any size that is amenable to amplification. One particularly valuable use of the methods and compositions provided herein involves the identification of short fragments, such as siRNAs and miRNAs. Another particularly valuable use is for samples where the nucleic acid may have been fragmented and / or degraded, such as fixed samples or samples exposed to the environment. Thus, the method can be used, for example, for biopsy tissue and forensic DNA. The target can be purified or not. The target can be produced in vitro (e.g., cDNA target) or found in a biological sample (e.g., RNA or genomic DNA (gDNA) target). The biological sample can be used without treatment, or the biological sample can be treated to remove substances that may interfere with the methods disclosed herein.

[0201] The probes provided herein may be used in diagnostic methods, such as SNP detection, identification of specific biomarkers, etc., whereby the probes are complementary to sequences (e.g., genomes) of infectious agents of human diseases, including, but not limited to, viruses, bacteria, parasites, and fungi, thereby diagnosing the presence of an infectious agent in a sample having nucleic acid from a patient. The target nucleic acid may be genomic or cDNA or mRNA or synthetic, human or animal, or microbial, etc. In other embodiments, the probes may be used to diagnose or prognose diseases or disorders not caused by infectious agents. For example, the probes may be used to diagnose or prognose cancer, autoimmune diseases, psychiatric disorders, genetic disorders, etc., by identifying the presence of mutations, polymorphisms, or alleles in samples from humans or animals. In some embodiments, the probes include mutations or polymorphisms. Additionally, the probes may be used to evaluate or track the progress of treatment of a disease or disorder.

[0202] Another field that benefits from multiplex analysis is the use of genetic markers in the field of human identification. Genetic markers are generally a set of polymorphic loci with alleles in genomic DNA with a characteristic to be analyzed, such as DNA typing, where individuals are differentiated based on DNA mutations. Most DNA typing methods are designed to detect and analyze differences in length and / or sequence of one or more regions of a DNA marker known to appear in at least two different forms or alleles in a population. Such mutations are called "polymorphisms" and the regions of DNA where such mutations occur are called "polymorphic loci". One possible way to perform DNA typing involves coupling PCR amplification techniques (KB Mullis, U.S. Pat. No. 4,683,202) with the analysis of length mutation polymorphisms. Short tandem repeats (STRs), minisatellites, and variable number of tandem repeats (VNTRs) are examples of length mutation polymorphisms. STRs, which contain repeating units of approximately 3 to 7 nucleotides, are short enough to be useful as genetic markers in PCR applications because amplification protocols can be designed to generate products that are smaller than those possible from other variable-length regions of DNA.

[0203] Several such systems containing multiple STR loci have been described, see, e.g., AMPFLSTR® SGMPLUS™ PCR AMPLIFICATION KIT USER'S MANUAL, Applied Biosystems, pp. ix and 1-1 to 1-16 (2001), AMPFLSTR® IDENTIFILER® PCR AMPLIFICATION KIT USER'S MANUAL, Applied Biosystems, pp. ix and 1-1 to 1-10 (2001), JW Schumm et al., U.S. Patent No. 7,008,771.

[0204] The methods of the present teachings contemplate selecting a suitable set of loci, primers, and amplification protocols to generate amplified alleles (amplicons) from multiple co-amplified loci, labeled so that they do not overlap in size and / or so that alleles at different loci that overlap in size can be distinguished. Additionally, the methods contemplate the selection of multiple STR loci that are compatible for use within a single amplification protocol.

[0205] In addition to those disclosed herein, successful combinations can be generated, for example, by trial and error combinations of loci, selection of primer pair sequences, and adjustment of primer concentrations to identify equilibria that can amplify all loci for analysis. Given the disclosure of the methods and materials of these teachings, various methods of selecting loci, primer pairs, and amplification techniques for use in the methods and kits of these teachings will likely be suggested to those of skill in the art. All such methods are intended to be within the scope of the appended claims.

[0206] Any of a number of different techniques can be used to select a set of loci for use in accordance with the present teachings. Regardless of which method may be used to select the loci to be analyzed by the methods of the present teachings, the loci selected for multiplex analysis in various embodiments share one or more of the following characteristics: (1) produce sufficient amplification products to allow allelic evaluation of the DNA; (2) cause few, if any, artifacts during the process of multiplex amplification due to incorporation of additional bases during extension of valid target loci or during generation of non-specific amplicons; (3) cause few, if any, artifacts due to premature termination of the amplification reaction by the polymerase. See, e.g., JW Schumm et al. (1993), FORTH INTERNATIONAL SYMPOSIUM ON HUMAN IDENTIFICATION, pp. 177-187, Promega Corp.

[0207] Generally, oligonucleotide primers can be chemically synthesized. Primer design and selection are routine steps in PCR optimization. Those skilled in the art can easily design specific primers for amplifying the target locus of interest, or can obtain primer sets from the references listed herein. All of these primers are within the scope of the present teachings.

[0208] As an example, primers can be selected by using any of a variety of software programs available and known in the art for developing amplification and / or multiplex systems. See, for example, Primer Express® software (Applied Biosystems, Foster City, Calif.). In an example of using a software program, sequence information from a region of a locus of interest can be imported into the software. The software then uses a variety of algorithms to select primers that best fit the user's specifications.

[0209] Samples of genomic DNA can be prepared for use in the methods of the present teachings using any procedure for sample preparation that is compatible with subsequent DNA amplification, many of which are known to those of skill in the art. Some examples are DNA purification by phenol extraction (J. Sambrook et al. (1989), MOLECULAR CLONING: A LABORATORY MANUAL, SECOND EDITION, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 9.14-9.19), and partial purification by salt precipitation (S. Miller et al. (1988), NUCL. ACIDS REs. 16:1215) or chelex (P.S. Walsh et al. (1991), BIOTECHNIQUES 10:506-513; C.T. Corney et al. (1994), J. FORENSIC Ser, 39:1254) and release of crude material using untreated blood (J. Burckhardt (1994), PCR METHODS AND APPLICATIONS 3:239-243; R.B.E. McCabe (1991), PCR METHODS AND APPLICATIONS 1:99-106; Nordvag (1992), BIOTECHNIQUES 12:4 pp.490-492).

[0210] Where at least one DNA sample analyzed using the methods of the present teachings is human genomic DNA, the DNA can be prepared from a tissue sample, such as one or more of blood, semen, vaginal cells, hair, saliva, urine, bone, buccal samples, amniotic fluid containing placental cells or fetal cells, chorionic villi, and / or mixtures of any of these or other tissues.

[0211] Samples, including blood or buccal samples, can also be processed directly from FTA® paper (Whatman Inc., Piscataway, NJ), Bode Buccal Collector, or cotton swabs. Examples of swabs include, but are not limited to, Copan 4N6 Forensic Flocked Swab (Copan, P / N 3520CS01, Murrieta, CA), Omi Swab (Whatman Inc., P / N 10005), and Puritan Cotton swab (Puritan, P / N 25-806 1WC EC, various medical suppliers).

[0212] Once the genomic DNA samples are prepared, the target loci can be co-amplified in the multiplex amplification process of the present teachings. Any of a number of different amplification methods can be used to amplify the loci, such as PCR (RK Saiki et al. (1985), SCIENCE 230:1350-1354), transcription-based amplification (DY Kwoh and TJ Kwoh (1990), AMERICAN BIOTECHNOLOGY LABORATORY, October 1990), and strand displacement amplification (SDA) (GT Walker et al. (1992), PROC. Natl. Acad. Ser., USA 89:392-396). In some embodiments of the present teachings, multiplex amplification can be performed via PCR, where the DNA sample is amplified using primer pairs specific for each locus in the multiplex. Standard PCR chemical components generally include a solvent, a DNA polymerase, deoxyribonucleoside triphosphates ("dNTPs"), oligonucleotide primers, divalent metal ions, and a DNA sample expected to contain the target(s) for PCR amplification. Water can generally be used as a typical PCR solvent, including buffers and non-buffering salts such as KCL. The buffers can be any buffer known in the art, such as but not limited to Tris-HCl, and can vary with routine experimentation to optimize PCR results. One of skill in the art can easily determine optimal buffer conditions. PCR buffers can be optimized depending on the particular enzyme used for amplification.

[0213] In PCR, the enzyme that polymerizes nucleotide triphosphates into amplification products can be any DNA polymerase. The DNA polymerase can be, for example, any thermostable polymerase known in the art. Some examples of polymerases that can be used in the present teachings are DNA polymerases from organisms such as Thermus aquaticus, Thermus thermophilus, Thermococcus litoralis, Bacillus stearothermophilus, Thermotoga maritima, and Pyrococcus sp. The enzyme can be obtained by any of several possible methods, such as, for example, isolated from a bacterial source, produced by recombinant DNA technology, or purchased from a commercial source. Some examples of such commercially available DNA polymerases include AmpliTaq Gold® DNA polymerase, AmpliTaq® DNA polymerase, AmpliTaq® DNA polymerase Stoffel Fragment, rTth DNA Polymerase, and rTth DNA Polymerase, XL, all manufactured by Applied Biosystems, Foster City, Calif. Other examples of suitable polymerases include Tne from Bacillus stearothermophilus, Bst DNA polymerase large fragment, Vent and Vent Exo- from Thermococcus litoralis, Tma from Thermotoga maritima, Deep Vent and Deep Vent Exo- from Pyrococcus sp., and mutants and derivatives of the above.

[0214] When using fluorescent labeling of primers in a multiplex reaction, typically at least three different labels can be used to label different primers. When using size markers to evaluate the products of a multiplex reaction, the primers used to prepare the size markers can be labeled with a different label than the primers that amplify the locus of interest in the reaction. With the advent of automated fluorescent imaging and analysis, more rapid detection and analysis of multiplex amplification products can be achieved.

[0215] In some embodiments of the present teachings, a fluorophore can be used to label at least one primer of a multiplex amplification, for example, by covalently attaching it to the primer to create a fluorescently labeled primer. In some embodiments, primers for different target loci in a multiplex can be labeled with different fluorophores, each producing a different colored product depending on the emission wavelength of the fluorophore. These differently labeled primers can be used in the same multiplex reaction, and then the respective amplification products are analyzed together. Either the forward or reverse primer of a pair amplifying a particular locus can be labeled, although the forward is often more frequently labeled.

[0216] PCR products can be analyzed in sieved or non-sieved media. In some embodiments of these teachings, for example, PCR products can be analyzed by electrophoresis, e.g., capillary electrophoresis as described in H. Wenz et al. (1998), GENOME RE. 8:69-80 (see also E. Buel et al. (1998), J. FORENSIC SCI. 43:(1), pp. 164-170), or slab gel electrophoresis as described in M. Christensen et al. (1999), SCAND. J. CLIN. LAB. INVEST. 59(3):167-177, or denaturing polyacrylamide gel electrophoresis (see, e.g., J. Sambrook et al. (1989), in MOLECULAR CLONING: A LABORATORY MANUAL, SECOND EDITION, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 13.45-13.57). The separation of DNA fragments in electrophoresis is primarily based on the discrimination of fragment sizes. Amplification products can be analyzed by chromatography, for example, size exclusion chromatography (SEC).

[0217] Once the amplified alleles are separated, they and other DNA can be visualized and analyzed, for example in a gel or capillary (e.g., DNA size markers or allele ladders). Often, the detection method for multiplex loci is by fluorescence. See, for example, JW Schumm et al. PROCEEDINGS FROM THE EIGHTH INTERNATIONAL SYMPOSIUM ON HUMAN IDENTIFICATION, pub. 1998, Promega Corporation, pp. 78-84; E. Buel et al. (1998) supra. When fluorescently labeled primers are used to detect each locus in a multiplex reaction, amplification can be followed by detection of the labeled products using a fluorescent detector.

[0218] The size of the alleles present at each locus of the DNA sample can be determined by comparison with size standards in electrophoresis, such as DNA markers of known size. Markers for evaluation of multiplex amplification involving two or more polymorphic STR loci can also include locus-specific allelic ladders or combinations of allelic ladders for each locus evaluated. See, for example, C. Puers et al. (1993), AM. J. HUM. GENET. 53:953-958; C. Puers et al. (1994), GENOMICS 23:260-264. See also U.S. Patent Nos. 5,599,666, 5,674,686, and 5,783,406 for a description of several allelic ladders suitable for use in detecting STR loci, and several methods of ladder construction disclosed herein. Following construction of allelic ladders for individual loci, the ladders can be electrophoresed simultaneously with the amplification products. Each allelic ladder comigrates with the alleles from the corresponding locus.

[0219] The products of the multiplex reactions of the present teachings can also be evaluated using internal lane standards, i.e., a special type of size marker configured to be electrophoresed in the same capillary as the amplification products, for example. The internal lane standards can include a series of fragments of known length. The internal lane standards can also be labeled with a fluorescent dye that can be distinguished from other dyes in the amplification reaction. The lane standards can be mixed with the amplified samples or size standards / allelic ladders to compare migration in different lanes of gel electrophoresis or different capillaries of capillary electrophoresis. Variations in migration of the internal lane standards serve to indicate variations in the performance of the separation medium. Quantification of this difference and correlation with the allelic ladder can provide a calibration of the amplification products electrophoresed in different lanes or capillaries, and a correction in the size determination of alleles in unknown samples.

[0220] If fluorescent dyes are used to label the amplification products, the electrophoresed and separated products can be analyzed using a fluorescence detection device, such as an ABI PRISM® 310 or 3130xl genetic analyzer, or an ABI PRISM® 37 DNA Sequencer (Applied Biosystems, Foster City, Calif.); or a Hitachi FMBIO™ II Fluorescent Scanner (Hitachi Software Engineering America, Ltd., South San Francisco, Calif.). In various embodiments of the present teachings, PCR products can be analyzed by capillary gel electrophoresis protocols in combination with an electrophoresis instrument such as an ABI PRISM® 3130xl genetic analyzer (Applied Biosystems), and allelic analysis of the electrophoretic amplification products can be performed, for example, by Applied Biosystems' GeneMapper® ID Software v3.2. In other embodiments, the amplification products can be separated by electrophoresis in, for example, about 4.5%, 29:1 acrylamide:bisacrylamide, 8M urea gels prepared for an ABI PRISM® 377 Automated Fluorescence DNA Sequencer.

[0221] The present teachings are also directed to kits that utilize the above process. In some embodiments, the basic kit can include a container with one or more loci and specific primers. The kit can also optionally include instructions for use. The kit can include any other kit components, such as one or more allelic ladders directed to each of the specific loci, sufficient amounts of enzyme for amplification, an amplification buffer to facilitate amplification, a divalent cation solution to facilitate enzyme activity, dNTPs for chain extension during amplification, a loading solution for preparing the amplified material for electrophoresis, genomic DNA as a template control, size markers to ensure that the material migrates as expected in the separation medium, and protocols and manuals to educate the user and limit errors in use. The amounts of various reagents in the kit can also be varied depending on many factors, such as the optimal sensitivity of the process. It is within the scope of these teachings to provide test kits for use in manual applications or for use with automated detectors or analyzers.

[0222] In clinical settings, STR markers can be used, for example, to monitor the degree of engraftment of donors in bone marrow transplants. In hospitals, these markers also aid in matching and tracking specimens. These markers have also entered other areas of science, such as population biology of human racial and ethnic group differences (DB Goldstein et al. (1995), PROC. NATL. ACAD. Ser. USA 92:6723-6727), evolution and species diversity, and changes in plant and animal taxonomy (MW Bruford et al. (1993), CURR. BIOL. 3:939-943).

[0223] Amplification of mini-STRs (locuses less than about 200 base pairs) has allowed for profiling analysis of highly degraded DNA, as evidenced by MD Coble (2005), J. FORENSIC SCI 50(1):43-53, which is incorporated herein by reference. Table 1 (see U.S. Patent Application No. 61 / 413,946, filed November 15, 2010, and U.S. Patent Application No. 61 / 526,195, filed August 22, 2011 for Table 1) also provides loci that can be considered mini-STRs depending on the position of the primers used to amplify the STR markers within the primer amplification set.

[0224] DNA concentration can be measured prior to use in the methods of the present teachings using standard methods of DNA quantification known to those skilled in the art. Such quantification methods include, for example, spectrophotometric measurements as described by J. Sambrook et al., (1989), Appendix E.5, supra, or fluorometric measurements using measurement techniques such as those described by CF Brunk et al. (1979), ANAL. BIOCHEM. 92:497-500. DNA concentration can be measured by comparing the amount of hybridization of DNA standards with a human-specific probe as described by JS Waye et al. (1991), J. FORENSIC SCI. 36:1198-1203 (1991). Using too much template DNA in the amplification reaction can result in amplification artifacts that do not represent the correct allele.

[0225] In multiplex reactions, when fluorescent labels of primers are used, typically at least three different labels, at least four different labels, at least five different labels, at least six different labels are used. For example, an existing commercial assay uses six unique dye labels (VeriFiler™ Plus PCR Amplification Kit, Thermo Fisher Scientific). The instruments used to analyze multiplex fluorescent dye-based reactions are limited in the wavelengths they can emit to excite the fluorescent dyes, and are limited in the wavelengths of light emitted from the dyes that they can detect. Designing a multiplex assay using at least eight labels, at least ten labels, or at least sixteen labels requires a set of dyes with unique spectral emission characteristics so that the peak emission peaks are well resolved without overlap. Furthermore, all fluorescent dye labels must be detectable on an instrument that can generate a specific set of excitation wavelengths and has a specific range of detectable emission wavelengths. The class of rhodamine derivatives described herein provides dye labels with unique spectral properties not available in existing dye compounds, thus opening the possibility of increasing the number of fluorescent dye labels used in multiplex reactions with up to 8, 10, 12, 16 or more different labels using existing laser technology commonly used in current multiplex assays. With improvements in instrument performance, it is envisioned that multiplex assays running more than 8 labels (e.g., at least 10, at least 12, or at least 16 different labels) can be used to label different primers. When using size markers to evaluate the products of multiplex reactions, the primers used to prepare the size markers can be labeled with a different label than the primers that amplify the locus of interest in the reaction. With the advent of automated fluorescent imaging and analysis, more rapid detection and analysis of multiplex amplification products can be achieved.

[0226] Below are some examples of possible fluorophores that are well known in the art and are suitable for use in combination with the compounds described in the present teachings to provide assays using multiple fluorescent labels. This list is intended to be illustrative and not exhaustive. Some possible fluorophores include fluorescein (FL), which absorbs at a maximum of 492 nm and emits at a maximum of 520 nm; N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA™), which absorbs at a maximum of 555 nm and emits at a maximum of 580 nm; 5-carboxyfluorescein (5-FAM™), which absorbs at a maximum of 495 nm and emits at a maximum of 525 nm; 2',7'-dimethoxy-4',5'-dichloro-6-carboxyfluorescein (JOE™), which absorbs at a maximum of 525 nm and emits at a maximum of 555 nm; 6-carboxy-X-rhodamine (ROX™), which absorbs at a maximum of 585 nm and emits at a maximum of 605 nm; CY3™, which absorbs at a maximum of 552 nm and emits at a maximum of 570 nm; CY5™, which absorbs at a maximum of 643 nm and emits at a maximum of 667 nm; tetrachloro-fluorescein (TET™), which absorbs at a maximum of 521 nm and emits at a maximum of 536 nm; and hexachloro-fluorescein (HEX™), which absorbs at a maximum of 535 nm and emits at a maximum of 556 nm; NED™, which absorbs at a maximum of 546 nm and emits at a maximum of 575 nm; 6-FAM™, which emits at a maximum of about 520 nm; VIC™, which emits at a maximum of about 550 nm; PET™, which emits at a maximum of about 590 nm; and LIZ™, which emits at a maximum of about 650 nm. See U.S. Patent No. 6,780,588 by SR Coticone et al.; AMPFLSTR® IDENTIFILER™ PCR AMPLIFICATION KIT USER'S MANUAL, pp. 1-3, Applied Biosystems (2001). Note that the emission and / or absorption wavelengths listed above are typical and can be used for general guidance purposes only; actual peak wavelengths may vary depending on the application and conditions.Additional fluorophores can be selected for desired absorption and emission spectra and colors, as known to those of skill in the art and provided below.

[0227] [Table 3] JPEG2025060648000030.jpg142170

[0228] [Table 4]

[0229] The asymmetric rhodamine compounds described herein can be used in combination with one or more additional fluorescent labels in a multiplex assay. Various embodiments of the present teachings can include a single multiplex reaction that includes at least eight different dyes. The at least eight dyes can include any eight of the dyes listed above. In certain embodiments, the set of eight dyes includes the asymmetric rhodamine compounds described herein with additional asymmetric rhodamine compounds as described in PCT / US2019 / 67925. In other embodiments, a single multiplex reaction that includes at least 10 or at least 12 different dyes can be used, and the number of dyes can be any number in these ranges.

[0230] Compositions, such as reaction mixtures or master mixes, that include the described probes are also provided. In one embodiment, a composition for PCR, such as real-time or quantitative PCR, or end-point PCR, includes at least one of the described probes. In one embodiment, a composition or reaction mixture or master mix for PCR (e.g., qPCR, or end-point PCR) includes a probe that allows detection of four target nucleic acids and a probe(s) that allows detection of at least one of a fifth and / or sixth target nucleic acid, each of the described probes consisting of a FRET donor moiety, i.e., a fluorophore, and a FRET acceptor moiety, i.e., a quencher, the fluorophore having an emission maximum of about 650-720 nm. The absorption maximum of the quencher described herein is 660-668 nm. The absorbance range of the quencher described herein is 530-730 nm. In an alternative embodiment, a labeling reagent is provided for conjugating the described fluorophores and quenchers to selected oligonucleotides.

[0231] Furthermore, such a composition or reaction mixture or master mix may comprise one or several compounds and reagents selected from the following list: a buffer applicable to polymerase chain reaction, deoxynucleoside triphosphates (dNTPs), a DNA polymerase with 5' to 3' exonuclease activity, at least one or several pairs of amplification primers and / or additional probes.

[0232] In some embodiments, the methods provided further comprise determining the genotype of the target polynucleotide using the amplification products. In some embodiments, the methods provided further comprise determining the copy number of the target polynucleotide using the amplification products.

[0233] References, patents, patent applications, scientific literature and other printed publications, and accession numbers to the GenBank database sequences cited herein, particularly PCT / US2019 / 67925, are all incorporated by reference in their entirety.

[0234] As those skilled in the art will appreciate, numerous changes and modifications can be made to the various embodiments of the present teachings without departing from the spirit of the present teachings, and all such changes are intended to be within the scope of these teachings.

[0235] Unless otherwise stated, the methods and techniques of the present embodiments are generally carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Loudon, Organic Chemistry, Fourth Edition, New York: Oxford University Press, 2002, pp. 360-361, 1084-1085; Smith and March, March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Fifth Edition, Wiley-Interscience, 2001.

[0236] The names of the compounds described herein were generally derived using the commercially available ACD / Name 2014 (ACD / Labs) or ChemBioDraw Ultra 13.0 (Perkin Elmer).

[0237] It is to be understood that, for clarity, certain features of the present disclosure described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, for brevity, various features of the present disclosure described in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments relating to chemical groups represented by the variables are specifically included in the present disclosure and disclosed herein as if each and every combination were individually and expressly disclosed herein, to the extent that such combinations include compounds that are stable compounds (i.e., compounds that can be isolated, characterized, and tested for biological activity). Furthermore, subcombinations of chemical groups listed in the embodiments describing such variables are also specifically included in the present disclosure and disclosed herein as if each and every such subcombination of chemical groups were individually and expressly disclosed herein.

[0238] chemical synthesis Exemplary chemicals useful in the described methods are described by reference to the illustrative synthetic schemes of the general preparations below and the specific examples below. To obtain the various compounds herein, one skilled in the art will appropriately select starting materials, with or without suitable protection to obtain the desired product, and carry out the final desired substitution through the reaction schemes. Alternatively, it may be necessary or desirable to carry out suitable groups through the reaction schemes and replace them with the desired substituents as necessary, in place of the final desired substitution. Furthermore, one skilled in the art will recognize that the transformations shown in the following schemes can be carried out in any order that is compatible with the functionality of the particular pendant groups.

[0239] All common chemicals were purchased from commercial chemical companies such as Fisher Scientific, Acros, or Alfa Aesar. Silica gel (220-400 mesh) from Fisher Scientific was used for normal phase flash chromatography. Reverse phase chromatography was performed using octadecyl-functionalized silica gel from JT Baker. All chromatographic solvent gradients were stepwise. Thin layer chromatography (TLC) was performed on aluminum-supported silica gel slides from EM Science. Reverse phase TLC was performed on HPTLC RP18F Uniplate plates from Analtech. Generated spots were visualized by both long and short wavelength UV irradiation.

[0240] NMR spectra were recorded on a Varian 400MHz NMR referenced to the solvent peak. HPLC was performed on an Agilent 1200 HPLC equipped with a diode array detector and multiple channel wavelengths. Typical elution was performed at 1ml / min through an Agilent Pursuit C8 150x4.6mm 5μ column with a gradient of acetonitrile and 0.1M triethylammonium acetate (TEAA). LCMS data was obtained using an Agilent 1200LC system interfaced to a PE Sciex API 150EX mass spectrometer. MS data was obtained by direct infusion into an API Sciex 4000 mass spectrometer.

[0241] Anhydrous solvents were operated under nitrogen using oven-dried syringes. As used herein, the term "aqueous workup" refers to the following steps: dissolving or diluting the reaction mixture in a specified organic solvent, washing with a specified aqueous solution or water, washing the combined organic layer once with saturated NaCl, and rinsing the solution with anhydrous NaCl. 2 SO 4 This refers to a purification method that includes the steps of drying at 200° C., filtering the drying agent, and removing the solvent in vacuo.

[0242] Example 1: Preparation of asymmetric rhodamine dyes. [ka]

[0243] Step 1: Preparation of 10-Methoxy-5,5,7-trimethyl-2,3-dihydro-1H,5H-pyrido[3,2,1-ij]quinoline, 2

[0244] 7-Methoxy-2,2,4-trimethyl-1,2-dihydroquinoline, 1 (8.00 g, 39.4 mmol, A. Rosowsky, E. J. Modest, JOC, 1965, 30, 1832.) was dissolved in acetonitrile (125 ml) and mixed with 1-bromo-3-chloropropane (24.8 g, 157 mmol), sodium iodide (47.2 g, 315 mmol), and sodium carbonate (8.35 g, 78.8 mmol). The mixture was refluxed for 23 h. The mixture was filtered and the filtrate was concentrated, washed with water in DCM, and worked up. The crude residue was purified by silica gel flash chromatography, eluting with 20% DCM / hexane to give 2 as an off-white solid (8.28 g, 86%). 1 H NMR (400 MHz, CD 2 Cl 2 ): Chemical shifts 6.85(d, 1H), 6.15(d, 1H), 5.15(s, 1H), 3.75(s, 3H), 3.22(m, 2H), 2.59(m, 2H), 1.90(s, 3H), 1.87(m, 2H), 1.28(s, 6H). MS:calcd 244.17, observed 244.15(MH + ).

[0245] Step 2: Preparation of 5,5,7-trimethyl-2,3-dihydro-1H,5H-pyrido[3,2,1-ij]quinolin-10-ol, 3

[0246] Compound 2 (8.28 g, 34.0 mmol) was refluxed with hydrobromic acid (50 ml) for 1 h. The solution was neutralized with sodium bicarbonate in portions. The mixture was extracted into EtOAc, and the EtOAc layer was washed with water and worked up to give 3 (7.49 g, 98%) as a pale orange solid. 1H NMR (400 MHz, CD 2 Cl 2 ): Chemical shifts 6.75 (d, 1H), 6.02 (d, 1H), 5.12 (s, 1H), 3.25 (m, 2H), 2.55 (m, 2H), 1.90 (m, 5H), 1.25 (s, 6H). MS: calcd 230.15, observed 230.13 (MH + ).

[0247] Step 3: Preparation of 2,2,4-trimethyl-1,2-dihydroquinolin-7-ol 4

[0248] 7-Methoxy-2,2,4-trimethyl-1,2-dihydroquinoline, 1 (10.00 g, 49.2 mmol) was refluxed with hydrobromic acid (50 ml) for 6 h. The mixture was cooled to room temperature and then cooled in an ice bath. The resulting solid was collected by suction filtration and washed with ice water. The solid was then mixed with 50% EtOAc / water and neutralized with sodium bicarbonate. The organic layer was retained, the aqueous layer was extracted twice with EtOAc, and the EtOAc layers were combined and worked up. The resulting solid was purified from hot DCM / hexane precipitation followed by silica gel flash chromatography eluting with 10%-20% EtOAc / hexane to give 4 (Koelmel, Dominik K. et al., Organic & Biomolecular Chemistry, 2013, 11(24), 3954-3962) as an off-white / light yellow solid (7.45 g, 80%). 1 H NMR (400 MHz, CD 2 Cl 2 ): Chemical shifts 6.95 (m, 1H), 6.11 (m, 1H), 5.95 (s, 1H), 5.22 (s, 1H), 1.97 (s, 3H), 1.28 (s, 6H).

[0249] Step 4: Preparation of 3,6-dichloro-2-(7-hydroxy-2,2,4-trimethyl-1,2-dihydroquinoline-6-carbonyl)-4-(isopropoxycarbonyl)benzoic acid, 6 and 7

[0250] 2,2,4-Trimethyl-1,2-dihydroquinolin-7-ol, 4, (9.37 g, 49.5 mmol) and 3,6-dichlorotrimethinoic acid isopropyl ester, 5, (18.01, 59.4 mmol; 5 prepared by the method described in WO 2002 / 30944, incorporated herein by reference for the preparation of 5) were mixed in toluene (95 ml) and refluxed for 3.5 h. After cooling, the toluene was removed and the resulting solid was semi-purified by silica gel flash chromatography, eluting with 5%-10% MeOH / DCM. The resulting solid was then further purified by silica gel flash chromatography, eluting with 2% TEA / 35% EtOAc / Hexane, followed by 100% EtOAc, followed by 10%-15% MeOH / DCM. The resulting solid was then dissolved in DCM, washed twice with 1N HCl and worked up to give 6 / 7 as a yellow-green solid (18.45 g, 71%). 1 H NMR (400 MHz, CD 2 Cl 2 ): Chemical shifts 7.90 (d, 1H), 6.68 (d, 1H) 5.89 (s, 1H), 5.25 (m, 2H), 1.75 (d, 3H) 1.40 (m, 6H), 1.33 (s, 6H), MS: calcd 492.10 observed 492.08 (MH + ).

[0251] Step 4: Preparation of asymmetric rhodamine dye 8

[0252] Compounds 6 and 7 (19.79 g, 37.42 mmol) were dissolved in chloroform (400 ml) and mixed with phosphorus oxychloride (10.5 ml, 112 mmol) at room temperature for 10 minutes. Compound 3 (8.58 g, 37.4 mmol) was dissolved in chloroform (200 ml) and added to the compound 6 / 7 / phosphorus oxychloride solution. A dark aqua green color formed immediately. The solution was then refluxed for 3.5 hours to obtain a dark blue color. The solution was concentrated and the dark blue-black solid was then refluxed with hydrobromic acid (570 ml) with vigorous stirring for 1 hour. The hot solution was poured onto ice to produce a fine blue solid, which was collected by centrifugation and filtration and washed with water. The solid was mixed with a large volume of 2M triethylammonium acetate (TEAA), filtered to remove fine insolubles, and the isomers were loaded onto a large reverse phase chromatography column and eluted with 50 mM TEAA followed by 60%-65%-70% MeOH / 50 mM TEAA. Fractions were analyzed by HPLC before combining. The pool of dye 8 (dye eluted by second HPLC and RP-TLC) was diluted with an equal volume of water and, after concentration and drying, dye 8 was desalted on a C18 gel pad to give a dark black-blue solid (8.79 g, 32%) as the TEA salt. 1 HNMR (400MHz, CD3OD): Chemical shifts 7.55(s, 1H), 6.92(s, 1H), 6.81(s, 1H), 6.61(s, 1H), 5.60(d, 2H), 3.61(t, 2H) , 3.16(q, 6H), 2.94(m, 2H), 2.02(m, 2H), 1.87(d, 6H), 1.50(m, 6H), 1.39(d, 6H), 1.27(t, 9H), maximum absorption wavelength 610 nm.

[0253] Step 5: Preparation of asymmetric rhodamine dye 9

[0254] Dye 8 (5.25 g, 7.05 mmol) was dissolved in anhydrous DCM (300 ml), mixed with TEA (13.8 ml, 98.7 mmol), placed under nitrogen and cooled in an ice bath. Trifluoroacetic anhydride (6.86 ml, 49.3 mmol) was added dropwise and the solution was stirred for 0.5 h. The now colorless solution was concentrated, redissolved in DCM, washed with sodium bicarbonate, 1N HCl and worked up to give compound 9.

[0255] Example 2: Preparation of Rhodamine Dye 9 Phosphoramidite [ka]

[0256] Step 1: Preparation of Rhodamine Dye 9 Activated Ester

[0257] Compound 9 was dissolved in anhydrous DCM (200 ml) and mixed with N-hydroxysuccinimide (1.62 g, 14.1 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC, 3.38 g, 17.6 mmol) and stirred for 1 h. The solution was diluted with DCM and washed with 1N HCl to give compound 10.

[0258] Step 2: Preparation of N-protected 6-amino-2-DMThexan-1-ol linker rhodamine dye, 11

[0259] Compound 10 was added dropwise to anhydrous DMC (200 ml) and 6-amino-2-((bis(4-methoxyphenyl)(phenyl)methoxy)methyl)hexan-1-ol (4.12 g, 9.17 mmol) and triethylamine (1.47 ml, 10.6 mmol, TEA). This was stirred at room temperature for 2 hours. The solution was diluted with DCM, washed with water and worked up. The crude solid was purified using reverse phase column chromatography, eluting with 95%-100% MeCN / water to give compound 11 as a green solid (5.91 g, 72%). 1 H NMR (400 MHz, CD 2 Cl 2): Chemical shifts 7.85(s, 1H), 7.41(m, 2H), 7.29(m, 6H), 7.21(m, 1H), 6.82(m, 5H), 6.62(s, 1H), 6.22(s, 1H), 6.21(m, 1H), 5.53(s, 1H), 5.20(s, 1H), 3.78(s, 6H), 3.64(m, 2H), 3.44(m, 2H), 3.32(m, 2H), 3.24(m, 1H), 3.09(m, 1H), 2.88(t, 2H), 2.10(broad t, 1H), 1.96(m, 2H), 1.85(d, 3H), 1.79(m, 1H), 1.72(d, 3H), 1.55(m, 6H), 1.25~1.42(m, 10H), MS:calcd 1170.40, observed 1170.34(MH + ).

[0260] Step 3: Preparation of Rhodamine Dye 9 Phosphoramidite

[0261] Compound 11 (5.97 g, 5.10 mmol) and tetrazole amine (0.44 g, 2.54 mmol) were dissolved in anhydrous DCM (150 ml), stirred and placed under nitrogen. 3A molecular sieves were added and stirred for an additional 10 min. To this was added 2-cyanoethyl N,N,N',N'-tetraisopropyl phosphorodiamidite (3.07 g, 10.2 mmol) and the mixture was stirred at room temperature for 1 h. The molecular sieves were filtered and the solution was concentrated to a solid. This was purified by reverse phase column chromatography where the column was pre-treated with 20% TEA / MeCN and then washed extensively with MeCN. The sample was then dissolved in MeCN and eluted with MeCN. The pooled product containing fractions were concentrated and dried to give TFA-N-protected NH-asymmetric rhodamine dye phosphoramidite 12 as a dark yellow solid (6.13 g, 88%). 1 H NMR (400 MHz, CD 2 Cl 2): Chemical shifts 7.77 (s, 1H), 7.43 (m, 2H), 7.30 (m, 6H), 7.20 (m, 1H), 6.82 (m, 5H), 6.66 (s, 1H), 6.31 (m, 1H), 6.24 (s, 1H), 5.53 (s, 1H), 5.21 (s, 1H), 3.78-3.64 (s and m, 10H), 3.57 (m, 2H), 3.43 (q, 2H), 3.32(t, 2H), 3.11(m, 2H), 2.89(t, 2H), 2.54(q, 2H), 1.97(m, 2H), 1.92~1.84(m, 4H) ), 1.73(s, 3H), 1.61(m, 2H), 1.51(d, 6H), 1.45(m, 2H), 1.40~1.28(s and m, 8H), 1.50(m, 12H), 31 P NMR (400 MHz, CD 2 Cl 2 ): Chemical shift 147.4 (s, 1P), MS calcd 1370.51, observed 1370.57 (MH + ).

[0262] Example 3: Solid-phase synthesis of Big Dye asymmetric rhodamine-labeled oligonucleotides [ka]

[0263] Oligonucleotides labeled with N-protected asymmetric rhodamine phosphoramidite synthesis reagents were synthesized on polystyrene solid supports using standard operating conditions on a Biolytic 3900 automated DNA synthesizer. N-protected asymmetric rhodamine phosphoramidite 12 was dissolved in acetonitrile solvent for the coupling reaction, and the N-protected asymmetric rhodamine dye adducts are stable to repeated synthesis cycles using removal of DMT with trichloroacetyl acid, addition of other special phosphoramidites, coupling with acetic anhydride, and oxidation with iodine to generate internucleotide phosphodiester bonds. This class of asymmetric rhodamines was also found to be stable to the conditions used to deprotect and cleave the synthetic labeled oligonucleotides from the solid support (treatment with a solution containing t-butylamine / methanol / water at 65° C. for 5 hours). The overall scheme used to synthesize the labeled oligonucleotides is shown in the scheme above. By this process, mono TFA-asymmetric rhodamine DMT phosphoramidite 12 was attached to the 5'-hydroxyl of a support-bound oligonucleotide to give phosphodiester intermediate 13 after oxidation and removal of the DMT group. A PEG dimer phosphoramidite was attached to the free hydroxyl of intermediate 13 to give intermediate 14 after oxidation, capping, and removal of the DMT. A fluorescein phosphoramidite (ThermoFisher) was attached to the free hydroxyl of intermediate 14. The resulting labeled oligo was oxidized, capped, cleaved, and deprotected from the support to give labeled oligonucleotide 15. Oligo 15 was purified using standard chromatographic protocols.

[0264] The disclosure can be further described by the following numbered clauses:

[0265] 1. A compound comprising:

[0266] wherein R1, R2, R3, R6, R7, R8, R11, R12, R13, and R14, when taken alone and independently of one another, are selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, -Rb, or -(CH2)n-Rb; or R1 and R2 and / or R6 and R7 together with the carbon atom to which they are attached form an optionally substituted benzo group;

[0267] R4, alone, is selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, or R4 and one of R2 or R3, together with the atom to which they are attached, form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group;

[0268] R5 is H or a protecting group;

[0269] R9, when taken alone, is selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, or R7 and R9, taken together with the atom to which they are attached, form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group;

[0270] R10 is H or a protecting group, or R8 and R10 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group;

[0271] At least one of R7 and R9 or R8 and R10, together with the atom to which they are attached, form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group; and optionally, one of R4 and R2 or R3, together with the atom to which they are attached, form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group, with the proviso that the compound has the formula

[0272] -or-, but not

[0273] each Ra is independently selected from lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, -CX3, and 6-20 membered heteroarylalkyl;

[0274] Each Rb is independently selected from X, -OH, -ORa, -SH, -SRa-NH2, -NHRaNRcRc, N+RcRcRc, perhalo lower alkyl, trihalomethyl, trifluoromethyl, -P(O)(OH)2, P(O)(ORa)2, P(O)(OH)(ORa), -OP(O)(OH)2, OP(O)(ORa)2, -OP(O)(ORa)(O H), -S(O)2OH, S(O)2Ra, -C(O)H, C(O)Ra, -C(S)X, -C(O)ORa, -C(O)OH, -C(O)NH2, -C(O)NHRa, selected from C(O)NRcRc, C(S)NH2, C(O)NHRa, -C(O)NRcRc, -C(NH)NH2, -C(NH)NHRa, and C(NH)NRcRc,

[0275] each Rc is independently Ra, or two Rc bound to the same nitrogen atom together with the nitrogen atom form a 5-8 membered saturated or unsaturated ring which may optionally contain one or more of the same or different ring heteroatoms selected from O, N, and S;

[0276] each Rd and Re, when taken alone, is independently selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, -Rb, or -(CH2)n-Rb;

[0277] A compound of the formula, wherein X is halo and n is an integer ranging from 1 to 10.

[0278] 2. A compound according to clause 1, in which the spirolactone ring is in the open acid form and the amine group is unprotected. In certain embodiments, the open acid form of the compound is fluorescent (or exhibits increased fluorescence) compared to the closed spirolactone form of the compound. The amine group of the compounds described herein can be protected in the closed spirolactone form and used as and phosphoramidite for high yield and purity of labeling of nucleic acids. Thus, provided herein are fluorescently labeled nucleic acid probes and primers comprising the deprotected compound of clause 1. Representative examples of compounds of clause 1 in the open lactone form after deprotection of the amine group and cleavage of the nucleic acid from the solid support are shown in Figures 8 and 9.

[0279] Thermo Fisher Scientific offers reagents for labeling nucleic acids with five reporter dyes (i.e., FAM, VIC, TED, TAZ, and SID) and size standard LIZ (NGM Detect™ PCR Amplification Kit). Certain dyes provided herein have unique spectral properties that complement existing dye sets and can be used to expand the number of reporter dyes that can be included in HID applications. In particular, certain asymmetric rhodamines described in clause 1 have been found to exhibit peak emission wavelengths and narrow spectral widths such that they can be resolved from other dyes in existing commercially available dye sets. For example, a representative compound exhibiting a peak emission wavelength (e.g., ∼634 nm) belongs to the class of asymmetric rhodamine compounds shown in structure D.1. Applicant has further discovered that by replacing VIC with two new dyes, the existing HID dye set can be expanded to include seven or more reporter dyes. In certain embodiments, asymmetric rhodamines having structures described in PCT / US2019 / 67925 (Cmpd A) and TET (~536 nm) are used as substitutes for VIC in a kit further comprising asymmetric rhodamines of structure D.1 and FAM, TED, TAZ, and SID (Cmpd B) (Figure 10). Such compounds are well separated from the adjacent emission peaks of SID (~617) and LIZ (~653 nm) of the proposed dye set. Thus, certain kits provided herein include nucleic acids (or reagents for labeling nucleic acids) labeled with compounds described in clause 1 (e.g., compounds having structure D.1) that emit at ~634 nm, FAM, TET, TED, TAZ, and SID.

[0280] 3. An oligonucleotide containing a labeling moiety prepared by reacting an oligonucleotide attached to a solid support with a reagent has the structure:

[0281] LM-L-PEP

[0282] where PEP is a phosphate precursor group, L is an optional linker attaching a labeling moiety to the PEP group, and LM comprises an N-protected NH-rhodamine moiety of formula (I),

[0283] wherein R1, R2, R3, R6, R7, R8, R11, R12, R13, and R14, when taken alone and independently of one another, are selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, -Rb, or -(CH2)n-Rb; or R1 and R2 and / or R6 and R7 together with the carbon atom to which they are attached form an optionally substituted benzo group, and one of R2, R3, R7, R8, R12, or R13 comprises a group of formula -Y-, where Y is selected from the group consisting of -C(O)-, S(O)2-, -S-, and -NH-;

[0284] R4, alone, is selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, or R4 and one of R2 or R3, together with the atom to which they are attached, form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group;

[0285] R5 is H or a protecting group;

[0286] R9, when taken alone, is selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, or R7 and R9, taken together with the atom to which they are attached, form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group;

[0287] R10 is H or a protecting group, or R8 and R10 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group;

[0288] At least one of R7 and R9 or R8 and R10, together with the atom to which they are attached, form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group; and optionally, one of R4 and R2 or R3, together with the atom to which they are attached, form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group, with the proviso that the compound has the formula

[0289] -or-, but not

[0290] each Ra is independently selected from lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, -CX3, and 6-20 membered heteroarylalkyl;

[0291] Each Rb is independently selected from X, -OH, -ORa, -SH, -SRa-NH2, -NHRa-NRcRc, N+RcRcRc, perhalo lower alkyl, trihalomethyl, trifluoromethyl, -P(O)(OH)2, P(O)(ORa)2, P(O)(OH)(ORa), -OP(O)(OH)2, OP(O)(ORa)2, -OP(O)(ORa)( OH), -S(O)2OH, S(O)2Ra, -C(O)H, C(O)Ra, -C(S)X, -C(O)ORa, -C(O)OH, -C(O)NH2, -C(O)NHRa, selected from C(O)NRcRc, C(S)NH2, C(O)NHRa, -C(O)NRcRc, -C(NH)NH2, -C(NH)NHRa, and C(NH)NRcRc,

[0292] each Rc is independently Ra, or two Rc bound to the same nitrogen atom together with the nitrogen atom form a 5-8 membered saturated or unsaturated ring which may optionally contain one or more of the same or different ring heteroatoms selected from O, N, and S;

[0293] each Rd and Re, when taken alone, is independently selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, -Rb, or -(CH2)n-Rb;

[0294] A compound of the formula, wherein X is halo and n is an integer ranging from 1 to 10.

[0295] 4. The oligonucleotide according to clause 3, wherein the spirolactone ring is in the open acid form and the amine group is unprotected.

[0296] 5. A reagent useful for labeling oligonucleotides, comprising a compound of the structural formula:

[0297] LM-L-PEP(XX)

[0298] where LM represents a labeling moiety comprising an N-protected NH-rhodamine moiety, PEP is a phosphate precursor group comprising a phosphoramidite group or an H-phosphonate group, L is an optional linker attaching the labeling moiety to the phosphate precursor group, and is an N-protected NH-rhodamine moiety of the formula:

[0299] wherein R1, R2, R3, R6, R7, R8, R11, R12, R13, and R14, when taken alone and independently of one another, are selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, -Rb, or -(CH2)n-Rb; or R1 and R2 and / or R6 and R7 together with the carbon atom to which they are attached form an optionally substituted benzo group, and one of R2, R3, R7, R8, R12, or R13 comprises a group of formula -Y-, where Y is selected from the group consisting of -C(O)-, S(O)2-, -S-, and -NH-;

[0300] R4, alone, is selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, or R4 and one of R2 or R3, together with the atom to which they are attached, form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group;

[0301] R5 is H or a protecting group;

[0302] R9, when taken alone, is selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, or R7 and R9, taken together with the atom to which they are attached, form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group;

[0303] R10 is H or a protecting group, or R8 and R10 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group;

[0304] At least one of R7 and R9 or R8 and R10, together with the atom to which they are attached, form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group; and optionally, one of R4 and R2 or R3, together with the atom to which they are attached, form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group, with the proviso that the compound has the formula

[0305] -or-, but not

[0306] each Ra is independently selected from lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, -CX3, and 6-20 membered heteroarylalkyl;

[0307] Each Rb is independently selected from X, -OH, -ORa, -SH, -SRa-NH2, -NHRa-NRcRc, N+RcRcRc, perhalo lower alkyl, trihalomethyl, trifluoromethyl, -P(O)(OH)2, P(O)(ORa)2, P(O)(OH)(ORa), -OP(O)(OH)2, OP(O)(ORa)2, -OP(O)(ORa)( OH), -S(O)2OH, S(O)2Ra, -C(O)H, C(O)Ra, -C(S)X, -C(O)ORa, -C(O)OH, -C(O)NH2, -C(O)NHRa, selected from C(O)NRcRc, C(S)NH2, C(O)NHRa, -C(O)NRcRc, -C(NH)NH2, -C(NH)NHRa, and C(NH)NRcRc,

[0308] each Rc is independently Ra, or two Rc bound to the same nitrogen atom together with the nitrogen atom form a 5-8 membered saturated or unsaturated ring which may optionally contain one or more of the same or different ring heteroatoms selected from O, N, and S;

[0309] each Rd and Re, when taken alone, is independently selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, -Rb, or -(CH2)n-Rb;

[0310] A compound of the formula, wherein X is halo and n is an integer ranging from 1 to 10.

[0311] 6. The reagent according to clause 5, wherein the spirolactone ring is in the open acid form and the amine group is unprotected.

Claims

1. A compound of the formula: 【Chemistry 1】 During the ceremony, R 1 , R 2 , R 3 , R 6 , R 7 , R 8 , R 11 , R 12 , R 13 , and R 14 are each independently hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, -R b or -(CH2) n -R b Alternatively, R 1 and R 2 and / or R 6 and R 7 together with the carbon atom to which they are attached form an optionally substituted benzo group, R 4 is selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, or R 4 and R 2 Or R 3 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group; R 5 is H or a protecting group, R 9 is selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, or R 7 and R 9 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group; R 10 is H or a protecting group, or R 8 and R 10 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group; R 7 and R 9 Or R 8 and R 10 at least one of, together with the atom to which they are attached, forms an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group, and optionally, R 4 and R 2 Or R 3 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group, provided that the compound has the formula 【Chemistry 2】 Not a compound of Each R a is lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, -CX 3 and 6- to 20-membered heteroarylalkyl; Each R b are independently -X, -OH, -OR a , -SH, -SR a , -NH 2 , -NHR a , N.R. c R c , N + R c R c R c , perhalo lower alkyl, trihalomethyl, trifluoromethyl, -P(O)(OH) 2 , P(O)(OR a ) 2 , P(O)(OH)(OR a ), -OP(O)(OH) 2 , OP(O)(OR a ) 2 , -OP(O)(OR a )(OH), -S(O) 2 OH, S(O) 2 R a , -C(O)H, C(O)R a , -C(S)X, -C(O)OR a , -C(O)OH, -C(O)NH 2 , -C(O)NHR a , -C(O)NR c R c , C(S)NH 2 , C(O)NHR a , -C(O)NR c R c , -C(NH)NH 2 , -C(NH)NHR a and C(NH)NR c R c is selected from Each R c are independently R a or two R c together with the nitrogen atom form a 5-8 membered saturated or unsaturated ring which may optionally contain one or more of the same or different ring heteroatoms selected from O, N, and S; Each R d and R e when alone, independently represents hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, -R b , or -(CH 2 ) n -R b is selected from X is a halo; A compound wherein n is an integer ranging from 1 to 10.

2. The compound has the formula (II.1) 【Chemistry 3】 In the formula, each R f , R g , R h , R i , and R j are each independently hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, -R b or -(CH2) n -R b 2. The compound of claim 1 , selected from:

3. The compound has the formula (II.2) 【Chemistry 4】 In the formula, each R f , R g , R h , R i , and R j are each independently hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, -R b or -(CH2) n -R b 2. The compound of claim 1 , selected from:

4. The compound has the formula (II.3) 【Chemistry 5】 In the formula, R h , R i , and R j are each independently hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, -R b or -(CH2) n -R b 2. The compound of claim 1 , selected from:

5. The compound has the formula (II.4), 【Chemistry 6】 In the formula, each R f , R g , R h , R i , and R j are each independently hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, -R b or -(CH2) n -R b 2. The compound of claim 1 , selected from:

6. Each R 11 and R 14 The compound of any one of claims 1 to 5, wherein is halo.

7. The compound of claim 6 , wherein said halo is fluoro or chloro.

8. The compound of claim 6 , wherein said halo is chloro.

9. R 13 -C(O)H, -C(O)R a -C(S)X, C(O)O - , -C(O)OH, -C(O)NH 2 , -C(O)NHR a , -C(O)NR c R c , -C(S)NH 2 , -C(O)NHR a , C(O)NR c R c , -C(NH)NH 2 , -C(NH)NHR a or -C(NH)NR c R c The compound according to any one of claims 1 to 8,

10. R 13 But C(O)O - or -C(O)OH.

11. R 5 The compound according to any one of claims 1 to 10, wherein is a protecting group.

12. The protecting group is —C(O)R 15 Wherein R 15 is hydrogen, lower alkyl, -CX 3 , -CHX 2 , -CH 2 X, -CH 2 -OR d and lower alkyl, -X, -OR d , phenyl optionally monosubstituted with a cyano or nitro group, d 12. The compound of claim 11, wherein is selected from the group consisting of lower alkyl, phenyl and pyridyl, and each X is a halo group.

13. If present, R 10 The compound according to any one of claims 1 to 12, wherein is a protecting group.

14. The protecting group is —C(O)R 15 Wherein R 15 is hydrogen, lower alkyl, -CX 3 , -CHX 2 , -CH 2 X, -CH 2 -OR d and lower alkyl, -X, -OR d , phenyl optionally monosubstituted with a cyano or nitro group, d 14. The compound of claim 13, wherein is selected from the group consisting of lower alkyl, phenyl and pyridyl, and each X is a halo group.

15. R 15 But, -CX 3 , -CHX 2 , -CH 2 15. The compound of claim 12 or 14, wherein X is

16. R 15 But, -CX 3 16. The compound of claim 15,

17. A compound according to any one of claims 12 to 16, wherein said X in said protecting group, when present, is fluoro or chloro.

18. The compound according to any one of claims 12 to 16, wherein said X in said protecting group, when present, is fluoro.

19. R 1 and R 2 and together with the carbon atom to which they are attached form an optionally substituted benzo group.

20. R 6 and R 7 and together with the carbon atom to which they are attached form an optionally substituted benzo group.

21. R 7 and R 9 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group.

22. R 8 and R 10 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group.

23. R 4 and R 2 Or R 3 and one of, taken together with the atom to which they are attached, forms an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group.

24. The optional substitutions on the benzo ring, the heterocycloalkyl, the heterocycloalkenyl, or the heteroaryl group, when present, are -X, -OH, -OR a , -SH, -SR a -NH 2 , -NHR a , -NR c R c , N + R c R c R c , perhalo lower alkyl, trihalomethyl, trifluoromethyl, -P(O)(OH) 2 , P(O)(OR a ) 2 , P(O)(OH)(OR a ), -OP(O)(OH) 2 , OP(O)(OR a ) 2 , -OP(O)(OR a )(OH), -S(O) 2 OH, S(O) 2 R a , C(O)H, C(O)R a , -C(S)X, -C(O)OR a , -C(O)OH, -C(O)NH 2 , -C(O)NHR a , C(O)NR c R c , C(S)NH 2 , C(O)NHR a , -C(O)NR c R c , -C(NH)NH 2 , -C(NH)NHR a and C(NH)NR c R c Each R a is, independently of the others, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, -CX 3 and 6- to 20-membered heteroarylalkyl, each R c are each independently R a or two R c can be taken together with the nitrogen atom to form a 5-8 membered saturated or unsaturated ring which may optionally contain one or more of the same or different ring heteroatoms selected from O, N, and S.

25. The optional substitutions on the benzo ring, the heterocycloalkyl, the heterocycloalkenyl, or the heteroaryl group, when present, are selected from -X, -C1-C6 alkyl, -C2-C6 alkenyl, -C2-C6 alkynyl, -OH, OR a -SH,SR a -NH 2 , -NHR a -NR c R c , N + R c R c R c , perhalo lower alkyl, trihalomethyl, trifluoromethyl, C(O)H, -C(O)R a , -C(S)X,C(O)OR a , -C(O)OH, -C(O)NH 2 , -C(O)NHR a , C(O)NR c R C , C(S)NH 2 , -C(O)NHR a , -C(O)NR c R c , -C(NH)NH 2 , -C(NH)NHR a or C(NH)NR c R c The compound according to any one of claims 1 or 19 to 23, comprising at least one of:

26. The optional substitutions on the benzo ring, the heterocycloalkyl, the heterocycloalkenyl, or the heteroaryl group are -X, -C1 to C6 alkyl, -C2 to C6 alkenyl, -C2 to C6 alkynyl, -OH, -OR a -SH, -SR a N.H. 2 , N.H.R. a -NR c R c , N + R c R c R c , perhalo lower alkyl, trihalomethyl, trifluoromethyl, C(O)H, C(O)R a , -C(S)X,C(O)OR a , -C(O)OH, -C(O)NH 2 , -C(O)NHR a , -C(O)NR c R C , C(S)NH 2 , C(O)NHR a , -C(O)NR c R c , -C(NH)NH 2 , -C(NH)NHR a or -C(NH)NR c R c The compound according to any one of claims 1 or 19 to 25, comprising at least two of:

27. 27. The compound of any one of claims 1 or 19-26, wherein the optional substitution on the benzo ring, the heterocycloalkyl, the heterocycloalkenyl, or the heteroaryl group comprises at least two -C1-C6 alkyl.

28. 1. An oligonucleotide containing a labeling moiety prepared by reacting a solid support-bound oligonucleotide with a reagent having the structure: LM-L-PEP wherein PEP is a phosphate precursor group, L is an optional linker attaching the labeling moiety to the PEP group, and LM comprises an N-protected NH-rhodamine moiety of formula (I): 【Chemistry 7】 During the ceremony, R 1 , R 2 , R 3 , R 6 , R 7 , R 8 , R 11 , R 12 , R 13 , and R 14 are each independently hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, -R b or -(CH2) n -R b Alternatively, R 1 and R 2 and / or R 6 and R 7 together with the carbon atom to which they are attached form an optionally substituted benzo group; R 2 , R 3 , R 7 , R 8 , R 12 , or R 13 one of which contains a group of formula -Y-, where Y is -C(O)-, S(O) 2 selected from the group consisting of -, -S- and -NH-; R 4 is selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, or R 4 and R 2 Or R 3 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group; R 5 is H or a protecting group, R 9 is selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, or R 7 and R 9 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group; R 10 is H or a protecting group, or R 8 and R 10 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group; R 7 and R 9 Or R 8 and R 10 at least one of, together with the atom to which they are attached, forms an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group, and optionally, R 4 and R 2 Or R 3 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group, provided that the compound has the formula 【Chemistry 8】 Not a compound of Each R a is lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, -CX 3 and 6- to 20-membered heteroarylalkyl; Each R b are independently X, -OH, -OR a -SH, -SR a -NH 2 , -NHR a N.R. c R c , N + R c R c R c , perhalo lower alkyl, trihalomethyl, trifluoromethyl, -P(O)(OH) 2 , P(O)(OR a ) 2 , P(O)(OH)(OR a ), -OP(O)(OH) 2 , OP(O)(OR a ) 2 , -OP(O)(OR a )(OH), -S(O) 2 OH, S(O) 2 R a , -C(O)H, C(O)R a , -C(S)X, -C(O)OR a , -C(O)OH, -C(O)NH 2 , -C(O)NHR a , C(O)NR c R c , C(S)NH 2 , C(O)NHR a , -C(O)NR c R c , -C(NH)NH 2 , -C(NH)NHR a and C(NH)NR c R c is selected from Each R c are independently R a or two R c together with the nitrogen atom form a 5-8 membered saturated or unsaturated ring which may optionally contain one or more of the same or different ring heteroatoms selected from O, N, and S; Each R d and R e when alone, independently represents hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, -R b , or -(CH 2 ) n -R b is selected from X is a halo; An oligonucleotide, wherein n is an integer ranging from 1 to 10.

29. said N-protected NH-rhodamine moiety having the formula (II.1), 【Chemistry 9】 In the formula, each R f , R g , R h , R i , and R j are each independently hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, -R b , or -(CH 2 ) n -R b 29. The oligonucleotide of claim 28, selected from:

30. said N-protected NH-rhodamine moiety having the formula (II.2), 【Chemistry 10】 In the formula, each R f , R g , R h , R i , and R j are each independently hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, -R b , or -(CH 2 ) n -R b 29. The oligonucleotide of claim 28, selected from:

31. said N-protected NH-rhodamine moiety having the formula (II.3), 【Chemistry 11】 In the formula, R h , R i , and R j are each independently hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, -R b or -(CH2) n -R b 29. The oligonucleotide of claim 28, selected from:

32. said N-protected NH-rhodamine moiety having the formula (II.4), 【Chemistry 12】 In the formula, each R f , R g , R h , R i , and R j are each independently hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, -R b or -(CH2) n -R b 29. The oligonucleotide of claim 28, selected from:

33. Each R 11 and R 14 The oligonucleotide of any one of claims 28 to 32, wherein is halo.

34. 34. The oligonucleotide of claim 33, wherein said halo is fluoro or chloro.

35. 34. The oligonucleotide of claim 33, wherein said halo is chloro.

36. R 12 -C(O)H, -C(O)R a -C(S)X, C(O)O - , -C(O)OH, -C(O)NH 2 , -C(O)NHR a , -C(O)NR c R C , -C(S)NH 2 , -C(O)NHR a , -C(O)NR c R c , -C(NH)NH 2 , -C(NH)NHR a or -C(NH)NR c R c The oligonucleotide according to any one of claims 28 to 35,

37. R 12 But -C(O)O - or -C(O)OH.

38. R 5 The compound according to any one of claims 28 to 37, wherein is a protecting group.

39. The protecting group is -C(O)R 10 Wherein R 10 is hydrogen, lower alkyl, -CX 3 , -CHX 2 , -CH 2 X, -CH 2 -OR d and lower alkyl, -X, -OR d , phenyl optionally monosubstituted with a cyano or nitro group, d 39. The compound of claim 38, wherein is selected from the group consisting of lower alkyl, phenyl and pyridyl, and each X is a halo group.

40. If present, R 10 The oligonucleotide of any one of claims 28 to 39, wherein is a protecting group.

41. The protecting group is -C(O)R 15 Wherein R 15 is hydrogen, lower alkyl, -CX 3 , -CHX 2 , -CH 2 X, C.H. 2 -OR d and lower alkyl, -X, -OR d , phenyl optionally monosubstituted with a cyano or nitro group, d 41. The oligonucleotide of claim 40, wherein X is selected from the group consisting of lower alkyl, phenyl and pyridyl, and each X is a halo group.

42. R 15 But, -CX 3 , -CHX 2 , -CH 2 42. The oligonucleotide of claim 39 or 41, wherein

43. R 15 But, -CX 3 43. The oligonucleotide of claim 42,

44. 44. The oligonucleotide of any one of claims 39 to 43, wherein X in the protecting group, if present, is fluoro or chloro.

45. 44. The oligonucleotide of any one of claims 39 to 43, wherein X in the protecting group, if present, is fluoro.

46. R 1 and R 2 together with the carbon atom to which they are attached form an optionally substituted benzo group.

47. R 6 and R 7 together with the carbon atom to which they are attached form an optionally substituted benzo group.

48. R 7 and R 9 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group.

49. R 8 and R 10 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group.

50. R 4 and R 2 Or R 3 30. The oligonucleotide of claim 28, wherein one of:

51. The optional substitutions on the benzo ring, the heterocycloalkyl, the heterocycloalkenyl, or the heteroaryl group, if present, are each independently selected from R b At least one of the groups is independently -X, -OH, OR a , -SH, -SR a -NH 2 , -NHR a , -NR c R c , N + R c R c R c , perhalo lower alkyl, trihalomethyl, trifluoromethyl, -P(O)(OH) 2 , P(O)(OR a ) 2 , P(O)(OH)(OR a ), -OP(O)(OH) 2 , OP(O)(OR a ) 2 , OP(O)(OR a )(OH), -S(O) 2 OH, S(O) 2 R a , C(O)H, C(O)R a , -C(S)X, -C(O)OR a , -C(O)OH, C(O)NH 2 , -C(O)NHR a , -C(O)NR c R c , C(S)NH 2 , C(O)NHR a , -C(O)NR c R c , -C(NH)NH 2 , -C(NH)NR a and C(NH)NR c R c , and -C(NH)NHR c R c Each R a is, independently of the others, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, -CX 3 and 6- to 20-membered heteroarylalkyl, each R c is, independently of others, R a or two R c can be taken together with the nitrogen atom to form a 5-8 membered saturated or unsaturated ring which may optionally contain one or more of the same or different ring heteroatoms selected from O, N and S.

52. The optional substitutions on the benzo ring, the heterocycloalkyl, the heterocycloalkenyl, or the heteroaryl group, when present, are selected from -X, -C1-C6 alkyl, -C2-C6 alkenyl, -C2-C6 alkynyl, -OH, OR a -SH,SR a -NH 2 , -NHR a -NR c R c , N + R c R c R c , perhalo lower alkyl, trihalomethyl, trifluoromethyl, C(O)H, -C(O)R a , -C(S)X,C(O)OR a , -C(O)OH, -C(O)NH 2 , -C(O)NHR a , -C(O)NR c R C , C(S)NH 2 , -C(O)NHR a , -C(O)NR c R c , -C(NH)NH 2 , -C(NH)NHR a or C(NH)NR c R c The oligonucleotide according to any one of claims 28 or 46 to 51, comprising at least one of:

53. The optional substitutions on the benzo ring, the heterocycloalkyl, the heterocycloalkenyl, or the heteroaryl group are -X, -C1 to C6 alkyl, -C2 to C6 alkenyl, -C2 to C6 alkynyl, -OH, OR a -SH,SR a -NH 2 , -NHR a -NR c R c , N + R c R c R c , perhalo lower alkyl, trihalomethyl, trifluoromethyl, -C(O)H, C(O)R a , -C(S)X,C(O)OR a , -C(O)OH, -C(O)NH 2 , -C(O)NHR a , C(O)NR c R C , C(S)NH 2 , C(O)NHR a , -C(O)NR c R c , -C(NH)NH 2 , -C(NH)NHR a or -C(NH)NR c R c The oligonucleotide according to any one of claims 28 or 46 to 52, comprising at least two of:

54. 54. The oligonucleotide of any one of claims 28 or 46-53, wherein the optional substitutions on the benzo ring, the heterocycloalkyl, the heterocycloalkenyl, or the heteroaryl group comprise at least two -C1-C6 alkyl.

55. The N-protected NH-rhodamine moiety has the formula 【Chemistry 13】 29. The oligonucleotide of claim 28, having the following structure:

56. The oligonucleotide of any one of claims 28 to 55, wherein the label moiety is attached to the 3'- or 5'-hydroxyl of the oligonucleotide.

57. The oligonucleotide of any one of claims 28 to 56, wherein the labeling moiety is attached to a nucleobase of the oligonucleotide.

58. The oligonucleotide of any one of claims 28 to 57, wherein the oligonucleotide is further labeled with a donor and / or acceptor moiety for the N-protected NH-rhodamine moiety.

59. The oligonucleotide of any one of claims 28 to 57, wherein the oligonucleotide is further labeled with a quencher moiety.

60. The oligonucleotide of any one of claims 28 to 57, wherein the oligonucleotide is further labeled with a minor groove binding moiety.

61. 58. The oligonucleotide of any one of claims 28 to 57, wherein the labeling moiety further comprises the donor or acceptor moiety for the N-protected NH-rhodamine moiety.

62. 62. The oligonucleotide of claim 61, wherein the labeling moiety further comprises a donor moiety for the N-protected NH-rhodamine moiety.

63. 63. The oligonucleotide of claim 62, wherein the donor moiety comprises an N-protected NH-rhodamine moiety or an O-protected fluorescein moiety.

64. 64. The oligonucleotide of claim 63, wherein the 2'-, 2''-, 4'-, 5'-, 7'-, 7''-, 5-, or 6-position of the donor moiety is linked to the 2'-, 2''-, 4'-, 5'-, 7'-, 7''-, 5-, or 6-position of the N-protected NH-rhodamine moiety.

65. 64. The oligonucleotide of claim 63, wherein the donor moiety and the N-protected NH-rhodamine moiety are linked in a head-to-head orientation.

66. 64. The oligonucleotide of claim 63, wherein the donor moiety and the N-protected NH-rhodamine moiety are linked in a head-to-tail orientation.

67. 64. The oligonucleotide of claim 63, wherein the donor moiety and the N-protected NH-rhodamine moiety are linked in a tail-to-tail orientation.

68. 64. The oligonucleotide of claim 63, wherein the donor moiety and the N-protected NH-rhodamine moiety are linked in a side-to-side orientation.

69. 64. The oligonucleotide of claim 63, wherein the donor moiety and the N-protected NH-rhodamine moiety are linked in a side-to-head orientation.

70. 64. The oligonucleotide of claim 63, wherein the donor moiety and the N-protected NH-rhodamine moiety are linked in a side-to-tail orientation.

71. The labeling moiety has structural formula (VI): A-Z 1 -Sp-Z 2 -D(VI) wherein A represents the N-protected NH-rhodamine moiety, D represents the donor moiety, and Z 1 and Z 2 may be the same or different, and the functional group F z and Sp represents a spacing moiety.

72. A is said N-protected NH-rhodamine moiety and D is selected from the group consisting of moieties having the structural formulas D.1, D.2, D.3, D.4, D.5, D.6, D.7, D.8, D.9, D.10, D.11, and D.12; 【Chemistry 14】 【change】 【change】 【change】 In each of D.1 to D.12, Each R 1’ , R 2’ , R 2’’ , R 4’ , R 4’’ , R 5’ , R 5’’ , R 7’ , R 7’’ , and R 8’ when alone, independently represents hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, R b and -(CH 2 ) x -R b where x is an integer having a value from 1 to 10; b -X, -OH, -OR a -SH, -SR a -NH 2 , -NHR a -NR c R c , N + R c R c R c , perhalo lower alkyl, trihalomethyl, trifluoromethyl, -P(O)(OH) 2 , P(O)(OR a ) 2 , P(O)(OH)(OR a ), -OP(O)(OH) 2 , OP(O)(OR a ) 2 , -OP(O)(OR a )(OH), -S(O) 2 OH, S(O) 2 R a , -C(O)H, C(O)R a , -C(S)X, -C(O)OR a , -C(O)OH, -C(O)NH 2 , -C(O)NHR a , C(O)NR c R c , -C(S)NH 2 , C(O)NHR a , -C(O)NR c R c , -C(NH)NH 2 , -C(NH)NHR a and -C(NH)NR c R c wherein X is halo and each R a is independently selected from the group consisting of lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, and 6-20 membered heteroarylalkyl; c is independently R a or two R c can be combined with the nitrogen atom to form a 5-8 membered saturated or unsaturated ring which may optionally contain one or more of the same or different ring heteroatoms selected from the group consisting of O, N, and S; Or, R 1’ and R 2’ Or R 7’ and R 8’ together with the carbon atoms to which they are attached form an optionally substituted (C6-C14) aryl bridge, and / or R 4’ and R 4’’ and / or R 5’ and R 5’’ together with the carbon atom to which they are attached form a benzo group, Each R 4 , R 5 , R 6 , and R 7 are each independently hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 6- to 14-membered heteroaryl, 7- to 20-membered heteroarylalkyl, -R b and -(CH 2 ) x -R b is selected from E 1 is -NHR 9 , -NR 9 R 10 and -OR 9b is selected from the group consisting of E 2 is -NHR 9 , -NR 9 R 10 and -OR 9b is selected from the group consisting of R 9 is selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, or R 7 and R 9 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group; R 10 is H or a protecting group, or R 8 and R 10 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group; R 9b is R 9 and Each Y 1a , Y 1b , Y 2a , Y 2b , Y 3a and Y 3b are independently -O-, -S-, -NH-, -C(O-) and -S(O) 2 is selected from the group consisting of However, each E 1 and E 2 Ga-OR 9b If R 1’ and R 2’ and / or R 7’ and R 8’ may form, together with only the carbon atoms to which they are attached, an optionally substituted (C6-C14) aryl bridge.

73. The L of the reagent is -Z-(CH 2 ) 3-6 -O-, -Z-(CH 2 ) a - [(Ar) b - (CH 2 ) a ] c -O-, -Z-(CH 2 ) a −[C≡C−(CH 2 ) a ] c -O-, -Z-(CH 2 ) a -[C≡C-(Ar) b ] c - (CH 2 ) a -O-, -Z-(CH 2 ) d -NH-C(O)-[(CH 2 ) a -(Ar)-(CH 2 ) a -C(O)-NH] c - (CH 2 ) d -O-, and -Z-[CH 2 (CH 2 ) e O] f -CH 2 (CH 2 ) e O-, wherein Each Z is independently a functional group F z represents the fraction of bonds contributed by each a represents, independently of the others, an integer ranging from 0 to 4; each b represents, independently of the others, an integer ranging from 1 to 2; each c represents, independently of the others, an integer ranging from 1 to 5; each d represents, independently of the others, an integer ranging from 1 to 10; each e represents, independently of the others, an integer ranging from 1 to 4; each f, independently of the others, represents an integer ranging from 1 to 10; 30. The oligonucleotide of claim 28, wherein each Ar represents, independently of the other, an optionally substituted monocyclic or polycyclic cycloalkylene, cycloheteroalkynene, arylene, or heteroarylene group.

74. 74. The oligonucleotide of claim 73, wherein each Ar of L is, independently of the others, a group derived from cyclohexane, piperazine, benzene, naphthalene, phenol, furan, pyridine, piperidine, imidazole, pyrrolidine, or oxadizole.

75. 29. The oligonucleotide of claim 28, wherein the reagent further comprises a suitably protected synthesis handle comprising a reactive group selected from the group consisting of amino, hydroxyl, thiol, and aldehyde, and a) a protecting group configured to be removed during the course of the oligonucleotide synthesis to provide the reactive group for addition of an additional moiety, or b) a protecting group that is stable during the course of the oligonucleotide synthesis and configured to be removed after the oligonucleotide synthesis to provide the reactive group for addition of an additional moiety.

76. The reagent is a compound of structural formula (VIII): ( k _________________________________________ In the formula, R k 76. The oligonucleotide of claim 75, wherein: represents an acid labile protecting group; each L, independently of the other, represents an optional linker; LM represents the labeling moiety; and PEP represents the phosphate precursor group.

77. The reagent is a compound of structural formula (IX): 【Chemistry 15】 In the formula, R k is an acid labile protecting group.

78. The reagent is a compound of structural formula (IX.1), 【Chemistry 16】 In the formula, -Z- is a functional group F z , Sp 1 , Sp 2 and Sp 3 wherein G represents a portion of the bond contributed by, which may be the same or different, and each represents a spacing moiety; and G represents CH, N, or a group comprising arylene, phenylene, heteroarylene, lower cycloalkylene, cyclohexylene, and / or lower cycloheteroalkylene.

79. Sp 1 , Sp 2 and Sp 3 each independently represents an alkylene chain containing 1 to 10 carbon atoms, (CH 2 ) a [(Ar) b - (CH 2 ) a ] c -, -(CH 2 ) a −[C≡C−(CH 2 ) a ] c -, -(CH 2 ) a -[C≡C-(Ar) b ] c - (CH 2 ) a -, (CH 2 ) d NH-C(O)-[(CH 2 ) a -(Ar)-(CH 2 ) a -C(O)-NH] c - (CH 2 ) d - and -[CH 2 (CH 2 ) e O] f -CH 2 (CH 2 )-, each a independently represents an integer ranging from 0 to 4; each b represents, independently of the others, an integer ranging from 1 to 2; each c represents, independently of the others, an integer ranging from 1 to 5; each d represents, independently of the others, an integer ranging from 1 to 10; each e represents, independently of the others, an integer ranging from 1 to 4; 79. The oligonucleotide of claim 78, wherein each f, independently of the others, represents an integer in the range of 1 to 10, and Ar, independently of the others, represents an optionally substituted monocyclic or polycyclic cycloalkylene, cycloheteroalkylene, arylene, or heteroarylene group.

80. the reagent is a compound of structural formula (IX.2), (IX.3), (IX.4) or (IX.5), 【Chemistry 17】 wherein B represents a suitably protected nucleobase; L 2 represents a linker connecting the labeling moiety LM to the nucleobase B, and in structure (IX.4), R 16 represents a protecting group, 76. The oligonucleotide of claim 75, wherein the nucleobase is selected from adenine, 7-deazaguanine, guanine, 7-deazaguanine, cytosine, uracil, thymine, inosine, xanthene, and hypoxanthene.

81. The reagent B is A iBu , A Pac , C Ac , G iPr-Pac 81. The oligonucleotide of claim 80, wherein the amino acid sequence is selected from the group consisting of , T and U.

82. L of the reagent 2 But -C≡C-CH 2 -NH-, -C≡C-C(O)-, -CH=CH-NH-, -CH=CH-C(O)-, -C≡C-CH 2 -NH-C(O)-(CH 2 ) 1-6 -NH-, -CH=CH-C(O)-NH-(CH 2 ) 1-6 -NH-C(O)-, -C=CH-CH 2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 -NH-, -C≡C-C≡C-CH 2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 -NH-, -C≡C-(Ar) 1-2 -C≡C-CH 2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 -NH-, -C≡C-(Ar) 1-2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 -NH- and -C≡C-(Ar) 1-2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 81. The oligonucleotide of claim 80, wherein Ar is selected from -NH-, where Ar is, independently of the other, an optionally substituted monocyclic or polycyclic cycloalkylene, cycloheteroalkynene, arylene, or heteroarylene group.

83. 29. The oligonucleotide of claim 28, wherein the PEP group comprises a phosphoramidite group and an H-phosphonate group.

84. said phosphate precursor group comprising a phosphoramidite of formula (P.1) 【Chemistry 18】 During the ceremony, R 20 is a linear, branched or cyclic, saturated or unsaturated alkyl containing 1 to 10 carbon atoms, 2-cyanoethyl, aryl containing 6 to 10 ring carbon atoms, and arylalkyl containing 6 to 10 ring carbon atoms and 1 to 10 alkylene carbon atoms; Each R 21 and R 22 are each independently a linear, branched or cyclic, saturated or unsaturated alkyl having 1 to 10 carbon atoms, an aryl having 6 to 10 carbon atoms, and an arylalkyl having 6 to 10 carbon atoms and 1 to 10 alkylene carbon atoms, or alternatively R 21 and R 22 The oligonucleotide of claim 83, wherein, together with the nitrogen atom to which they are attached, form a saturated or unsaturated ring containing 5 to 6 ring atoms, one or two of which, in addition to the exemplified nitrogen atom, can be a heteroatom selected from O, N and S.

85. R 20 is β-cyanoethyl, R 21 and R 22 The oligonucleotide of claim 83, wherein each of is isopropyl.

86. The synthetic handle has the formula -OR k wherein R k 86. The oligonucleotide of claim 85, wherein is an acid labile protecting group.

87. 87. The oligonucleotide of claim 86, wherein the acid labile protecting group is selected from the group consisting of triphenylmethyl (trityl), 4-monomethoxytrityl, 4,4'-dimethoxytrityl, 4,4',4''-trimethoxytrityl, bis(p-anisyl)phenylmethyl, naphthyldiphenylmethyl, p-(p'-bromophenacyloxy)phenyldiphenylmethyl, 9-anthryl, 9-(9-phenyl)xanthenyl, and 9-(9-phenyl-10-oxo)anthryl.

88. A reagent useful for labeling oligonucleotides, which is a compound of the structural formula: LM-L-PEP(XX) wherein LM represents a labeling moiety comprising an N-protected NH-rhodamine moiety, PEP is a phosphate precursor group comprising a phosphoramidite group or an H-phosphonate group, L is an optional linker attaching the labeling moiety to the phosphate precursor group, and the N-protected NH-rhodamine moiety is of the formula: 【Chemistry 19】 During the ceremony, R 1 , R 2 , R 3 , R 6 , R 7 , R 8 , R 11 , R 12 , R 13 , and R 14 are each independently hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, -R b or -(CH2) n -R b Alternatively, R 1 and R 2 and / or R 6 and R 7 together with the carbon atom to which they are attached form an optionally substituted benzo group; R 2 , R 3 , R 7 , R 8 , R 12 , or R 13 one of which contains a group of formula -Y-, where Y is -C(O)-, S(O) 2 selected from the group consisting of -, -S- and -NH-; R 4 is selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, or R 4 and R 2 Or R 3 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group; R 5 is H or a protecting group, R 9 is selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, or R 7 and R 9 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group; R 10 is H or a protecting group, or R 8 and R 10 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group; R 7 and R 9 Or R 8 and R 10 at least one of, together with the atom to which they are attached, forms an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group, and optionally, R 4 and R 2 Or R 3 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group, provided that the compound has the formula 【Chemistry 20】 Not a compound of Each R a is lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, -CX 3 and 6- to 20-membered heteroarylalkyl; Each R b are independently X, -OH, -OR a -SH, -SR a -NH 2 , -NHR a N.R. c R c , N + R c R c R c , perhalo lower alkyl, trihalomethyl, trifluoromethyl, -P(O)(OH) 2 , P(O)(OR a ) 2 , P(O)(OH)(OR a ), -OP(O)(OH) 2 , OP(O)(OR a ) 2 , -OP(O)(OR a )(OH), -S(O) 2 OH, S(O) 2 R a , -C(O)H, C(O)R a , -C(S)X, -C(O)OR a , -C(O)OH, -C(O)NH 2 , -C(O)NHR a , C(O)NR c R c , C(S)NH 2 , C(O)NHR a , -C(O)NR c R c , -C(NH)NH 2 , -C(NH)NHR a and C(NH)NR c R c is selected from Each R c are independently R a or two R c together with the nitrogen atom form a 5-8 membered saturated or unsaturated ring which may optionally contain one or more of the same or different ring heteroatoms selected from O, N, and S; Each R d and R e when alone, independently represents hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, -R b , or -(CH 2 ) n -R b is selected from X is a halo; A reagent, wherein n is an integer ranging from 1 to 10.

89. The L of the reagent is -Z-(CH 2 ) 3-6 -O-, -Z-(CH 2 ) a - [(Ar) b - (CH 2 ) a ] c -O-, -Z-(CH 2 ) a −[C≡C−(CH 2 ) a ] c -O-, -Z-(CH 2 ) a -[C≡C-(Ar) b ] c - (CH 2 ) a -O-, -Z-(CH 2 ) d -NH-C(O)-[(CH 2 ) a -(Ar)-(CH 2 ) a -C(O)-NH] c - (CH 2 ) d -O-, and -Z-[CH 2 (CH 2 ) e O] f -CH 2 (CH 2 ) e O-, wherein Each Z is independently a functional group F z represents the fraction of bonds contributed by each a represents, independently of the others, an integer ranging from 0 to 4; each b represents, independently of the others, an integer ranging from 1 to 2; each c represents, independently of the others, an integer ranging from 1 to 5; each d represents, independently of the others, an integer ranging from 1 to 10; each e represents, independently of the others, an integer ranging from 1 to 4; each f, independently of the others, represents an integer ranging from 1 to 10; 90. The reagent of claim 88, wherein each Ar represents, independently of the other, an optionally substituted monocyclic or polycyclic cycloalkylene, cycloheteroalkynene, arylene, or heteroarylene group.

90. 74. The oligonucleotide of claim 73, wherein each Ar of L is, independently of the others, a group derived from cyclohexane, piperazine, benzene, naphthalene, phenol, furan, pyridine, piperidine, imidazole, pyrrolidine, or oxadizole.

91. A compound of structural formula (VII.1) 【Chemistry 21】 wherein B represents a suitably protected nucleobase; L 2 89. The reagent of claim 88, wherein represents a linker connecting the nucleobase B to the labeling moiety LM.

92. 92. The reagent of claim 91, wherein the nucleobase is selected from adenine, 7-deazaguanine, guanine, 7-deazaguanine, cytosine, uracil, thymine, inosine, xanthene, and hypoxanthene.

93. B is A iBu , A Pac , C Ac , G iPr-Pac 93. The reagent of claim 92, wherein the amino acid sequence is selected from the group consisting of , T and U.

94. L 2 But -C≡C-CH 2 -NH-, -C≡C-C(O)-, -CH=CH-NH-, -CH=CH-C(O)-, -C≡C-CH 2 -NH-C(O)-(CH 2 ) 1-6 -NH-, -CH=CH-C(O)-NH-(CH 2 ) 1-6 -NH-C(O)-, -C≡CH-CH 2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 -NH-, -C≡C-C≡C-CH 2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 -NH-, -C≡C-(Ar) 1-2 -C≡C-CH 2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 -NH-, -C≡C-(Ar) 1-2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 -NH- and -C≡C-(Ar) 1-2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 92. The reagent of claim 91, wherein Ar is selected from -NH-, and Ar is as defined in claim 89.

95. -B-L 2 -but, 【Chemical 22】 【change】 92. The reagent of claim 91, selected from the group consisting of:

96. 90. The reagent of claim 89, further comprising a suitably protected synthetic handle.

97. Formula (VIII) ( k _________________________________________ [In the formula, R k represents an acid labile protecting group; each L, independently of the other, represents an optional linker; LM represents said labeling moiety; and PEP represents said phosphate ester precursor group.

98. The reagent is a compound of structural formula (IX): 【Chemistry 23】 In the formula, R k 97. The reagent of claim 96, wherein represents an acid labile protecting group.

99. The reagent is a compound of structural formula (IX.1), 【Chemistry 24】 In the formula, -Z- is a functional group F z , Sp 1 , Sp 2 and Sp 3 which may be the same or different and each represents a spacing moiety; and G represents CH, N, or a group comprising arylene, phenylene, heteroarylene, lower cycloalkylene, cyclohexylene, and / or lower cycloheteroalkylene.

100. Sp 1 , Sp 2 and Sp 3 each independently represents an alkylene chain containing 1 to 10 carbon atoms, -(CH 2 ) a - [(Ar) b - (CH 2 ) a ] c -, -(CH 2 ) a −[C≡C−(CH 2 ) a ] c -, -(CH 2 ) a -[C≡C-(Ar) b ] c - (CH 2 ) a -, -(CH 2 ) d -NH-C(O)-[(CH 2 ) a -(Ar)-(CH 2 ) a -C(O)-NH] c - (CH 2 ) d - and -[CH 2 (CH 2 ) e O] f -CH 2 (CH 2 )-, each a independently represents an integer ranging from 0 to 4; each b represents, independently of the others, an integer ranging from 1 to 2; each c represents, independently of the others, an integer ranging from 1 to 5; each d represents, independently of the others, an integer ranging from 1 to 10; each e represents, independently of the others, an integer ranging from 1 to 4; 100. The reagent of claim 99, wherein each f, independently of the others, represents an integer in the range of 1 to 10, and Ar, independently of the others, represents an optionally substituted monocyclic or polycyclic cycloalkylene, cycloheteroalkylene, arylene, or heteroarylene group.

101. The reagent is a compound of structural formula (IX.2), (IX.3), (IX.4) or (IX.5), 【Chemistry 25】 wherein B represents a suitably protected nucleobase; L 2 represents a linker that attaches the labeling moiety LM to the nucleobase B, and in structure (IX.4), R 16 99. The reagent of claim 98, wherein represents a protecting group.

102. 102. The reagent of claim 101, wherein the nucleobase is selected from adenine, 7-deazaguanine, guanine, 7-deazaguanine, cytosine, uracil, thymine, inosine, xanthene, and hypoxanthene.

103. The reagent B is A iBu , A Pac , C Ac , G iPr-Pac , T and U.

104. L of the reagent 2 But -C≡C-CH 2 -NH-, -C≡C-C(O)-, -CH=CH-NH-, -CH=CH-C(O)-, -C≡C-CH 2 -NH-C(O)-(CH 2 ) 1-6 -NH-, -CH=CH-C(O)-NH-(CH 2 ) 1-6 -NH-C(O)-, -C=CH-CH 2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 -NH-, -C≡C-C≡C-CH 2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 -NH-, -C≡C-(Ar) 1-2 -C≡C-CH 2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 -NH-, -C≡C-(Ar) 1-2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 -NH- and -C≡C-(Ar) 1-2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 102. The reagent of claim 101, wherein Ar is selected from -NH-, where Ar is, independently of the other, an optionally substituted monocyclic or polycyclic cycloalkylene, cycloheteroalkynene, arylene, or heteroarylene group.

105. 【Catalogue 26】 【change】 The reagent of claim 101, selected from the group consisting of:

106. 106. The reagent of any one of claims 89 to 105, wherein the phosphate precursor groups include phosphoramidite groups and H-phosphonate groups.

107. said phosphate precursor group comprising a phosphoramidite of formula (P.1) 【Chemistry 27】 During the ceremony, R 20 is a linear, branched or cyclic, saturated or unsaturated alkyl containing 1 to 10 carbon atoms, 2-cyanoethyl, aryl containing 6 to 10 ring carbon atoms, and arylalkyl containing 6 to 10 ring carbon atoms and 1 to 10 alkylene carbon atoms; Each R 21 and R 22 are each independently a linear, branched or cyclic, saturated or unsaturated alkyl containing 1 to 10 carbon atoms, an aryl containing 6 to 10 carbon atoms, and an arylalkyl containing 6 to 10 carbon atoms and 1 to 10 alkylene carbon atoms, or alternatively R 21 and R 22 The reagent of claim 106, wherein, together with the nitrogen atom to which they are attached, form a saturated or unsaturated ring containing 5 to 6 ring atoms, one or two of which, in addition to the exemplified nitrogen atom, may be a heteroatom selected from O, N and S.

108. R 20 is β-cyanoethyl, R 21 and R 22 The reagent of claim 107, wherein each of is isopropyl.

109. 90. The reagent of claim 88, further comprising a solid support and a suitably protected synthetic handle.

110. A compound of structural formula (X): 【Chemistry 28】 where LM represents a labeling moiety, L represents an optionally cleavable linker, and R k The reagent of claim 106, wherein represents an acid labile protecting group.

111. A compound of structural formula (X.1), 【Chemical 29】 In the formula, Z, G, Sp 1 , Sp 2 and W are as previously defined in claim 14; Sp 4 represents a selectively cleavable spacing moiety.

112. Sp 1 and Sp 2 are each independently an alkylene chain containing 1 to 10 carbon atoms, -(CH 2 ) a - [(Ar) b - (CH 2 ) a ] c -, -(CH 2 ) a −[C≡C−(CH 2 ) a ] c -, -(CH 2 ) a -[C≡C-(Ar) b ] c - (CH 2 ) a -, -(CH 2 ) d -NH-C(O)-[(CH 2 ) a -(Ar)-(CH 2 ) a -C(O)-NH] c - (CH 2 ) d - and [CH 2 (CH 2 ) e O] f -CH 2 (CH 2 )-, in which a, b, c, d, e, f and Ar are as defined in claim 4, and Sp 4 The reagent of claim 106, wherein said compound comprises an ester bond.

113. Compounds of structural formula (X.2), (X.3), (X.4) or (X.5), 【Chemistry 30】 In the formula, B, L 2 and R 16 The reagent according to claim 110, wherein is as previously defined in claim 16.

114. 114. The reagent of claim 113, wherein the nucleobase is selected from adenine, 7-deazaguanine, guanine, 7-deazaguanine, cytosine, uracil, thymine, inosine, xanthene, and hypoxanthene.

115. B is A iBu , A Pac , C Ac , G iPr-Pac 114. The reagent of claim 113, wherein said compound is selected from the group consisting of:

116. L 2 But -C≡C-CH 2 -NH-, -C≡C-C(O)-, -CH=CH-NH-, -CH=CH-C(O)-, -C≡C-CH 2 -NH-C(O)-(CH 2 ) 1-6 -NH-, -CH=CH-C(O)-NH-(CH 2 ) 1-6 -NH-C(O)-, -C≡CH-CH 2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 -NH-, -C≡C-C≡C-CH 2 -O-CH 2 CH 2 [O-CH 2 CH 2 ] 0-6 -NH-, -C≡C-(Ar) 1-2 -C≡C-CH 2 -O-CH 2 CH 2 [O-CH 2 CH 2 ] 0-6 -NH-, -C≡C-(Ar) 1-2 -O-CH 2 CH 2 [O-CH 2 CH 2 ] 0-6 -NH- and -C≡C-(Ar) 1-2 -O-CH 2 CH 2 -[O-CH 2 CH 2 ] 0-6 114. The reagent of claim 113, wherein Ar is selected from -NH-, and Ar is as defined in claim 4.

117. -B-L 2 The reagent of claim 113, wherein - is selected from the following: 【Chemistry 31】 【change】

118. R k The reagent according to any one of claims 97 to 117, wherein is 4',4''-dimethoxytrityl.

119. wherein the N-protected NH-rhodamine moiety comprises a structure selected from the formula: 【Chemistry 32】 In the formula, R f , R g , R h , R i , and R j each, when alone, independently of the other, is hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5- to 14-membered heteroaryl, 6- to 20-membered heteroarylalkyl, -R b or -(CH 2 ) n -R b R' is R 3’ and hydrogen.

120. the N-protected NH-rhodamine moiety comprises a structure selected from structural formulas (III.1), (III.2), (III.3), and (III.4): 【Chemical 33】 In the formula, R 1~ R 14 , R a ~R j and Y are as defined above.

121. the N-protected NH-rhodamine moiety (i) Y is -C(O)-, -S(O) 2 -, -S-, and -NH-; (ii) R 11 and R 14 are chloro, (iii) R 1 and R 6 are hydrogen, (iv) R 1 and R 2 Or R 6 and R 6 together form a benzo group, (v) R 2 and R 7 are each hydrogen or lower alkyl, (vi) R 5 is a protecting group, (vii) R 4 and R 9 taken together with the substituents on the adjacent carbon atoms, represents -CH 2 CH 2 --, --CH 2 CH 2 CH 2 -, -C(CH 3 ) 2 CH=C(CH 3 ) -, C(CH 3 ) 2 CH=CH, -CH 2 C (CH 3 ) 2 - and 【Chemical 34】 forming a group selected from 121. The reagent of claim 119 or 120, having one or more applicable features selected from:

122. R 5 is represented by the formula -C(O)R 15 where R 10 is hydrogen, lower alkyl, methyl, -CX 3 , -CHX 2 , C.H. 2 X, C.H. 2 OR d and lower alkyl, methyl, -X, -OR d phenyl optionally monosubstituted with a cyano or nitro group, d The reagent of claims 116 to 119, wherein X is selected from lower alkyl, phenyl and pyridyl, and each X is a halo group.

123. R15 The reagent of claim 122, wherein is selected from methyl and trifluoromethyl.

124. The reagent of any one of claims 88 to 123, wherein the label moiety further comprises a donor and / or acceptor moiety of the N-protected NH-rhodamine moiety.

125. 122. The reagent of any one of claims 88 to 121, wherein the labeling moiety further comprises a donor moiety of the N-protected NH-rhodamine moiety.

126. 126. The reagent of claim 125, wherein the donor moiety comprises an N-protected NH-rhodamine moiety or an O-protected fluorescein moiety.

127. The 2'-, 2''-, 4'-, 5'-, 7'-, 7''-, 5- or 6-position of the donor moiety is R 3 , R 12 , or R 13 The reagent of claim 126, which is bound to

128. 128. The reagent of claim 127, wherein the donor moiety and the N-protected NH-rhodamine moiety are linked in a head-to-head orientation.

129. 128. The reagent of claim 127, wherein said donor moiety and said N-protected NH-rhodamine moiety are linked in a head-to-tail orientation.

130. 128. The reagent of claim 127, wherein the donor moiety and the N-protected NH-rhodamine moiety are linked in a tail-to-tail orientation.

131. 128. The reagent of claim 127, wherein the donor moiety and the N-protected NH-rhodamine moiety are attached in a side-to-side orientation.

132. 128. The reagent of claim 127, wherein the donor moiety and the N-protected NH-rhodamine moiety are linked in a side-to-head orientation.

133. 128. The reagent of claim 127, wherein the donor moiety and the N-protected NH-rhodamine moiety are linked in a side-to-tail orientation.

134. the labeling moiety comprises structural formula (VI), A-Z 1 -Sp-Z 2 -D(VI) wherein A represents the N-protected NH-rhodamine moiety, D represents the donor moiety, and Z 1 and Z 2 may be the same or different, and the functional group F z and Sp represents a spacing moiety.

135. A is an N-protected NH-rhodamine moiety and D is selected from the structural formulas D.1, D.2, D.3, D.4, D.5, D.6, D.7, D.8, D.9, D.10, D.11 and D.12; 【Chemistry 35】 【change】 【change】 【change】 In each of D.1 to D.12, Each R 1’ , R 2’ , R 2’’ , R 4’ , R 4’’ , R 5’ , R 5’’ , R 7’ , R 7’’ , and R 8’ when alone, independently represents hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, R b and -(CH 2 ) x -R b where x is an integer having a value from 1 to 10; b -X, -OH, -OR a -SH, -SR a -NH 2 , -NHR a -NR c R c , N + R c R c R c , perhalo lower alkyl, trihalomethyl, trifluoromethyl, -P(O)(OH) 2 , P(O)(OR a ) 2 , P(O)(OH)(OR a ), -OP(O)(OH) 2 , OP(O)(OR a ) 2 , -OP(O)(OR a )(OH), -S(O) 2 OH, S(O) 2 R a , -C(O)H, C(O)R a , -C(S)X, -C(O)OR a , -C(O)OH, -C(O)NH 2 , -C(O)NHR a , C(O)NR c R c , C(S)NH 2 , C(O)NHR a , -C(O)NR c R c , -C(NH)NH 2 , -C(NH)NHR a and -C(NH)NR c R c wherein X is halo and each R a is selected from the group consisting of lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, and 6-20 membered heteroarylalkyl; each R c is independently R a or two R c together with the nitrogen atom form a 5-8 membered saturated or unsaturated ring which may optionally contain one or more of the same or different ring heteroatoms selected from the group consisting of O, N, and S; Or, R 1’ and R 2’ Or R 7’ and R 8’ together with the carbon atoms to which they are attached form an optionally substituted (C6-C14) aryl bridge, and / or R 4’ and R 4’’ and / or R 5’ and R 5’’ together with the carbon atom to which they are attached form a benzo group, Each R 4 , R 5 , R 6 , and R 7 are each independently hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 6- to 14-membered heteroaryl, 7- to 20-membered heteroarylalkyl, -R b and -(CH 2 ) x -R b is selected from E 1 is -NHR 9 , -NR 9 R 10 and -OR 9b is selected from the group consisting of E 2 is -NHR 9 , -NR 9 R 10 and -OR 9b is selected from the group consisting of R 9 is selected from hydrogen, lower alkyl, (C6-C14)aryl, (C7-C20)arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, or R 7 and R 9 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group; R 10 is H or a protecting group, or R 8 and R 10 together with the atom to which they are attached form an optionally substituted heterocycloalkyl group, an optionally substituted heterocycloalkenyl group, or an optionally substituted heteroaryl group; R 9b is R 9 and Each Y 1a , Y 1b , Y 2a , Y 2b , Y 3a and Y 3b are independently -O-, -S-, -NH-, -C(O)- and -S(O) 2 is selected from the group consisting of However, each E 1 and E 2 Ga-OR 9b If R 1’ and R 2’ and / or R 7’ and R 8’ may form an optionally substituted (C6-C14) aryl bridge only with the carbon atoms to which they are attached.

136. D.1 to D.12 are (i) Y 1a , Y 2a and Y 3a are each independently -C(O)- and S(O) 2 - selected from Y 1b , Y 2b and Y 3b is -NH-, (iii) R 4 and R 7 are chloro, (iv) R 1’ and R 8 are hydrogen, (v) R 1’ and R 2’ Or R 7’ and R 8’ together form a benzo group, and (vi) R 2’ and R 7’ is hydrogen or lower alkyl, where applicable.

137. Y 1a , Y 2a and Y 3a is -NH-, and Y 1b , Y 2b and Y 3b is -C(O)- and -S(O) 2 -, Z 1 is -C(O)- and -S(O) 2 -, Z 2 is -NH- and Sp is -(CH 2 ) a - [(Ar) b - (CH 2 ) a ] c -, -(CH 2 ) a −[C≡C−(CH 2 ) a ] c -, -(CH 2 ) a -[C≡C-(Ar) b ] c - (CH 2 ) a -, -(CH 2 ) d -NH-C(O)-[(CH 2 ) a -(Ar)-(CH 2 ) a -C(O)-NH] c - (CH 2 ) d - and -[CH 2 (CH 2 ) e O] f -CH 2 (CH 2 136. The reagent of claim 135, wherein a, b, c, d, e, f, and Ar are as previously defined in claim 4.

138. In structural formulas D.1 to D.12, R 1’ and R 2’ and / or R 7’ and R 8’ together with the carbon atom to which they are attached form a benzo bridge.

139. E 1 and E 2 are -OR 9b The reagent according to claims 135 to 138,

140. R 9b is represented by the formula -C(O)R 15 R 15 The reagent of claim 139, wherein is lower alkyl.

141. R 15 The reagent of claim 140, wherein is t-butyl.

142. R 9 is represented by the formula -C(O)R 15 where R 15 is hydrogen, lower alkyl, methyl, -CX 3 , -CHX 2 , C.H. 2 X, C.H. 2 OR d and lower alkyl, methyl, -X, -OR d , phenyl optionally monosubstituted with a cyano or nitro group, d The reagent of claims 135-141, wherein X is selected from lower alkyl, phenyl and pyridyl, and each X is a halo group.

143. R 15 The reagent of claim 142, wherein is selected from methyl and trifluoromethyl.

144. 1. A method for simultaneously amplifying and analyzing multiple genetic loci, comprising:

30. A method comprising amplifying a nucleic acid sample using a plurality of amplification primer pairs to form a plurality of amplification products, at least one of each of the primer pairs comprising a labeled nucleotide of claim 28, and the amplification products comprising different loci.

145. 145. The method of claim 144, wherein the labeled nucleotide of claim 28 is used to label at least three primer pairs.

146. The method of claim 145, wherein the labeled nucleotide of claim 28 is used to label from 3 to 8 primer pairs.

147. The method of claim 144, wherein the nucleic acid sample is isolated from whole blood, tissue biopsy, lymph, bone, bone marrow, teeth, skin, skin cells, e.g., found in fingerprints, bone, teeth, amniotic fluid containing placental cells and amniotic fluid containing fetal cells, hair, skin, semen, anal secretions, feces, urine, vaginal secretions, sweat, saliva, buccal swabs, various environmental samples (e.g., agricultural, water, and soil), general research samples, general purification samples, and lysed cells.

148. 30. A kit comprising oligonucleotide primers for co-amplifying a set of loci of at least one nucleic acid sample to be analyzed, the set of loci being capable of being co-amplified, at least one of the primers comprising a labeled nucleotide according to claim 28, the primers being in one or more containers.

149. 149. The kit of claim 148, wherein all of the oligonucleotide primers in the kit are contained in a single container.

150. 149. The kit of claim 148, further comprising at least one container having reagents for at least one multiplex amplification reaction and at least one size standard.

151. 149. The kit of claim 148, wherein at least one of said oligonucleotide primers comprises a labeled nucleotide, said labeled nucleotide having different spectral properties than the labeled nucleotide of claim 28.

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