NZ748730B2 - Coumarin compounds and their uses as fluorescent labels - Google Patents

Abstract

The present application relates to new coumarin compounds of formula (I) and their uses as fluorescent labels. The compounds may be used as fluorescent labels for nucleotides in nucleic acid sequencing applications.

Description

(12) Granted patent caon (19) NZ (11) 748730 (13) B2 (47) Publicaon date: 2021.12.24 (54) COUMARIN COMPOUNDS AND THEIR USES AS FLUORESCENT LABELS (51) Internaonal Patent Classificaon(s): C07D 491/04 G01N 33/52 (22) Filing date: (73) Owner(s): 2017.12.15 ILLUMINA CAMBRIDGE LIMITED (23) Complete specificaon filing date: (74) Contact: 2017.12.15 Wrays Pty Ltd (30) Internaonal Priority Data: (72) Inventor(s): US 62/438,006 2016.12.22 ROMANOV, Nikolai (86) aonal Applicaon No.: (87) Internaonal Publicaon number: WO/2018/114710 (57) Abstract: The present applicaon s to new coumarin compounds of formula (I) and their uses as fluorescent labels. The compounds may be used as fluorescent labels for nucleodes in nucleic acid sequencing applicaons.

NZ 748730 B2 COUMARIN COMPOUNDS AND THEIR USES AS FLUORESCENT LABELS BACKGROUND Field The present application relates to coumarin compounds. The compounds may be used as fluorescent labels, particularly for nucleotide ng in nucleic acid sequencing applications.

Background Non-radioactive detection of nucleic acids utilizing fluorescent labels is an ant logy in molecular y. Many procedures employed in recombinant DNA technology previously relied on the use of tides or polynucleotides ctively labeled with, for example 32P. Radioactive compounds permit sensitive detection of nucleic acids and other molecules of interest. However, there are serious tions in the use of radioactive isotopes such as their e, limited shelf life and more importantly safety considerations. Eliminating the need for radioactive labels enhances safety whilst reducing the environmental impact and costs associated with, for example, reagent disposal. Methods amenable to non-radioactive cent detection include by way of non-limiting example, ted DNA sequencing, hybridization s, real-time detection merase-chain-reaction products and assays.

For many applications it is desirable to employ multiple spectrally distinguishable fluorescent labels in order to e independent detection of a plurality of spatially overlapping analytes. In such multiplex methods the number of reaction vessels may be reduced to simplify experimental protocols and tate the production of application-specific reagent kits. In multi-color automated DNA sequencing systems for example, multiplex fluorescent detection allows for the analysis of l different nucleotide bases in a single electrophoresis lane. This increases throughput over detection systems using a single-color, and also can reduce the uncertainties associated with inter-lane electrophoretic mobility variations.

However, multiplex fluorescent detection can be problematic and there are a number of important factors, which constrain selection of fluorescent labels. First, it is difficult to find dye compounds whose absorption and emission a are suitably spectrally resolved. In addition, when several fluorescent dyes are used together, simultaneous excitation may be difficult because the absorption bands of the dyes for different spectral regions may be widely separated.

Many excitation methods use high power lasers and therefore the dye must have sufficient photo- stability to withstand such laser excitation. A final consideration of particular importance in molecular biology methods is that the fluorescent dyes must be compatible with the reagent chemistries. Thus, for example the fluorescent dyes used in DNA synthesis or sequencing reactions must be compatible with the solvents and reagents, buffers, rase enzymes and ligase enzymes used in those reactions. In one example, PCT Publication No. WC 2007/135368 describes a class of rhodamine compounds used as fluorescent labels.

Coumarin dyes family has ted attention ofchemists due to their remarkable spectral properties. Nevertheless, there are only a few photo-stable fluorescent dyes with large Stokes shifts (LSS) that are commercially ble. Most of these dyes also contain the coumarin nt as a scaffold. For example, most of the dyes from Dyomics are coumarin derivatives absorbing at about 480-520 nm, and emitting in the region of 560-630. Other examples of this class of coumarin dyes include orylated coumarin based dyes as disclosed in US. Publication No. 2014/0220588 and commercially available dyes Star44OSXP and Star 47OSXP from Abberior.

Another practically usefill coumarin dye is AlexaFluorTM 430 with absorption and emission maxima at 434 nm and 539 nm respectively. Other LSS fluorescent dyes include Pacific OrangeTM (abs. 390 nm, emission 540 nm; Stokes shift 150 nm, Invitrogen) and BD HorizonTM V500 (abs. 415 nm, emission 500 nm; Stokes shift 85 nm, BD Biosciences) ChromeoTM 494 (abs. 494 nm, emission 628 nm, Stokes Shift 134 nm, Active Motive).

SUMMARY bed herein are novel coumarin derivatives and their use as bio-molecule , ularly as labels for tides used in nucleic acid sequencing. When such dyes are used for the preparation of bio-molecule conjugates, improvements can be seen in the length, intensity and quality of sequencing read obtainable due to the use of these new fluorescent compounds.

Some embodiments bed herein are d to coumarin nds of Formula (I), salts or mesomeric forms thereof: xfl _ (D \ GDN—R10a \ I wherein R1 is EE/AN 319 Rlob or R100 and wherein R1 , , , is optionally substituted with one or more substituents selected from the group consisting of alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, halo, cyano, y, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido, o, nitro, yl, sulfo, , ate, S-sulfonamido, N—sulfonamido, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and optionally substituted heterocyclyl; each R2, R3, R4, R5, and R9 is independently selected from the group consisting of H, alkyl, substituted alkyl, , alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, halo, cyano, hydroxy, yalkyl, alkyl, C-carboxy, O-carboxy, o, N—amido, nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonamido, N—sulfonamido, optionally substituted carbocyclyl, optionally tuted aryl, optionally substituted aryl and optionally substituted heterocyclyl; each R6, Rloa, R10b and R100 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, alkynyl, aminoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfonyl hydroxide, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl; each R7 and R8 is independently selected from the group ting of H, alkyl, substituted alkyl, alkoxy, alkenyl, l, haloalkyl, koxy, alkoxyalkyl, amino, aminoalkyl, halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, oxy, C-amido, N—amido, nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonamido, N—sulfonamido, optionally substituted carbocyclyl, optionally tuted aryl, optionally substituted heteroaryl and optionally substituted heterocyclyl; alternatively, R6 and R7 together with the atoms to which they are attached form a ring or ring system selected from the group consisting of optionally substituted 5-10 membered heteroaryl or optionally substituted 5-10 membered heterocyclyl; X is selected from the group consisting of O, S, NR", and Se; R11 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, alkynyl, aminoalkyl, carboxyalkyl, sulfonatoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfo, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl; and the bond represented by a solid and dashed line: is selected from the group consisting of a single bond and a double bond, provided that when : is a double bond, then R3 is absent.

Some embodiments described herein are related to fluorescent compounds of Formula (II) with a Stokes shift at least about 60 nm, salts, or mesomeric forms thereof: (11) wherein RHat is a 5 to 10 membered heteroaryl optionally substituted with one or more R10; each R1, R2, R3, R4, and R5 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, alkenyl, l, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido, N—amido, nitro, sulfonyl, sulfo, sulfino, sulfonate, onamido, N—sulfonamido, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl and optionally tuted cyclyl; R6 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, alkynyl, aminoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfonyl hydroxide, ally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl; each R7 and R8 is independently selected from the group consisting of H, alkyl, substituted alkyl, , alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-amido, N—amido, nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonamido, N—sulfonamido, ally substituted carbocyclyl, optionally substituted aryl, optionally tuted heteroaryl and optionally substituted heterocyclyl; atively, R6 and R7 together with the atoms to which they are attached form a ring or ring system selected from the group consisting of optionally substituted 5-10 membered heteroaryl or optionally substituted 5-10 membered heterocyclyl; each R10 is independently selected from the group consisting of alkyl, substituted alkyl, alkenyl, alkynyl, aminoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfonyl ide, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally tuted heterocyclyl; the bond ented by a solid and dashed line: is selected from the group consisting of a single bond and a double bond, ed that when : is a double bond, then R3 is absent.

Some embodiments described herein are d to nucleotide or oligonucleotide labeled with a compound of Formula (I) or Formula (II).

Some embodiments described herein are related to kits ning one or more nucleotides where at least one nucleotide is a labeled nucleotide described herein.

WO 14710 Some further embodiments described herein are related to methods of sequencing including orating a labeled nucleotide described herein in a sequencing assay, and detecting the d nucleotide.

BRIEF DESCRIPTION OF THE DRAWINGS illustrates the usability of the A-nucleotide labeled with the new coumarin dye I-16 as described herein for sequencing analysis. illustrates the usability of the A-nucleotide labeled with a commercial fluorescent dye DYS lOXL with long Stokes shift for sequencing analysis. rates the usability of the eotide labeled with a commercial fluorescent dye Chromeo494 with long Stokes shift for sequencing analysis.

DETAILED DESCRIPTION Embodiments described herein relate to new coumarin dyes and their tives of the structure of Formula (I) for use as fluorescent labels. Further embodiments relate to fluorescent nd of the structure of Formula (II) with a Stokes shift of at least about 60 nm.

These new fluorescent dyes may be used as fluorescent labels, particularly for nucleotide labeling in nucleic acid sequencing applications. Methods of preparing these fluorescent dyes and downstream sequencing applications utilizing these dyes are also exemplified.

Surprisingly, it has been discovered that the cence intensities of the new dyes and their bio-conjugates are nearly equal when irradiated with either blue or green light sources. For example, when the dyes are excited with 460 nm (blue) and 540 nm (green) laser or LED, the fluorescence intensities are about the same in some cases. As described below, this property holds true in solution and on flow cells, enabling simplified sequencing analysis with high quality. nds of Formula (I) Some embodiments described herein are related to new coumarin derivatives of a (I), or salts, mesomeric forms thereof: R2 R3 R8 R9 ,{XL@ 3%? QED/ 210 \ 1oa T9 N \ ®N—R \ wherein R1 is / Rlob or R100 and wherein R1 , , , , is optionally substituted with one or more substituents selected from the group consisting of alkyl, substituted alkyl, , alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, oxy, do, N—amido, nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonamido, N—sulfonaniido, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted cycloalkyl, and optionally substituted heterocyclyl; each R2, R3, R4, R5, and R9 is independently ed from the group consisting of H, alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, C-carboxy, O-carboxy, C-aniido, N—amido, nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonaniido, N—sulfonaniido, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted heterocyclyl; each R6, Rloa, R10b and R100 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, alkynyl, aminoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfonyl ide, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally tuted heterocyclyl; each R7 and R8 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, kyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, halo, cyano, hydroxy, hydroxyalkyl, alkyl, C-carboxy, O-carboxy, C-aniido, N—aniido, nitro, sulfonyl, sulfo, sulfino, sulfonate, onaniido, N—sulfonaniido, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted aryl and optionally tuted heterocyclyl; alternatively, R6 and R7 together with the atoms to which they are attached form a ring or ring system selected from the group consisting of optionally substituted 5-10 membered heteroaryl or optionally substituted 5-10 membered heterocyclyl; X is selected from the group consisting of O, S, NR", and Se; R11 is selected from the group ting of H, alkyl, substituted alkyl, alkenyl, alkynyl, aminoalkyl, carboxyalkyl, sulfonatoalkyl, haloalkyl, alkyl, alkoxyalkyl, sulfo, ally substituted aryl, optionally substituted aryl, ally substituted carbocyclyl, and optionally substituted heterocyclyl; and the bond represented by a solid and dashed line: is selected from the group consisting of a single bond and a double bond, provided that when : is a double bond, then R3 is absent.

In some embodiments of the compounds of Formula (I), the alkyl or substituted alkyl sed herein is C142 alkyl, or more preferably C1_6 alkyl. In some embodiments, the alkoxy sed herein is C142 alkoxy, or more preferably C1_6 alkoxy. In some ments, the alkenyl and alkynyl groups disclosed herein are C2-6 alkenyl and C26 l. In some embodiments, the haloalkyl, haloalkoxy, aminoalkyl, yalkyl, heteroalkyl groups sed herein are C142 kyl, C142 haloalkoxy, C142 aminoalkyl, C142 hydroxyalkyl and C142 heteroalkyl; more preferably C1_6 haloalkyl, C1_6 haloalkoxy, C1_6 aminoalkyl, C1_6 hydroxyalkyl and C1_6 heteroalkyl.

In some embodiments, the alkoxyalkyl group sed herein is C1-6 alkoxy(C1_6 alkyl). In some embodiments, the optionally tuted aryl disclosed herein is optionally substituted C6—10 aryl, for example, phenyl. In some embodiments, the optionally substituted heteroaryl disclosed herein is optionally substituted 5-10 membered heteroaryl; more preferably, optionally substituted 5-6 membered heteroaryl. In some embodiments, the optionally substituted carbocyclyl disclosed herein is optionally substituted 3-7 membered carbocyclyl, in particular 3-7 membered cycloalkyl.

In some embodiments, optionally substituted heterocyclyl sed herein are optionally substituted 3-7 membered heterocyclyl, more preferably 5-6 membered heterocyclyl.

In some embodiments of the compounds of Formula (I), any of R2 through R9 may be selected from an alkyl substituted with one or more substituents selected from carboxyl (- CO2H) or carboxylate (CO2_), sulfo (SO3H) or sulfonate (803—) groups. In some such embodiments, the compounds of Formula (I) are also represented by its salt form a (I’) with an organic or inorganic cation: (I’), wherein K is an organic or inorganic cation, and n is an integer selected from 1 to 20.

In some embodiments of the compounds of a (I) or (I’), R1 is . In some such embodiments, X is O. In some other embodiments, X is O. In some such embodiments, the compounds of Formula (I) are also represented by Formula (Ia): R6 R7 (Ia). In some embod1ments, R1. . . . 1s substituted With one or more substituents selected from the group consisting of alkyl, halo, and C-carboxy. In one ment, R1 is substituted with a chloro (i.e., -Cl). In another embodiment, R1 is substituted with a carboxyl (i.e., -C(O)OH).

In some embodiments of the compounds of a (I) or (I’), R1 is / R . In some other embodiments, R1 is . It is understood that when Rloa, R10b or R100 is connected to a pyridyl group bearing a positive charge on the nitrogen atom, Rloa, R10b or R100 may contain a negative charge so that R1 as a whole is charge neutral. Alternatively, when Rloa, R10b or R100 is connected to a pyridyl group bearing a positive charge on the nitrogen atom, the compound described herein may contain a rion so that the compound as a whole is charge neutral. In some such embodiments, Rloa, R10b or R100 is a substituted alkyl, for e, substituted C1, C2, C3, C4, C5, or C6 alkyl. In some such embodiment, the alkyl is substituted with carboxyl (-C02H), ylate (C02_), sulfo (SO3H), or sulfonate (803—). In some such embodiments, the compounds of Formula (I) are also represented by Formula (Ib) and (lo) or their salt Formula (Ib’) and (Ic’): (10) wherein Y is an anion that is capable of forming a charge neutral compound with Ib. In some embodiment, Y is an anion derived from c or inorganic acid. In some embodiments, Y is a halogen anion.

In some embodiments of the compounds of Formula (I), (I’), (Ia), (Ib), (Ib’), (Ic), or (Ic’), the bond represented by a solid and dashed line : is a double bond and the compounds are also presented by Formula (I-l), (I’-l), (Ia-l), , (Ib’-l), , or (Ic’-l): R2 R8 R9 R9 R8 R2 (Ic’-l).

In some embodiments of the compounds of Formula (I), (I-l), (I’-l), (Ia-l), (Ib- l), (Ib’-l), (Ic-l), or (Ic’-l), R2 is alkyl. In one embodiment, R2 is methyl. In some other ments, R2 is H.

In some embodiments of the compounds of a (I), (I-l), (I’-l), (Ia-l), (Ib- l), (Ib’-l), (Ic-l), or (Ic’-l), at least one of R4 and R5 is alkyl. In some such embodiments, each R4 and R5 is alkyl. In one embodiment, both R4 and R5 are methyl. In some alternative embodiments, at least one of R4 and R5 is H. In one such embodiment, both R4 and R5 are H.

In some embodiments of the compounds of Formula (I), (I’), (Ia), (lb) or (Ib’), the bond ented by a solid and dashed line : is a single bond and the compounds are also presented by Formula (I-2), (I’-2), (Ia-2), (Ib-2), (Ib’-2), (Ic-2), or (Ic’-2): R2 R3 R8 R9 R9 R8 R2 R3 R 1 R1 \ / R4 R5 [i] O O O O R5 1}] R4 R6 R7 R _ K+ (1-2), (CH2)nCOZ (122), In some embodiments of the compounds of Formula (I), (I-2), (I’-2), (Ia-2), (Ib- 2), (Ib’-2), (Io-2), or (Ic’-2), at least one of R2 and R3 is alkyl. In some further embodiments, both R2 and R3 are alkyl. In one embodiment, both R2 and R3 are methyl. In some other embodiments, at least one of R2 and R3 is H. In one embodiment, both R2 and R3 are H.

In some ments of the compounds ofFormula (I), (I-2), (I’-2), , (Ib- 2), (Ib’-2), (Io-2), or (Ic’-2), at least one of R4 and R5 is H. In one such ment, both R4 and R5 are H. In some alternative embodiments, at least one of R4 and R5 is alkyl. In some such embodiments, each R4 and R5 is alkyl. In one embodiment, both R4 and R5 are methyl.

In some embodiments of the compounds of Formula (I), (1’), (1-1), (I’-l), (Ia-l), (Ib-l), (Ib’-l), (Ic-l), (Ic’-l), (I-2), (I’-2), (Ia-2), (Ib-2), (Ib’-2), (Io-2), or ), R6 is a substituted alkyl, for example, substituted C1, C2, C3, C4, C5, or C6 alkyl. In one embodiment, R6 is alkyl tuted with carboxyl. In some ments, R6 is an alkyl substituted with —C(O)OR12, and wherein R12 is selected from the group consisting of optionally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl, and optionally substituted 3 to 7 membered cycloalkyl. In one such embodiment, R12 is an alkyl, for example, methyl, ethyl, or t-butyl. In some further embodiments, R6 is an alkyl substituted with R13R14, and wherein each R13 and R14 is independently selected from H, ally substituted alkyl, optionally substituted aryl, optionally tuted heteroaryl and optionally substituted 3 to 7 membered cycloalkyl. In some further embodiments, R13 and R14 is independently selected from an alkyl substituted with one or more substituents selected from the group consisting of carboxyl, carboxylate, —C(O)OR", sulfo and sulfonate.

In some embodiments of the compounds of Formula (I), (1’), (1-1), (I’-l), (Ia-l), , (Ib’-l), (Ic-l), (Ic’-1), (I-2), , (Ia-2), (Ib-2), (Ib’-2), (Ic-2), or (Ic’-2), R7 is H.

In some alternative embodiments of the compounds of Formula (I), (1’), (1-1), (I’-l), , (Ib-l), (Ib’-l), (I-2), (I’-2), (Ia-2), (Ib-2), or (Ib’-2), R6 and R7 are joined together with the atoms to which they are attached to form an optionally substituted 3 to 10 ed heterocyclyl, for example, an optionally substituted 6 membered cyclyl. In some such embodiments, the optionally substituted heterocyclyl contains one heteroatom. In some such embodiments, the optionally substituted heterocyclyl is substituted with one or more alkyls, for example, methyl.

In some embodiments of the compounds of Formula (I), (1’), (1-1), (I’-l), (Ia-l), (Ib-l), (Ib’-l), (Ic-l), (Ic’-1), (I-2), (I’-2), (Ia-2), (Ib-2), (Ib’-2), (Ic-2), or (Ic’-2), R8 is H.

In some embodiments of the nds of a (I), (1’), (1-1), (I’-l), (Ia-l), (Ib-l), ), , (Ic’-1), (I-2), (I’-2), (Ia-2), (Ib-2), (Ib’-2), (Ic-2), or (Ic’-2), R9 is H.

In some c embodiments, exemplary compounds of Formula (I) include Compounds I-1 through 1-20 and Compounds 1-22 through 1-32 as shown in Table 1 below: Table 1.

SQ oQ \ \N N N o 0 [\II | (CH2)3002H (CH2)3COOC(CH3)3 1'2 so 1'12 +-(CH2)5002H \ \ \\ N O N o 0 [\IJ O (CH2)3COOH 1'3 so 1'13 +-(CH2)5002H \ \ // \\ N N O O N O 0 (CH2)3COOC(CH3)3 -1]- 1'4 1'14 \N+«(CH2)5COZH 1'5 1'15 \N+-(CH2)4sos' N O O [T] O O (CH2>3cooc3 (CH2)3COZC(CH3)3 1'6 Q 1-16 \ +-3002H \(CH2)3803H N\ O (CH2)3SO3H 1-22 Q 1-23 0 O/é > N O O N O O (C':H2>3cozH (CH2)3COOH WO 14710 1'24 1+0ch0 1'25 C|—< 2&0 o o N (CH2)3COOH 1-26 \ 1-27 \ I I / Ni N+ \ (CH2)4303_ \ 4sos' [T] O O [i] O O C02H (CH2)3C02C(CH3)3 1-28 \ N+. 803' 1—29 \ N+«(CH2)4SO3' [\II 0 O I}! O O (CH2)3COZH (CH2)3COZH 1-30 \ I-3l \ I I /N+ /N+ \ 3303' \ \(CH2)4303' I}! O O [T] O O (CH2)3C02H (CH2)3COZH 1-32 \ \ \(CH2)4303' [T] O O (CH2)3COZH In some embodiments of the compounds of Formula (I), the compound is covalently attached to a nucleotide or oligonucleotide Via R6, and wherein R6 is a tuted alkyl, for example, a substituted C1, C2, C3, C4, C5, or C6 alkyl. In one embodiment, R6 is an alkyl substituted with carboxyl.

In some alternative embodiments, the compound is covalently attached to a nucleotide or oligonucleotide via R8, and wherein R8 is a substituted alkyl, for example, a substituted C1, C2, C3, C4, C5, or C6 alkyl. In one embodiment, R8 is an alkyl substituted with carboxyl.

In some alternative embodiments, the compound is covalently attached to a nucleotide or oligonucleotide via Rloa, RIOb, or R10", and wherein each of Rloa, RIOb, or R100 is a substituted alkyl, for example, a substituted C1, C2, C3, C4, C5, or C6 alkyl. In one embodiment, each of Rloa, RIOb, or R100 is an alkyl substituted with carboxyl.

In some ments, the structure of compound of Formula (I) is represented in one or more mesomeric forms: R3 R8 R9 R2 R3 R8 R9 Compounds of Formula (II) Some embodiments described herein are related to fluorescent compounds of Formula (II) with a Stokes shift at least about 60 nm, or salts, mesomeric forms thereof: (11) wherein RHat is a 5 to 10 membered heteroaryl ally substituted with one or more R10; each R1, R2, R3, R4, and R5 is independently selected from the group consisting of H, alkyl, tuted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, aminoalkyl, halo, cyano, y, hydroxyalkyl, heteroalkyl, oxy, O-carboxy, C-amido, N—amido, nitro, sulfonyl, sulfo, sulfino, sulfonate, S-sulfonamido, N—sulfonamido, optionally substituted carbocyclyl, optionally tuted aryl, optionally tuted aryl and optionally substituted heterocyclyl; R6 is selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, alkynyl, aminoalkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfonyl hydroxide, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl; each R7 and R8 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, alkoxyalkyl, amino, lkyl, halo, cyano, hydroxy, hydroxyalkyl, heteroalkyl, oxy, O-carboxy, C-amido, N—amido, nitro, sulfonyl, sulfo, sulfino, sulfonate, onamido, N—sulfonamido, optionally substituted carbocyclyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted heterocyclyl; alternatively, R6 and R7 together with the atoms to which they are attached form a ring or ring system selected from the group consisting of ally substituted 5-10 membered heteroaryl or optionally substituted 5-10 membered heterocyclyl; each R10 is independently selected from the group consisting of alkyl, substituted alkyl, alkenyl, alkynyl, lkyl, haloalkyl, heteroalkyl, alkoxyalkyl, sulfonyl hydroxide, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted carbocyclyl, and optionally substituted heterocyclyl; the bond represented by a solid and dashed line: is selected from the group consisting of a single bond and a double bond, ed that when : is a double bond, then R3 is absent.

In some ments, the fluorescent compounds of Formula (II) have a Stokes shift ranging from about 60 nm to about 100 nm, or from about 60 nm to about 90 nm.

In some embodiments of the compounds of a (II), the alkyl or substituted alkyl disclosed herein is C142 alkyl, or more preferably C1_6 alkyl. In some embodiments, the alkoxy disclosed herein is C142 alkoxy, or more preferably C1_6 alkoxy. In some embodiments, the alkenyl and alkynyl groups disclosed herein are C2-6 alkenyl and C26 alkynyl. In some embodiments, the haloalkyl, haloalkoxy, aminoalkyl, hydroxyalkyl, heteroalkyl groups disclosed herein are C142 haloalkyl, C142 haloalkoxy, C142 aminoalkyl, C142 hydroxyalkyl and C142 heteroalkyl; more preferably C1_6 haloalkyl, C1_6 haloalkoxy, C1_6 aminoalkyl, C1_6 hydroxyalkyl and C1_6 heteroalkyl.

In some embodiments, the alkoxyalkyl group disclosed herein is C1_6 alkoxy(C1_6 alkyl). In some embodiments, the optionally substituted aryl disclosed herein is optionally substituted C6—10 aryl, for example, phenyl. In some embodiments, the ally substituted heteroaryl disclosed herein is optionally substituted 5-10 ed heteroaryl; more preferably, optionally substituted 5-6 membered aryl. In some embodiments, the optionally substituted yclyl disclosed herein is optionally tuted 3-7 membered carbocyclyl, in particular 3-7 membered cycloalkyl.

In some embodiments, optionally substituted heterocyclyl disclosed herein are ally substituted 3-7 membered cyclyl, more preferably 5-6 membered heterocyclyl.

In some embodiments of the compounds of Formula (II), any of R1 h R8 may be selected from an alkyl substituted with one or more substituents selected from carboxyl (- CO2H) or ylate (CO2_), sulfo (SO3H) or sulfonate (803—) groups. In some such embodiments, the substituted alkyl is a substituted C1, C2, C3, C4, C5, or C6 alkyl. In some such embodiments, the compounds of Formula (II) are also ented by its salt form Formula (II’) with an organic or inorganic cation: (11’), n K is an organic or inorganic cation, and n is an integer selected from 1 to 20.

In some embodiments of the compounds of Formula (II) or (II’), RHat is a 9 membered heteroaryl optionally substituted with one or more R10. In some such ments, RHat is selected from optionally substituted benzothiazolyl or benzoxazolyl, for example, 2- benzothiazolyl or 2-benzoxazolyl . In some other embodiments, RHat is a 6 membered heteroaryl ally substituted with one or more R10. In one such embodiment, RHat is an substituted pyridyl, _ fl 3‘0 for exam 1 4— id 1 ith thesrucue.t t r - _§@N_Rm \Rm Flew pe, pyr y w or It is , . understood that when R10 is connected to a pyridyl group bearing a ve charge on the nitrogen atom, R10 may contain a negative charge so that RHat as a whole is charge neutral. Alternatively, when R10 is ted to a l group bearing a ve charge on the nitrogen atom, the compound bed herein may contain a counterion so that the compound as a whole is charge neutral. In some such embodiments, R10 is a substituted alkyl, for example, substituted C1, C2, C3, C4, C5, or C6 alkyl. In some such embodiment, R10 is substituted with carboxyl (-COzH), carboxylate (C02_), sulfo (SOgH), or sulfonate (803—).

In some embodiments of the compounds of Formula (II) or (II’), the bond represented by a solid and dashed line : in Formula (II) is a double bond and the compounds are also represented by Formula (11-1) or (II’-l): R2 R8 R1 R1 R8 R2 "WeRHet/ \ RHet/ \ 0 O N R55 R6 R7 R7 (11—1), CO2' K+(1121).

In some embodiments of the compounds of Formula (II), (11’), (11-1) or (II’-l), R2 is alkyl. In one embodiment, R2 is methyl. In some other embodiments, R2 is H.

In some embodiments of the compounds of Formula (II), (11’), (11-1) or (II’-l), at least one of R4 and R5 is alkyl. In some such embodiments, each R4 and R5 is alkyl. In one embodiment, both R4 and R5 are methyl. In some alternative embodiments, at least one of R4 and R5 is H. In one such embodiment, both R4 and R5 are H.

In some embodiments of the compounds of Formula (11) or (11’), the bond represented by a solid and dashed line: is a single bond and the compounds are also presented by Formula (11-2) or ): R2 R3 R8 R1 R1 R8 R3 R2 RHet RHet \ / R4 R5 N C) C) (D C) |R5 $ |Q4 R6 R7 R _ W (11-2), (CH2)n002 (1122).

In some embodiments of the compounds of Formula (11), (11’), (11-2) or (11’-2), at least one of R2 and R3 is alkyl. In some further embodiments, both R2 and R3 are alkyl. In one embodiment, both R2 and R3 are methyl. In some other embodiments, at least one of R2 and R3 is H. In one embodiment, both R2 and R3 are H.

In some embodiments of the compounds of Formula (11), (11’), (11-2) or (11’-2), at least one of R4 and R5 is H. In one such embodiment, both R4 and R5 are H. In some alternative embodiments, at least one of R4 and R5 is alkyl. In some such embodiments, each R4 and R5 is alkyl. In one embodiment, both R4 and R5 are methyl.

In some embodiments ofthe nds ofFormula (11), (11’), (11-l), (11’-l), (11- 2) or (11’-2), R6 is a substituted alkyl, for example, substituted C1, C2, C3, C4, C5, or C6 alkyl. In one embodiment, R6 is alkyl substituted with carboxyl. In some embodiments, R6 is an alkyl substituted with —C(O)OR12, and wherein R12 is selected from the group consisting of optionally substituted alkyl, ally substituted aryl, optionally substituted heteroaryl, and optionally substituted 3 to 7 membered cycloalkyl. In one such embodiment, R12 is an alkyl, for example, methyl, ethyl, or t-butyl. In some further embodiments, R6 is an alkyl substituted with — C(O)NR13R14, and wherein each R13 and R14 is independently ed from H, ally substituted alkyl, optionally substituted aryl, optionally substituted heteroaryl and optionally substituted 3 to 7 membered cycloalkyl. In some fithher embodiments, R13 and R14 is independently ed from an alkyl substituted with one or more substituents selected from the group consisting of carboxyl, carboxylate, —C(O)OR", sulfo and sulfonate.

In some ments ofthe compounds of Formula (11), (11’), (11-l), (11’-l), (11- 2) or (11’-2), R7 is H.

In some alternative ments of the nds of Formula (11), (11’), , (11’-l), (11-2) or (11’-2), R6 and R7 are joined together with the atoms to which they are attached to form an optionally substituted 3 to 10 membered heterocyclyl, for example, an optionally substituted 6 membered heterocyclyl. In some such embodiments, the optionally substituted heterocyclyl contains one atom. In some such embodiments, the optionally substituted heterocyclyl is tuted with one or more alkyl, for example, methyl.

In some embodiments ofthe compounds of Formula (11), (11’), (11-l), ), (11- 2) or (11’-2), R1 is H.

In some embodiments ofthe compounds of Formula (11), (11’), (11-l), (11’-l), (11- 2) or (11’-2), R8 is H.

As tood by one of ordinary skill in the art, when a nd of Formula (1) or (11) contains positively or vely d substituent groups, it may also contains a negatively or positively charged counterion such that the compound as a whole is neutral.

Definition The section headings used herein are for zational purposes only and are not to be construed as limiting the subject matter described.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art. The use of the term "including" as well as other forms, such as "include", "includes," and "included," is not limiting.

The use of the term "having" as well as other forms, such as "have", "has," and "had," is not limiting. As used in this specification, whether in a transitional phrase or in the body of the claim, the terms "comprise(s)" and "comprising" are to be interpreted as having an open-ended meaning.

That is, the above terms are to be interpreted synonymously with the phrases "having at least" or "including at least." For example, when used in the context of a s, the term "comprising" means that the process includes at least the recited steps, but may include additional steps. When used in the context of a compound, composition, or device, the term "comprising" means that the compound, composition, or device includes at least the recited features or components, but may also include additional es or components.

As used , common organic abbreviations are defined as follows: Ac Acetyl AC2O Acetic anhydride aq. Aqueous BOC or Boc tert-Butoxycarbonyl BOP (Benzotriazo l- l -yloxy)tris(dimethylamino)phosphonium hexafluorophosphate cat. Catalytic oC Temperature in degrees Centigrade dATP Deoxyadenosine triphosphate dCTP ytidine triphosphate dGTP Deoxyguanosine triphosphate dTTP Deoxythymidine triphosphate ddNTP(s) Dideoxynucleotide(s) DCM Methylene chloride DMA Dimethylacetamide DMF Dimethylformamide Et Ethyl EtOAc Ethyl acetate ffC Fully fianctionalized Nucleotide Conjugate g ) h or hr Hour(s) IPA Isopropyl Alcohol LCMS Liquid chromatography-mass spectrometry MeCN Acetonitrile mL Milliliter(s) PG Protecting group Ph Phenyl ppt Precipitate PyBOP (Benzotriazo l- l -yloxy)tripyrrolidinophosphonium hexafluorophosphate RT, rt Room temperature SBS Sequencing by Synthesis TEA ylamine TEAB Tetraethylammonium bromide TFA roacetic acid TRIS Tris(hydroxymethyl)aminomethane Tert, t tertiary THF Tetrahydrofilran TLC Thin Layer Chromatography TSTU O-(N—Succinimidyl)-N,N,N’,N’-tetramethyluronium tetrafluoroborate uL Microliter(s) As used herein, the term "covalently attached" or "covalently bonded" refers to the g of a chemical g that is characterized by the sharing of pairs of electrons between atoms. For example, a covalently attached polymer g refers to a polymer coating that forms chemical bonds with a fimctionalized surface of a substrate, as compared to attachment to the 2017/083128 surface Via other means, for example, adhesion or electrostatic interaction. It will be appreciated that polymers that are ed covalently to a surface can also be bonded Via means in addition to covalent attachment.

The term "halogen" or "halo," as used herein, means any one of the stable atoms ofcolumn 7 ofthe Periodic Table ofthe Elements, 6.g. or iodine, , e, chlorine, bromine, with fluorine and chlorine being preferred.

As used herein, "alkyl" refers to a straight or branched hydrocarbon chain that is fiJlly saturated (i.e., contains no double or triple bonds). The alkyl group may have 1 to 20 carbon atoms ver it appears herein, a numerical range such as "l to 20" refers to each integer in the given range; 6.g. , "l to 20 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, eta, up to and including 20 carbon atoms, although the present definition also covers the occurrence of the term "alkyl" where no numerical range is designated).

The alkyl group may also be a medium size alkyl having 1 to 9 carbon atoms. The alkyl group could also be a lower alkyl having 1 to 6 carbon atoms. The alkyl group may be designated as "C1- 4 alkyl" or similar designations. By way of example only, "C1_6 alkyl" indicates that there are one to six carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like.

As used herein, "alkoxy" refers to the formula —OR wherein R is an alkyl as is defined above, such as "C1-9 alkoxy", including but not limited to y, , n-propoxy, l- methylethoxy opoxy), n-butoxy, iso-butoxy, sec-butoxy, and tert-butoxy, and the like.

As used herein, "alkenyl" refers to a ht or branched hydrocarbon chain containing one or more double bonds. The alkenyl group may have 2 to 20 carbon atoms, although the present definition also covers the occurrence of the term "alkenyl" where no numerical range is designated. The alkenyl group may also be a medium size alkenyl haVing 2 to 9 carbon atoms.

The alkenyl group could also be a lower l haVing 2 to 6 carbon atoms. The l group may be designated as "C26 alkenyl" or similar designations. By way of example only, "C26 alkenyl" indicates that there are two to six carbon atoms in the alkenyl chain, i.e., the alkenyl chain is selected from the group consisting of ethenyl, —l-yl, propen—2-yl, propen—3-yl, buten—l- yl, butenyl, buten—3-yl, buten—4-yl, l-methyl-propen-l-yl, 2-methyl-propen-l-yl, l-ethyl-ethen- l-yl, 2-methyl-propenyl, buta-l,3-dienyl, buta-l,2,—dienyl, and ,2-dienyl. Typical alkenyl groups include, but are in no way limited to, ethenyl, propenyl, butenyl, yl, and hexenyl, and the like.

As used herein, "alkynyl" refers to a straight or branched arbon chain containing one or more triple bonds. The alkynyl group may have 2 to 20 carbon atoms, although the present definition also covers the occurrence of the term "alkynyl" where no numerical range is designated. The alkynyl group may also be a medium size alkynyl haVing 2 to 9 carbon atoms.

The alkynyl group could also be a lower alkynyl haVing 2 to 6 carbon atoms. The alkynyl group may be designated as "C2-6 alkynyl" or similar designations. By way of example only, "C2_6 alkynyl" indicates that there are two to six carbon atoms in the alkynyl chain, i.e., the alkynyl chain is selected from the group ting of ethynyl, propyn-l-yl, propynyl, butyn-l-yl, butynyl, butynyl, and nyl. Typical alkynyl groups include, but are in no way limited to, ethynyl, propynyl, butynyl, pentynyl, and hexynyl, and the like.

As used herein, "heteroalkyl" refers to a ht or branched hydrocarbon chain containing one or more atoms, that is, an element other than carbon, including but not limited to, nitrogen, oxygen and sulfur, in the chain backbone. The heteroalkyl group may have 1 to 20 carbon atom, although the t definition also covers the occurrence of the term "heteroalkyl" where no cal range is designated. The heteroalkyl group may also be a medium size heteroalkyl having 1 to 9 carbon atoms. The heteroalkyl group could also be a lower heteroalkyl having 1 to 6 carbon atoms. The heteroalkyl group may be designated as "C1-6 heteroalkyl" or similar designations. The alkyl group may contain one or more heteroatoms. By way of example only, "C1_6 heteroalkyl" indicates that there are one to six carbon atoms in the heteroalkyl chain and additionally one or more heteroatoms in the backbone of the chain.

The term "aromatic" refers to a ring or ring system haVing a conjugated pi electron system and includes both carbocyclic ic (e.g., phenyl) and heterocyclic aromatic groups (e.g., pyridine). The term includes clic or fiJsed-ring polycyclic (i.e., rings which share adjacent pairs of atoms) groups provided that the entire ring system is aromatic.

As used herein, "aryl" refers to an ic ring or ring system (i.e., two or more fiased rings that share two adjacent carbon atoms) containing only carbon in the ring backbone.

When the aryl is a ring system, every ring in the system is aromatic. The aryl group may have 6 to 18 carbon atoms, gh the present definition also covers the occurrence ofthe term "aryl" where no numerical range is designated. In some embodiments, the aryl group has 6 to 10 carbon atoms.

The aryl group may be designated as "C640 aryl," "C6 or C10 aryl," or similar designations.

Examples ofaryl groups include, but are not d to, phenyl, yl, azulenyl, and anthracenyl.

An "aralkyl" or "arylalkyl" is an aryl group connected, as a substituent, Via an alkylene group, such as "C744 aralkyl" and the like, including but not limited to benzyl, 2- phenylethyl, 3-phenylpropyl, and naphthylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., a C1-6 alkylene group). -2]- As used herein, "heteroaryl" refers to an aromatic ring or ring system (i.e., two or more fiJsed rings that share two adjacent atoms) that n(s) one or more heteroatoms, that is, an element other than carbon, including but not d to, nitrogen, oxygen and sulfur, in the ring backbone. When the heteroaryl is a ring system, every ring in the system is aromatic. The aryl group may have 5-18 ring members (i.e., the number of atoms making up the ring backbone, including carbon atoms and heteroatoms), although the present ion also covers the occurrence of the term "heteroaryl" where no numerical range is designated. In some embodiments, the heteroaryl group has 5 to 10 ring members or 5 to 7 ring members. The aryl group may be designated as "5-7 membered heteroaryl," "5-10 ed heteroaryl," or similar designations. Examples of aryl rings e, but are not limited to, filryl, thienyl, azinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolinyl, isoquinlinyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothienyl.

A "heteroaralkyl" or oarylalkyl" is heteroaryl group connected, as a tuent, Via an alkylene group. Examples include but are not limited to 2-thienylmethyl, 3- thienylmethyl, furylmethyl, thienylethyl, pyrrolylalkyl, pyridylalkyl, isoxazollylalkyl, and imidazolylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., a C16 alkylene group).

As used herein, "carbocyclyl" means a non-aromatic cyclic ring or ring system containing only carbon atoms in the ring system backbone. When the carbocyclyl is a ring system, two or more rings may be joined together in a fused, bridged or spiro-connected fashion. yclyls may have any degree of saturation provided that at least one ring in a ring system is not aromatic. Thus, carbocyclyls include cycloalkyls, cycloalkenyls, and cycloalkynyls. The carbocyclyl group may have 3 to 20 carbon atoms, although the present definition also covers the occurrence of the term "carbocyclyl" where no numerical range is ated. The carbocyclyl group may also be a medium size carbocyclyl having 3 to 10 carbon atoms. The carbocyclyl group could also be a carbocyclyl haVing 3 to 6 carbon atoms. The carbocyclyl group may be designated as "C3—6 carbocyclyl" or similar designations. Examples of carbocyclyl rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydro-indene, bicycle[2.2.2]octanyl, adamantyl, and spiro[4.4]nonanyl.

As used herein, "cycloalkyl" means a fially ted carbocyclyl ring or ring system. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

As used herein, "heterocyclyl" means a non-aromatic cyclic ring or ring system containing at least one heteroatom in the ring ne. Heterocyclyls may be joined together in a filSGd, bridged or spiro-connected fashion. Heterocyclyls may have any degree of saturation provided that at least one ring in the ring system is not aromatic. The atom(s) may be present in either a non-aromatic or aromatic ring in the ring system. The heterocyclyl group may have 3 to ring members (i.e., the number of atoms making up the ring backbone, including carbon atoms and heteroatoms), although the present definition also covers the occurrence of the term "heterocyclyl" where no cal range is designated. The heterocyclyl group may also be a medium size heterocyclyl having 3 to 10 ring members. The heterocyclyl group could also be a heterocyclyl having 3 to 6 ring members. The heterocyclyl group may be designated as "3-6 membered heterocyclyl" or similar designations. In preferred six membered monocyclic heterocyclyls, the heteroatom(s) are selected from one up to three of O, N or S, and in preferred five membered monocyclic heterocyclyls, the heteroatom(s) are selected from one or two heteroatoms selected from O, N, or S. Examples of heterocyclyl rings include, but are not limited to, azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidinyl, pyrrolidonyl, pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, l,3-dioxinyl, oxanyl, l,4-dioxinyl, oxanyl, l,3-oxathianyl, l,4-oxathiinyl, l,4-oxathianyl, 2H—l,2— oxazinyl, trioxanyl, hexahydro-l,3,5-triazinyl, l,3-dioxolyl, l,3-dioxolanyl, l,3-dithiolyl, l,3- dithiolanyl, isoxazolinyl, isoxazolidinyl, oxazolinyl, oxazolidinyl, idinonyl, thiazolinyl, thiazolidinyl, l,3-oxathiolanyl, indolinyl, isoindolinyl, tetrahydrofiaranyl, ydropyranyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydro - l ,4-thiazinyl, thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, and tetrahydroquino line.

An "O-carboxy" group refers to a O)R" group in which R is selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2—6 alkynyl, C3-7 carbocyclyl, C6—10 aryl, 5-10 ed heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

A "C-carboxy" group refers to a "-C(=O)OR" group in which R is selected from en, C1-6 alkyl, C2-6 alkenyl, C2—6 alkynyl, C3-7 carbocyclyl, C6—10 aryl, 5-10 membered heteroaryl, and 3-10 membered cyclyl, as defined herein. A non-limiting example es carboxyl (i.e., -C(=O)OH).

A "cyano" group refers to a "-CN" group.

A "sulfonyl" group refers to an "-SOzR" group in which R is selected from hydrogen, C1-6 alkyl, C2-6 alkenyl, C2—6 l, C3-7 carbocyclyl, C6—10 aryl, 5-10 membered heteroaryl, and 3-10 membered cyclyl, as defined herein.

A "sulfo" or "sulfonyl ide" group refers to a "-S(=O)2-OH" group.

A "sulfmo" group refers to a "-S(=O)OH" group.

A "sulfonate" group refers to -SO;.

An "S-sulfonamido" group refers to a "-SOzNRARB" group in which RA and R13 are each ndently selected from hydrogen, C1_6 alkyl, C26 alkenyl, C26 alkynyl, C3_7 carbocyclyl, C6-1o aryl, 5-10 ed heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

An "N-sulfonamido" group refers to a "-N(RA)S02RB" group in which RA and R1, are each independently ed from hydrogen, C1_6 alkyl, C2_6 alkenyl, C2_6 alkynyl, C3_7 carbocyclyl, C6-1o aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

A "C-amido" group refers to a )NRARB" group in which RA and R13 are each independently selected from hydrogen, C1_6 alkyl, C26 alkenyl, C2-6 alkynyl, C3-7 carbocyclyl, C6-1o aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein.

An "N-amido" group refers to a "-N(RA)C(=O)RB" group in which RA and R13 are each independently selected from hydrogen, C1_6 alkyl, C26 alkenyl, C26 alkynyl, C3_7 carbocyclyl, C6-1o aryl, 5-10 membered aryl, and 3-10 membered heterocyclyl, as defined herein.

An "amino" group refers to a "-NRARB" group in which RA and R13 are each independently ed from hydrogen, C1_6 alkyl, C26 alkenyl, C26 alkynyl, C3_7 carbocyclyl, C6- aryl, 5-10 membered heteroaryl, and 3-10 membered heterocyclyl, as defined herein. A non- limiting example includes free amino (i.e., -NH2).

An "aminoalkyl" group refers to an amino group connected Via an ne group.

An "alkoxyalkyl" group refers to an alkoxy group connected Via an alkylene group, such as a "C24; alkoxyalkyl" and the like.

As used herein, a substituted group is derived from the tituted parent group in which there has been an exchange of one or more hydrogen atoms for another atom or group. Unless otherwise indicated, when a group is deemed to be "substituted," it is meant that the group is tuted with one or more substituents independently ed from C1-C6 alkyl, C1-C6 l, C1-C6 alkynyl, C1-C6 heteroalkyl, C3-C7 carbocyclyl (optionally substituted with halo, C1- C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), C3-C7-carbocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 3-10 membered heterocyclyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 3-10 membered heterocyclyl-C1-C6-alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), aryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), aryl(C1-C6)alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 5-10 membered heteroaryl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), 5-10 membered heteroaryl(C1-C6)alkyl (optionally substituted with halo, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), halo, cyano, hydroxy, C1-C6 , C1-C6 (C1-C6)alkyl (i.e., , aryloxy, sulfhydryl (mercapto), halo(C1-C6)alkyl (e.g., —CF3), halo(C1-C6)alkoxy (e.g., —OCF3), C1-C6 alkylthio, arylthio, amino, amino(C1-C6)alkyl, nitro, amyl, N—carbamyl, O- thiocarbamyl, N—thiocarbamyl, C-amido, N—amido, S-sulfonamido, N—sulfonamido, C-carboxy, O- carboxy, acyl, cyanato, isocyanato, thiocyanato, isothiocyanato, sulfinyl, sulfonyl, sulfo, sulfino, sulfonate, and oxo (=0). Wherever a group is described as "optionally tuted" that group can be substituted with the above substituents.

As understood by one of ordinary skill in the art, if a compound contains positively or negatively charged substituent groups, for example, 803—, it may also ns a negatively or positively charged counterion such that the compound as a whole is neutral.

It is to be understood that certain l naming tions can include either a mono-radical or a di-radical, depending on the context. For example, where a substituent requires two points of ment to the rest of the molecule, it is tood that the substituent is a di- radical. For example, a substituent identified as alkyl that requires two points of attachment includes di—radicals such as —CH2—, —CH2CH2—, —CH2CH(CH3)CH2—, and the like. Other radical naming conventions clearly indicate that the l is a di-radical such as "alkylene" or "alkenylene." When two "adjacent" R groups are said to form a ring "together with the atom to which they are attached," it is meant that the collective unit of the atoms, ening bonds, and the two R groups are the recited ring. For example, when the ing substructure is present: and R1 and R2 are defined as selected from the group consisting of hydrogen and alkyl, or R1 and R2 together with the atoms to which they are attached form an aryl or carbocyclyl, it is meant that R1 and R2 can be selected from hydrogen or alkyl, or alternatively, the substructure has structure: where A is an aryl ring or a carbocyclyl containing the depicted double bond.

Labeled Nucleotides The dye compounds described herein are suitable for attachment to substrate moieties. Substrate es can be virtually any molecule or substance to which the fluorescent dyes described herein can be conjugated and, by way of non-limiting example, may include nucleosides, nucleotides, polynucleotides, carbohydrates, ligands, particles, solid surfaces, organic and inorganic polymers and combinations or assemblages thereof, such as chromosomes, nuclei, living cells and the like. The dyes can be conjugated by an optional linker by a variety of means including hydrophobic attraction, ionic attraction and covalent attachment. Particularly the dyes are conjugated to the substrate by nt attachment. More particularly the covalent attachment is by means of a linker group. In some instances, such labeled nucleotides are also referred to as "modified nucleotides." A particular useful application ofthe new fluorescent dyes with long Stokes shift as described herein is for labeling biomolecules, for example, nucleotides or oligonucleotides.

Some embodiments ofthe present application are directed to a nucleotide or oligonucleotide labeled with the new fluorescent compounds as described herein.

Fluorescent dye molecules with improved cence properties (such as Stokes shift, fluorescence intensity, position of fluorescence maximum and shape of fluorescence band) can improve the speed and accuracy of nucleic acid sequencing. Stokes Shift is a key aspect in the detection of the fluorescence in biological applications. For example, the detection of emitted light can be difficult to distinguish from the excitation light when using fluorophores with absorption and fluorescence max very close to each other (i.e., small Stokes shift), because the excitation and emission wavelengths greatly p. In contrast, fluorophores with large Stokes shifts are easy to distinguish because ofthe greater separation between the tion and emission wavelengths. The Stokes shift is especially critical in multiplex fluorescence applications, because the emission wavelength of one fluorophore may overlap, and therefore excite, another fluorophore in the same sample. In addition, fluorescence signal intensity is especially important when measurements are made in water based biological s and/or at higher temperature as fluorescence of most dyes is significantly lower at such ions. Moreover, the nature of the base to which a dye is attached also affects the cence m, cence intensity and other spectral dye ties. The ce specific interactions between the cent dye and the nucleobase can be tailored by specific design of the cent dyes. Optimization of the structure of the cent dyes can improve their fluorescent properties and also improve the efficiency of nucleotide incorporation, reduce the level of sequencing errors and se the usage ofreagents in, and ore the costs of, nucleic acid sequencing. 2017/083128 The attachment to the biomolecules may be via R6, R8, Rloa, RIOb, R100 or R10 moiety ofthe nd of Formula (I) or Formula (II). In some embodiments, R6, R8, Rloa, RIOb, R100 or R10 is a substituted alkyl, for example alkyl substituted with yl (i.e., -C02H) or an activated form of carboxyl group, for e, amide or ester, which may be used for ment to the amino group of the biomolecules. In one embodiment, R6, R8, Rloa, RIOb, R100 or R10 may contain an activated ester or amide residue most suitable for fiarther peptide bond formation.

The term "activated ester" as used herein, refers to a carboxyl group derivative which is capable of reacting in mild conditions, for example, with a nd containing an amino group. Non- limiting examples of activated esters include but not limited to p-nitrophenyl, pentafluorophenyl and imido esters.

In some embodiments, the dye compounds may be covalently attached to oligonucleotides or nucleotides via the tide base. For example, the labeled nucleotide or oligonucleotide may have the label attached to the C5 position of a pyrimidine base or the C7 position ofa 7-deaza purine base through a linker moiety. The labeled nucleotide or oligonucleotide may also have a 3'-OH blocking group ntly attached to the ribose or deoxyribose sugar of the nucleotide.

Linkers The dye compounds as disclosed herein may include a reactive linker group at one of the substituent positions for covalent attachment of the compound to another molecule.

Reactive linking groups are moieties capable of forming a covalent bond. In a particular embodiment the linker may be a cleavable linker. Use of the term able linker" is not meant to imply that the whole linker is required to be removed. The cleavage site can be located at a position on the linker that ensures that part ofthe linker remains attached to the dye and/or substrate moiety after cleavage. Cleavable linkers may be, by way ofnon-limiting example, electrophilically cleavable linkers, nucleophilically cleavable linkers, photocleavable linkers, cleavable under reductive conditions (for example disulf1de or azide containing linkers), oxidative conditions, cleavable via use of -catch linkers and cleavable by elimination mechanisms. The use of a cleavable linker to attach the dye compound to a substrate moiety ensures that the label can, if ed, be removed after ion, avoiding any interfering signal in downstream steps.

Non-limiting examples of linker groups include those disclosed in PCT Publication No. /018493 (herein incorporated by reference), which connect the bases of nucleotides to labels such as, for e, the new fluorescent compounds described herein. These linkers may be cleaved using water-soluble phosphines or water-soluble transition metal catalysts formed from a transition metal and at least partially water-soluble ligands. In aqueous solution the latter form at least lly water-soluble transition metal complexes. Additional suitable linkers that may be used include those sed in PCT ation No. W02004/018493 and WO 2007/020457 (both of which are herein incorporated by references. It was discovered that by altering, and in particular increasing, the length of the linker between a fluorescent dye (fluorophore) and the guanine base, by introducing a polyethylene glycol spacer group, it is possible to increase the fluorescence ity compared to the same fluorophore attached to the e base through other linkages known in the art. The design of the linkers, and especially their sed length, also allows improvements in the brightness of hores attached to the guanine bases of guanosine nucleotides when incorporated into polynucleotides such as DNA.

Thus, when the dye is for use in any method of analysis which requires detection of a cent dye label attached to a e-containing nucleotide, it is advantageous if the linker comprises a spacer group of formula —((CH2)20)n—, wherein n is an integer between 2 and 50, as described in Nucleosides and tides may be labeled at sites on the sugar or nucleobase.

As understood by one of ordinary skill in the art, a "nucleotide" consists of a nitrogenous base, a sugar, and one or more phosphate groups. In RNA the sugar is ribose and in DNA is a deoxyribose, i.e. a sugar lacking a hydroxyl group that is present in ribose. The nitrogenous base is a derivative of purine or dine. The purines are adenine (A) and guanine (G), and the pyrimidines are cytosine (C) and thymine (T) or in the context of RNA, uracil (U). The C-l atom of deoxyribose is bonded to N-l of a pyrimidine or N-9 of a purine. A nucleotide is also a phosphate ester of a side, with esteriflcation ing on the hydroxyl group attached to the C-3 or C-5 of the sugar. Nucleotides are usually mono, di- or triphosphates.

A "nucleoside" is structurally similar to a nucleotide but is missing the phosphate moieties. An example of a nucleoside analog would be one in which the label is linked to the base and there is no ate group attached to the sugar molecule.

Although the base is usually referred to as a purine or pyrimidine, the skilled person will appreciate that derivatives and analogues are ble which do not alter the capability of the nucleotide or nucleoside to undergo Watson-Crick base pairing. "Derivative" or "analogue" means a compound or molecule whose core structure is the same as, or closely resembles that of a parent compound but which has a chemical or physical modification, such as, for example, a different or additional side group, which allows the derivative nucleotide or nucleoside to be linked to another molecule. For example, the base may be a deazapurine. The derivatives should be capable of undergoing Watson-Crick pairing. "Derivative" and "analogue" also mean a tic nucleotide or nucleoside derivative having modified base es and/or modified sugar moieties.

Such derivatives and analogues are discussed in, for example, Scheit, Nucleotide analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90:543-5 84, 1990. tide analogues can also comprise modified odiester linkages ing phosphorothioate, phosphorodithioate, alkyl-phosphonate, phosphoranilidate, phosphoramidate linkages and the like.

The dye may be attached to any position on the tide base, through a linker, provided that Watson-Crick base pairing can still be d out. Particular nucleobase labeling sites include the C5 on of a pyrimidine base or the C7 position of a 7-deaza purine base. As described above a linker group may be used to covalently attach a dye to the nucleoside or tide.

In particular embodiments the labeled nucleoside or nucleotide may be enzymatically incorporable and enzymatically extendable. Accordingly a linker moiety may be of sufficient length to connect the nucleotide to the nd such that the compound does not significantly interfere with the l binding and recognition of the nucleotide by a nucleic acid replication enzyme. Thus, the linker can also comprise a spacer unit. The spacer distances, for example, the nucleotide base fiom a cleavage site or label.

Nucleosides or nucleotides d with the new fluorescent dyes described herein may have the formula: B—L—Dye R!O R! I F where Dye is a dye compound, B is a nucleobase, such as, for e uracil, thymine, cytosine, adenine, guanine and the like and L is an optional linker group which may or may not be present. R' can be H, monophosphate, phate, triphosphate, thiophosphate, a phosphate ester , —0— attached to a reactive phosphorous containing group or —O— protected by a ng group. R" can be H, OH, a phosphoramidite or a 3'-OH blocking group and R'" is H or OH; where R" is phosphoramidite, R' is an acid-cleavable hydroxyl protecting group which allows subsequent monomer coupling under automated synthesis conditions.

In some instances, the blocking group is separate and independent of the dye compound, i.e. not attached to it. Alternatively, the dye may comprise all or part of the 3'-OH blocking group. Thus R" can be a 3'-OH blocking group which may or may not comprise the dye compound. In additional alternative embodiments, there is no blocking group on the 3' carbon of the pentose sugar and the dye (or dye and linker construct) attached to the base, for example, can be of a size or structure sufficient to act as a block to the incorporation of a further nucleotide from a point other than the 3' site. Thus the block can be due to steric hindrance or can be due to a combination of size, charge and structure.

The use of a blocking group allows polymerization to be controlled, such as by stopping extension when a modified nucleotide is incorporated. If the blocking effect is reversible, for example by way of non-limiting example by changing chemical conditions or by removal of a chemical block, extension can be d at certain points and then allowed to continue.

Non-limiting examples of 3'-OH blocking groups include those disclosed in and W02014/139596, which are hereby incorporated by references. For example the blocking group may be ethyl (-CH2N3) or substituted azidomethyl (e. g., F2)N3 or CH(CH2F)N3), or allyl.

In a particular embodiment the linker and blocking group are both present and are separate moieties which are both cleavable under substantially similar ions. Thus deprotection and deblocking processes may be more efficient since only a single treatment will be ed to remove both the dye compound and the blocking group.

The present disclosure also directs to assing polynucleotides incorporating dye compounds described herein. Such polynucleotides may be DNA or RNA comprised respectively of deoxyribonucleotides or cleotides joined in phosphodiester linkage. Polynucleotides may comprise naturally occurring nucleotides, non-naturally occurring (or modified) tides other than the labeled nucleotides bed herein or any combination thereof, provided that at least one nucleotide labeled with a dye compound, according to the present ation is present. Polynucleotides may also include non-natural backbone es and/or non- nucleotide chemical modifications. Chimeric structures comprised of mixtures of ribonucleotides and deoxyribonucleotides comprising at least one labeled nucleotide are also contemplated.

Non-limiting exemplary labeled nucleotides as described herein include: /N NH2 Dye\ » Dye\ N A A R o O Dye\ A N Dye ,/"\ NH / \ 2 H \\ / H \x \ N I \l AA R (j R O o Dye ‘/H\ o \L jr—NH H CE: Dye—L I b0 wherein: L represents a linker and R represents a sugar residue as bed above.

In some embodiments, non-limiting exemplary fluorescent dye conjugates are shown below: HO~C§LQ O HO’R’ HO\P/O Ho’§3 ffA-LN3-Dye N/\/N O (CH2>nDye O N3 // H OékN 0 9H O‘F=o 9H 0 O~ N3\/O \P\’\ 'E) O‘ _ Ho’ oC ffC-LN3-Dye 2017/083128 Kits Some embodiments disclosed herein are kits including nucleosides and/or nucleotides labeled with the new fluorescent dyes described herein. Such kits will generally include at least one nucleotide or nucleoside labeled with a dye together with at least one further component. The r component(s) may be further modified or unmodified nucleotides or nucleosides. For example, nucleotides labeled with dyes may be supplied in ation with unlabeled or native tides, and/or with fluorescently labeled tides or any combination f. Combinations of nucleotides may be provided as separate individual components or as nucleotide mixtures. In some embodiments, the kits comprise one or more tides wherein at least one tide is a nucleotide labeled with a new fluorescent nd described herein. The kits may comprise two or more labeled nucleotides. The nucleotides may be labeled with two or more cent labels. Two or more of the labels may be excited using a single excitation source, which may be a laser.

The kits may contain four nucleotides, where the first of four nucleotides is labeled with a compound as disclosed herein, and the second, third, and fourth nucleotides are each may be labeled with a different compound, wherein each nd has a distinct fluorescence maximum and each of the compounds is distinguishable from the other three compounds. The kits may be such that two or more of the compounds have a similar absorbance maximum but different Stokes shift.

The fluorescent dye compounds, labeled nucleotides or kits described herein may be used in sequencing, expression analysis, hybridization analysis, genetic analysis, RNA analysis or protein binding assays. The use may be on an automated sequencing instrument. The sequencing instrument may contain two lasers ing at different wavelengths.

Where kits comprise a ity, particularly two, more particularly four, nucleotides labeled with a dye compound, the different nucleotides may be labeled with different dye compounds, or one may be dark, with no dye compounds. Where the different nucleotides are labeled with different dye compounds it is a feature of the kits that said dye compounds are spectrally guishable fluorescent dyes. As used herein, the term "spectrally distinguishable fluorescent dyes" refers to fluorescent dyes that emit fluorescent energy at wavelengths that can be distinguished by fluorescent detection equipment (for example, a commercial capillary based DNA sequencing platform) when two or more such dyes are present in one sample. When two nucleotides labeled with fluorescent dye compounds are ed in kit form, the spectrally distinguishable fluorescent dyes can be excited at the same wavelength, such as, for example by the same laser in some embodiments. When four nucleotides labeled with cent dye compounds are supplied in kit form, two of the spectrally distinguishable fluorescent dyes can both be excited at one wavelength and the other two spectrally distinguishable dyes can both be excited at another wavelength in some embodiments. Particular excitation wavelengths are about 460 nm.

In some embodiments, at least one nucleotide may be labelled with a dye which excitable with two lasers with different wavelength.

In one embodiment a kit comprises a nucleotide labeled with a compound described herein and a second nucleotide labeled with a second dye wherein the dyes have a difference in absorbance m ofat least 10 nm, particularly 20 nm to 50 nm. More particularly the two dye compounds have Stokes shifts of between 15-40 nm or between 20-40 nm. As used herein, the term "Stokes shift" is the distance between the peak absorption and peak emission wavelengths.

In a further embodiment said kit further comprises two other nucleotides labeled with fluorescent dyes wherein said dyes are excited by the lasers at about 440 nm to about 560 nm.

In an alternative embodiment, the kits may contain nucleotides where the same base is labeled with two different compounds. A first nucleotide may be labeled with a compound bed herein. A second nucleotide may be labeled with a spectrally ct compound, for example a ‘red’ dye absorbing at r than 600 nm. A third nucleotide may be labeled as a mixture of the fluorescent dye compound described herein and the spectrally distinct compound, and the fourth nucleotide may be ‘dark’ and contain no label. In simple terms therefore the nucleotides 1-4 may be labeled ‘green’, ‘red’, ‘red/green’, and dark. To simplify the mentation fiarther, four nucleotides can be labeled with a two dyes excited with a single laser, and thus the ng of nucleotides l-4 may be ‘green 1’, ‘green 2’ ‘ green n 2’, and dark.

In other embodiments the kits may include a polymerase enzyme capable of catalyzing incorporation of the nucleotides into a polynucleotide. Other components to be included in such kits may include buffers and the like. The nucleotides labeled with the new fluorescent dyes bed herein, and other any tide components including mixtures of different tides, may be provided in the kit in a concentrated form to be diluted prior to use. In such embodiments a suitable dilution buffer may also be ed.

Methods of Sequencing Nucleotides (or nucleosides) comprising a new fluorescent dye described herein may be used in any method of is which requires detection of a fluorescent label attached to a nucleotide or nucleoside, whether on its own or incorporated into or associated with a larger lar structure or conjugate. Some embodiments of the present application are directed to methods of sequencing including: (a) incorporating at least one labeled tide as described herein into a polynucleotide; and (b) detecting the labeled nucleotide(s) incorporated into the polynucleotide by detecting the fluorescent signal fiom the new fluorescent dye attached to said modified nucleotide(s).

In some embodiments, at least one labeled nucleotide is incorporated into a polynucleotide in the synthetic step by the action of a rase enzyme. However, other methods ofincorporating labeled nucleotides to cleotides, such as chemical oligonucleotide synthesis or ligation of labeled oligonucleotides to unlabeled oligonucleotides, are not excluded. Therefore, the term porating" a tide into a polynucleotide encompasses polynucleotide synthesis by chemical methods as well as enzymatic methods.

In all ments of the s, the detection step may be carried out whilst the polynucleotide strand into which the labeled nucleotides are incorporated is annealed to a template strand, or after a denaturation step in which the two strands are separated. r steps, for example chemical or enzymatic reaction steps or purification steps, may be included between the synthetic step and the detection step. In particular, the target strand incorporating the labeled nucleotide(s) may be isolated or purified and then processed further or used in a subsequent analysis. By way of example, target polynucleotides labeled with modified nucleotide(s) as described herein in a synthetic step may be subsequently used as labeled probes or primers. In other embodiments the product of the synthetic step (a) may be subject to fiarther reaction steps and, if d, the product of these subsequent steps purified or isolated.

Suitable conditions for the synthetic step will be well known to those familiar with standard molecular y techniques. In one embodiment the tic step may be analogous to a standard primer extension reaction using nucleotide precursors, including modified tides according to the present disclosure, to form an extended target strand complementary to the template strand in the presence of a suitable polymerase enzyme. In other embodiments the synthetic step may itself form part ofan amplification reaction producing a labeled double stranded amplification product comprised of annealed mentary strands derived from copying ofthe target and template polynucleotide strands. Other exemplary "synthetic" steps include nick translation, strand displacement polymerization, random primed DNA labeling etc. The polymerase enzyme used in the synthetic step must be capable of catalyzing the incorporation of modified nucleotides according to the present disclosure. Otherwise, the precise nature of the rase is not particularly d but may depend upon the conditions of the synthetic reaction. By way of example, if the synthetic reaction is carried out using thermocycling then a thermostable polymerase is required, s this may not be essential for standard primer extension ons.

Suitable thermostable polymerases which are capable of incorporating the modified nucleotides according to the present disclosure include those bed in WO 2005/024010 or WC 2006/120433. In synthetic ons which are carried out at lower temperatures such as 37°C, WO 14710 2017/083128 polymerase enzymes need not necessarily be thermostable polymerases, therefore the choice of polymerase will depend on a number of factors such as on temperature, pH, strand-displacing activity and the like.

In specific non-limiting embodiments, the modified nucleotides or sides labeled with the new fluorescent dyes with longer Stokes shift according to the present application may be used in a method of nucleic acid sequencing, re-sequencing, whole genome sequencing, single nucleotide polymorphism scoring, any other application involving the detection of the modified nucleotide or nucleoside when incorporated into a polynucleotide, or any other application ing the use of polynucleotides labeled with the modified nucleotides comprising fluorescent dyes according to the present application.

In a particular embodiment the present application provides use of modified nucleotides comprising dye compounds bed herein in a cleotide "sequencing-by- synthesis" on. Sequencing-by-synthesis generally involves sequential addition ofone or more nucleotides or oligonucleotides to a growing cleotide chain in the 5' to 3' direction using a polymerase or ligase in order to form an extended polynucleotide chain complementary to the template nucleic acid to be ced. The identity of the base present in one or more of the added nucleotide(s) is determined in a detection or "imaging" step. The identity ofthe added base may be determined after each nucleotide incorporation step. The sequence of the template may then be inferred using conventional Watson-Crick base-pairing rules. The use of the modified nucleotides labeled with dyes according to the present disclosure for determination of the identity of a single base may be useful, for example, in the scoring ofsingle nucleotide polymorphisms, and such single base extension reactions are within the scope of this application.

In an embodiment, the sequence of a template polynucleotide is determined by detecting the incorporation of one or more nucleotides into a nascent strand complementary to the template polynucleotide to be sequenced through the ion of fluorescent label(s) attached to the incorporated nucleotide(s). Sequencing of the template polynucleotide is primed with a suitable primer (or prepared as a hairpin construct which will contain the primer as part of the hairpin), and the nascent chain is extended in a stepwise manner by addition of nucleotides to the 3' end of the primer in a rase-catalyzed reaction.

In particular embodiments each of the different nucleotide triphosphates (A, T, G and C) may be d with a unique fluorophore and also comprises a blocking group at the 3' on to t uncontrolled polymerization. Alternatively, one of the four nucleotides may be unlabeled (dark). The polymerase enzyme incorporates a nucleotide into the t chain complementary to the template polynucleotide, and the blocking group prevents further incorporation of nucleotides. Any unincorporated nucleotides are removed and the fluorescent signal from each incorporated tide is "read" optically by suitable means, such as a chargecoupled device using laser tion and le emission filters. The 3'-blocking group and fluorescent dye compounds are then removed (deprotected), particularly by the same chemical or enzymatic method, to expose the nascent chain for fiarther nucleotide incorporation. Typically, the identity of the incorporated nucleotide will be determined after each oration step but this is not strictly essential. rly, US. Pat. No. 5,302,509 discloses a method to sequence polynucleotides immobilized on a solid support. The method relies on the incorporation of fluorescently labeled, 3'-blocked nucleotides A, G, C and T into a growing strand complementary to the lized polynucleotide, in the presence of DNA polymerase. The polymerase incorporates a base complementary to the target polynucleotide, but is prevented from further addition by the 3'-blo cking group. The label ofthe incorporated nucleotide can then be determined and the blocking group d by chemical cleavage to allow further polymerization to occur.

The c acid template to be ced in a sequencing-by-synthesis reaction may be any polynucleotide that it is desired to sequence. The nucleic acid template for a sequencing reaction will typically comprise a double stranded region having a free 3' hydroxyl group which serves as a primer or tion point for the on of fiarther nucleotides in the sequencing reaction. The region of the template to be sequenced will overhang this free 3' hydroxyl group on the complementary strand. The nging region of the template to be sequenced may be single stranded but can be double-stranded, provided that a "nick is present" on the strand complementary to the template strand to be sequenced to provide a free 3' OH group for initiation ofthe sequencing reaction. In such embodiments sequencing may proceed by strand displacement. In certain embodiments a primer bearing the free 3' hydroxyl group may be added as a separate component (e.g. a short oligonucleotide) which hybridizes to a single-stranded region of the template to be sequenced. Alternatively, the primer and the template strand to be sequenced may each form part of a lly self-complementary nucleic acid strand capable of g an intra-molecular duplex, such as for example a hairpin loop structure. Hairpin polynucleotides and methods by which they may be attached to solid supports are disclosed in PCT Publication Nos. and WO 2005/047301. Nucleotides are added successively to the free 3'-hydroxyl group, resulting in sis of a polynucleotide chain in the 5' to 3' direction. The nature of the base which has been added may be determined, particularly but not necessarily after each nucleotide addition, thus providing sequence information for the nucleic acid template. The term poration" of a nucleotide into a nucleic acid strand (or polynucleotide) in this t refers to joining of the nucleotide to the free 3' hydroxyl group ofthe nucleic acid strand via formation of a phosphodiester linkage with the 5' phosphate group of the nucleotide.

WO 14710 The nucleic acid template to be sequenced may be DNA or RNA, or even a hybrid molecule comprised of deoxynucleotides and ribonucleotides. The nucleic acid template may comprise naturally occurring and/or non-naturally occurring nucleotides and natural or non- natural backbone es, provided that these do not prevent copying of the template in the sequencing reaction.

In certain embodiments the nucleic acid template to be sequenced may be attached to a solid support via any suitable linkage method known in the art, for example via covalent attachment. In certain embodiments te polynucleotides may be attached directly to a solid support (e.g. a silica-based support). r, in other embodiments the surface of the solid support may be d in some way so as to allow either direct covalent attachment oftemplate polynucleotides, or to immobilize the te polynucleotides through a hydrogel or polyelectrolyte ayer, which may itselfbe non-covalently attached to the solid support.

Arrays in which polynucleotides have been directly attached to silica-based ts are those for example disclosed in PCT Publication No. WO 06770, wherein polynucleotides are immobilized on a glass support by on between a pendant epoxide group on the glass with an internal amino group on the polynucleotide. In addition, PCT ation No.

W02005/04730l discloses arrays of polynucleotides attached to a solid support, e.g. for use in the preparation ofSMAs, by reaction ofa sulfur-based nucleophile with the solid t. A still fithher example of solid-supported template polynucleotides is where the template polynucleotides are attached to el supported upon silica-based or other solid supports. Silica-based supports are typically used to support hydrogels and hydrogel arrays as described in PCT Publication Nos. WO 00/31148, WO 01/01 143, W002/12566, WO 03/014392, WO 00/53812 and US. Pat. No. 6,465,178.

A particular surface to which template polynucleotides may be immobilized is a polyacrylamide hydrogel. Polyacrylamide hydrogels are described in the prior art, some ofwhich is discussed above. Specific els that may be used in the present application include those described in and US. Pub. No. 2014/0079923. In one embodiment, the hydrogel is PAZAM (poly(N-(5-azidoacetamidylpentyl) acrylamide-co-acrylamide)).

DNA template molecules can be attached to beads or microparticles for the es of sequencing; for example as described in US. Pat. No. 6,172,218. Further examples of the preparation of bead libraries where each bead contains different DNA sequences can be found in Margulies et al., Nature 437, 0 (2005); Shendure et al., Science. 309(5741):l728-l732 (2005). Sequencing of arrays of such beads using tides as described is within the scope of the present application.

The template(s) to be sequenced may form part of an "array" on a solid support, in which case the array may take any ient form. Thus, the method of the present disclosure is applicable to all types of "high density" , including single-molecule arrays, clustered arrays and bead arrays. Modified nucleotides labeled with dye compounds of the present application may be used for sequencing templates on essentially any type of array formed by immobilization of nucleic acid molecules on a solid t, and more particularly any type of ensity array.

However, the modified nucleotides labeled with the new fluorescent dyes described herein are particularly advantageous in the context of sequencing of clustered arrays.

In multi-polynucleotide or clustered arrays, distinct regions on the array comprise multiple polynucleotide te molecules. The term "clustered array" refers to an array wherein distinct regions or sites on the array comprise multiple polynucleotide molecules that are not dually resolvable by l means. ing on how the array is formed each site on the array may comprise multiple copies of one dual polynucleotide molecule or even multiple copies of a small number of different cleotide molecules (e. g. multiple copies of two complementary nucleic acid strands). Multi-polynucleotide or clustered arrays of nucleic acid molecules may be produced using techniques generally known in the art. By way of example, WO 98/44151 and WO 00/18957 both describe methods of amplification of nucleic acids wherein both the template and amplification products remain immobilized on a solid support in order to form arrays comprised of clusters or "colonies" of immobilized c acid molecules. The nucleic acid molecules present on the clustered arrays prepared according to these methods are suitable templates for cing using the modified nucleotides labeled with the new fluorescent dyes bed .

The modified nucleotides labeled with dye compounds ofthe present ation are also useful in sequencing of templates on single molecule arrays. The term "single molecule array" or "SMA" as used herein refers to a population of polynucleotide molecules, distributed (or arrayed) over a solid support, wherein the spacing of any individual polynucleotide from all others of the population is such that it is possible to effect individual resolution of the polynucleotides.

The target nucleic acid molecules immobilized onto the surface of the solid support should thus be capable of being resolved by optical means. This means that, within the resolvable area of the particular imaging device used, there must be one or more distinct signals, each representing one polynucleotide.

This may be ed wherein the spacing between adjacent polynucleotide molecules on the array is at least 100 nm, more particularly at least 250 nm, still more particularly at least 300 nm, even more particularly at least 350 nm. Thus, each molecule is individually resolvable and detectable as a single molecule fluorescent point, and fluorescence from said single molecule fluorescent point also exhibits single step photo-bleaching.

The terms "individually resolved" and "individual resolution" are used herein to specify that, when ized, it is possible to distinguish one molecule on the array from its neighboring molecules. Separation n individual molecules on the array will be determined, in part, by the ular technique used to resolve the individual molecules. The general features of single molecule arrays will be understood by reference to PCT Publication Nos. WO 2000/006770 and . gh one application of the modified nucleotides of the present disclosure is in sequencing-by-synthesis reactions, the utility of such labeled tides is not limited to such methods. In fact, the nucleotides may be used advantageously in any sequencing methodology which requires detection of cent labels attached to nucleotides incorporated into a polynucleotide.

In particular, the d tides labeled with dye compounds of the present application may be used in automated fluorescent sequencing protocols, particularly fluorescent dye-terminator cycle sequencing based on the chain termination cing method of Sanger and co-workers. Such methods generally use enzymes and cycle sequencing to incorporate fluorescently labeled ynucleotides in a primer extension sequencing reaction. So called Sanger sequencing methods, and related protocols r-type), rely upon randomized chain termination with labeled dideoxynucleotides.

Thus, the present disclosure also encompasses modified nucleotides d with dye compounds as described herein which are dideoxynucleotides lacking hydroxyl groups at both of the 3' and 2' positions, such modified dideoxynucleotides being suitable for use in Sanger type sequencing methods and the like.

EXAMPLES Additional embodiments are disclosed in further detail in the following examples, which are not in any way intended to limit the scope of the claims.

EXAMPLE 1 tert-Butyl 4-[3-(benzo[d]thiazolyl)-6,8,8-trimethyloxo-7,8-dihydro-2H-pyrano[3 ,2- g]quinolin-9(6H)-yl]butanoate (Compound I- 1) I SQ N OH O S@ —> + AOMN N o m0K [0 14 1] tert-Butyl 4-(6-formylhydroxy-2,2,4-trimethyl-3 ,4-dihydroquinolin-1(2H)- yl)butanoate (0.19 g) was dissolved in ethanol (2 mL). Ethyl 2-(benzo[d]thiazolyl)acetate (0.124 g) was added and the mixture was stirred at room temperature for 15 min. Piperidine (5 uL) was added and color ofthe reaction e turned to llow. Reaction mixture was left stirring at room temperature overnight. Next day the crude reaction mixture underwent aqueous workup, drying and purification by chromatography (silica gel with petroleum ether/ethyl acetate as eluent) to afford Compound I-l. Purity, structure and ition were confirmed by HPLC, NMR and LCMS. MS (DUIS): MW ated 518.22. Found: (-) 517 (M-l).

EXAMPLE 2 4- [3 -(Benzo [d]thiazo lyl)-6, 8 , 8-trimethyloxo-7,8-dihydro-2H-pyrano [3 ,2-g]quino lin-9(6H)- anoic acid (Compound I-2) \ \N \ N o o N O O + KL OK C02H Compound 1-1 (51.8 mg) was dissolved in DCM (5 mL) and trifluoroacetic acid (0.5 mL) was added via syringe and the reaction mixture was left stirring overnight at room temperature. Solvent was distilled off using rotary evaporator, the residue triturated with water (5 mL) solid filtered off and dried to afford Compound I-2. Purity, structure and composition were ed by HPLC, NMR and LCMS. MS : MW Calculated 462.16. Found: (-) 461 (M- tert-Butyl 4-[3-(benzo[d]thiazolyl)-6,8,8-trimethyloxo-2H-pyrano[3 uinolin—9(6H)- yl]butanoate (Compound I-3) [0 143] tert-Butyl 4-(6-formylhydroxy-2,2,4-trimethyl-quino lin- 1 (2H)-yl)butanoate (0.19 g) was dissolved in ethanol (2 mL). Ethyl 2-(benzo[d]thiazolyl)acetate (0.124 g,) was added and the mixture was stirred at room temperature for 15 min. Piperidine (5 uL) was added and color of the reaction mixture turned to red-yellow. Reaction e was left stirring at room temperature overnight and the crude reaction mixture underwent aqueous workup, drying and purification by chromatography (silica gel with petroleum ether/ethyl acetate as eluent) to afford Compound I-3. Purity, ure and composition were confirmed by HPLC, NMR and LCMS. MS (DUIS): MW Calculated 516.22. Found: (-) 515 (M-l); (+) 517 (M+1).

EXAMPLE 4 4- [3 -(Benzo [d]thiazo )-6, 8 , 8-trimethyloxo-2H-pyrano [3 ,2-g]quino lin-9(6H)-yl]butanoic acid (Compound I-4) S/Q S/Q / \ \N / \ \N N o o N 0 0 + KL OK COZH Compound I-3 (51.7 mg) was dissolved in DCM (6 mL) and trifluoroacetic acid (1 mL) was added via syringe and the reaction mixture was left ng overnight at room temperature. Solvent was led off using rotary evaporator, and the residue was triturated with water (5 mL). The formed solid was filtered off and dried to afford Compound 1-4. Purity, structure and composition were confirmed by HPLC, NMR and LCMS. MS (DUIS): MW Calculated 460.15.

Found: (+) 461 (M+1).

EXAMPLE 5 t-Butyl 4- [3 -(benzoxazolylyl)-6, 8 lin- , 8-trimethyloxo-7,8-dihydro-2H-pyrano [3 ,2-g]quino 9(6H)-yl]butanoate (Compound I-5) | O@ \ \N 7 ::N : :OH 0 O@ —> E + AOMN N o o O7< KKKo o K [0 145] tert-Butyl 4-(6-formylhydroxy-2,2,4-trimethyl-3 ,4-dihydroquinolin-1(2H)- yl)butanoate (0.18 g) was dissolved in ethanol (3 mL). Ethyl 2-(benzoxazolyl)acetate (0.124 g) was added and the mixture was stirred at room temperature for 15 min. Piperidine (5 uL) was added.

Color of the reaction mixture turned to red-yellow. Reaction mixture was lefi ng at room ature overnight. The crude reaction mixture underwent aqueous workup, drying and ation by chromatography (silica gel with petroleum ether/ethyl acetate as ) to afford Compound 1-5. Purity, structure and composition were confirmed by HPLC, NMR and LCMS. MS (DUIS): MW Calculated 502.25. Found: (-) 501 (M-l).

EXAMPLE 6 4-[3-Benzoxazol—2-yl)-6,8,8-trimethyloxo-7,8-dihydro-2H-pyrano[3 ,2-g]quinolin—9(6H)- anoic acid (Compound I-6) \ \ N O O N O O + KL OK 002H Compound 1-5 (50 mg) was dissolved in DCM (5 mL) and trifluoroacetic acid (0.5 mL) was added via syringe and the reaction mixture was left stirring overnight at room ature. Solvent was distilled offusing rotary evaporator, and the e triturated with water (5 mL). The formed solid was filtered off and dried to afford Compound I-6. Purity, structure and composition were confirmed by HPLC, NMR and LCMS. MS (DUIS): MW Calculated 446.16.

Found: (-) 445 (M-l). 2017/083128 EXAMPLE 7 Ethyl 4-[3-(benzoxazolyl)-6,8,8-trimethyloxo-7,8-2H-pyrano[3 ,2-g]quinolin—9(6H)- yl]butanoate (Compound I-7) [0 147] Ethyl 4- [6-formyl—7-hydroxy-2,2,4-trimethyl-3 ,4-quino lin- 1 (2H)-yl]butanoate (0.17 g) was dissolved in anhydrous l (2.5 mL). Ethyl 2-(benzoxazolyl)acetate (0.102 g) was added and the mixture was stirred at room temperature for 15 min. dine (5 uL) was added. Reaction mixture was left stirring at room temperature overnight. The crude reaction mixture ent aqueous workup, drying and purification by chromatography (silica gel with petroleum ether/ethyl acetate as eluent) to afford Compound I-7. Purity, structure and composition were confirmed by HPLC, NMR and LCMS. MS (DUIS): MW Calculated . Found: (+) 473 (M+1).

EXAMPLE 8 4-[3-(Benzoxazol—2-yl)-6,8,8-trimethyloxo-2H-pyrano[3 ,2-g]quinolin-9(6H)-yl]butanoic acid (Compound 1-8) / \ / \ N O O N O O —> KL O\/ COZH Compound I-7 (51.8 mg) was dissolved in acetic acid (2.5 mL) and hydrochloric acid (5 mL) was added and the reaction mixture was lefi stirring overnight at 60°C. Solvent was distilled off using rotary evaporator, and the residue was triturated with water (5 mL). Compound 1-8 was filtered off and dried in vacuum. Purity, structure and composition were confirmed by HPLC, NMR and LCMS. MS (DUIS): MW Calculated 444.17. Found: (+) 445 (M+1).

EXAMPLE 9 3 -(benzoxazo lyl)-6,8,8-trimethyloxo-2H-pyrano[3 ,2-g]quinolin-9(8H)-yl)-N—(4-(tert- butoxy)oxobutyl)butanamido]propanesulfonic acid (Compound 1-9) :@ _.1.PyBOP / O:/©\ \N 2- 803H (Hsc)3cozc\/\/N\H)OHossk OH KL0020(CH3)3 Compound 1-8 (50 mg, 112 umol) was dissolved in dimethylformamide (1 mL) and then solvent distilled off in vacuo. This operation was repeated two more times, then dried nd 1-8 was redissolved in DMF (1 mL) at room temperature. (Benzotriazol—l- yloxy)tripyrrolidinophosphonium hexafiuorophosphate (PyBOP, 1.5 eq., 64 mg, 169 umol) was added to the flask then N,N—Diisopropylethylamine (DIPEA, 3 eq., 336 umol, 43 mg, 58 uL) was added via micropipette. Reaction flask was sealed under nitrogen gas. After reaction was completed, the ted dye solution in DMF was mixed with 3-[(4-(tert-butoxy) oxobutyl)amino]propanesulfonate ( 1.5 eq., 224 umol, 63 mg). More DIPEA (3 eq., 336 umol, 43 mg, 58 uL) was added. Flask was again sealed under nitrogen gas and left overnight at room temperature. Reaction progress was monitored by TLC, HPLC and (LCMS). When reaction was te, water (2 mL) was added, the on mixture was stirred for 15 min and then solvent was led off from the reaction mixture in vacuum at room ature. Compound 1-9 was used in next step without any farther purification.

EXAMPLE 10 4-[4-(3-(Benzoxazolyl)-6,8,8-trimethyloxo-2H-pyrano[3 ,2-g]quinolin-9(8H)-yl)-N—(3- sulfopropyl)butanamido]butanoic acid (Compound I- 1 0) HO SK3 SEQ HO 8 (H30)3COZC\/\/N7‘/HO 3 k MHOZCWNWKHN O O The0dried crude compound 1-9 was re-dissolved in dichloromethane (2 mL).

Trifiuoroacetic acid (0.5 mL) was added and the reaction was left stirring overnight at room temperature. The reaction was quenched with water concentrated in vacuum and then purified by preparative-HPLC. Purity, structure and composition of the product were confirmed by HPLC, NMR and LCMS. MS (DUIS): MW Calculated 651.23. Found: (-) 650 (M-l).

WO 14710 EXAMPLE ll 4-[3-(Benzoxazolyl)-8,8-dimethyloxo(sulfomethyl)-2H-pyrano[3 ,2-g]quinolin—9(8H)- yl)butanoic acid (Compound L1 1) m)?"o@\N O O KkWOH Sulfuric acid (2.5 mL) was cooled down to about 5°C then compound 1-8 (45 mg) was added and the reaction mixture was stirred at 20-25°C for 1h. Then reaction mixture was kept at room temperature overnight. on mixture was diluted slowly with anhydrous ether in presence of external g. Precipitate was filtered off, dissolved in a mixture of water (5 mL) and acetonitrile (5 mL). Solution was filtered and purified by preparative HPLC. Purity, structure and composition of the t were confirmed by HPLC, NMR and LCMS. MS (DUIS): MW Calculated 524.13. Found: (-) 623 (M-l).

EXAMPLE l2 1-[(5-carboxypentyl)( 1 1-oxo-2,3,6,7-tetrahydro-1H,5H,1 1H-pyrano[2,3-f]pyrido[3,2,1- ijjquino lin- 1 0-yl]pyridinium bromide (Compound I- 12) 0020sz / (CH2)5002 (CH2)5002H N+ / N+ \ \ \ \ (CH2)5COZH [0 1 52] B8-Hydroxy-2,3 ,6,7-tetrahydro- 1 H,5H-pyrido [3 ,2,1-ij]quinolinecarbaldehyde (0.217 g) was dissolved in ethanol (6 mL). To this solution 1-[(5-carboxypentyl)-(4- etxoxycarbonyl)methylpyridinium e (0.36 g) was added and the mixture was stirred at room temperature for 15 min. Piperidine acetate on prepared fiom piperidine (20 mg) and acetic acid (20 mg)/ in ethanol (5 mL) was added and this on mixture was stirred at 60°C for 5 hours then stirred overnight at room ature. Solvent was distilled off and the e was dissolved in a mixture of water (15 mL) and acetonitrile (15 mL). The resulting solution was filtered and purified by preparative HPLC. This compound was converted to more stable hydrobromide salt form to afford Compound I-12. Purity, structure and composition were confirmed by HPLC, NMR and LCMS. MS (DUIS): MW ated 432.20. Found: (+) 433 (M+l).

EXAMPLE l3 l-(5-Carboxypentyl)( l , l ,7,7-tetramethyl-l l-oxo-2,3 ,6,7-tetrahydro- l H,5H,l ano[2,3- fjpyrido [3 ,2, l -ijJquino lin- l 0-yl)pyridin- l -ium bromide (Compound I- l 3) C02C2H5 (I) / N+-(CH2)5C02' / N+-(CH2)5002H | | \ \ \ \ Br- 4) N OH N o O N O O (CH2)5C02H oxy-2,3 ,6,7-tetrahydro- l , l ,7,7-tetramethyl- lH,5H-pyrido[3 ,2, l - ij]quinolinecarbaldehyde (0.27 g) was dissolved in ethanol (7 mL). l-[(5-Carboxypentyl)-(4- carbonyl)methylpyridinium e (0.36 g) was added and the mixture was stirred at room temperature for 30 min. Solution ofpiperidine acetate prepared from piperidine (20 mg) and acetic acid (20 mg) in ethanol (5 mL) was added. This reaction mixture was stirred at 60°C for 5 hours then stirred overnight at room temperature. The solvent was distilled off and the red-colored residue was dissolved in a mixture of water (15 mL) and acetonitrile (15 mL). The resulting solution was filtered and purified by preparative HPLC. This nd was converted to the more stable hydrobromide salt form by reaction with HBr solution in acetic acid (10 %, 0.3 mL) to afford Compound I-l3.

Purity, ure and composition were confirmed by HPLC, NMR and LCMS. MS (DUIS): MW Calculated 488.27. Found: (+) 489 (M+l).

EXAMPLE l4 l-(5-Carboxypentyl)(9-ethyloxo-6,7,8,9-tetrahydro-2H-pyrano[3 ,2-g]quinolinyl)pyridin- l-ium bromide (Compound I- l 4) C02C2H5 (CH2)5C02 N+-(CH2)5CO2H (111+ fN/ja. (III/Q \ K[0154] O O (CH2)5C02H l-Ethylhydroxy-l,2,3,4-tetrahydroquinolinecarbaldehyde (0.2 g) was dissolved in ethanol (10 mL). l-[(5-Carboxypentyl)-(4-etxoxycarbonyl)methylpyridinium bromide (0.36 g) was added and the mixture was stirred at room temperature for 30 min. Solution of piperidine acetate prepared from piperidine (20 mg) and acetic acid (20 mg)/ in ethanol (5 mL) was added. This reaction mixture was stirred at 80°C for 2 hours then stirred overnight at room temperature. The solvent was distilled off and the e was dissolved in a mixture ofwater (10 mL) and acetonitrile (10 mL). The ing solution was d and purified by preparative HPLC.

This nd was converted to the more stable hydrobromide salt form by reaction with HBr solution in acetic acid (10 %, 0.3 mL) to afford Compound I-l4. Purity, structure and composition were confirmed by HPLC, NMR and LCMS. MS (DUIS): MW Calculated 420.20. Found: (+) 421 (M+1).

EXAMPLE 1 5 4- [4-(9-(4-(tert-Butoxy)oxobutyl)-6, 8 , 8-trimethyloxo-6,7, 8 rahydro-2H-pyrano [3 ,2- olin-3 -yl)pyridin— l -ium- l -yl]butane- l -sulfonate (Compound I- l 5) / N+—(CH2)4503' I | \ + ’ N OH | N o o KL N+ (CH2)4503' KL 0020(CH3)3 0020(CH3)3 [0 1 55] tert-Butyl 4-(6-formylhydroxy-2,2,4-trimethyl-3 ,4-dihydroquino lin- 1 (2H)- yl)butanoate (0.72 g) was dissolved in l (25 mL). 4-[4-(2-Ethoxyoxoethyl)pyridin-l-ium- l-yl)butane-l-sulfonate (0.60 g) was added and the mixture was stirred at room temperature for 30 min. Solution of dine acetate prepared from piperidine (50 mg) and acetic acid (50 mg)/ in ethanol (15 mL) was added. This reaction mixture was stirred at 80°C for 3 hours then stirred ght at room temperature. The solvent was distilled off and the e was dissolved in a mixture of water (20 mL) and acetonitrile (20 mL). The resulting solution was filtered and purified by preparative HPLC to afford Compound 1-15. Purity, structure and composition were confirmed by HPLC, NMR and LCMS. MS (DUIS): MW Calculated 598.20. Found: (+) 599 (M+1).

EXAMPLE l6 4-[4-(9-(3-Carboxypropyl)-6,8,8-trimethyloxo-6,7,8,9-tetrahydro-2H-pyrano[3 ,2-g]quinolin yl)pyridin- l -ium- l -yl]butane- l -sulfonate (Compound I- l 6) _ |N+(-CH2)4so- / 3 / N+-(CH2)4SO3 l \ \ \ ILL o o o o _> NKL CO2H COZC(CH3)3 Compound I-l5 was dissolved in DCM (5 ml) and TFA was added. on mixture was stirred overnight at room temperature. Solvent was distilled off in vacuum. To the residue water (1 .5 ml) and acetonitrile (10 mL) were added and the solvent was distilled off again to remove TFA. The remaining orange crystalline product was dissolved in a e of water (12 mL) and acetonitrile (12 mL). The resulting solution was filtered and purified by preparative HPLC. 2017/083128 Purity, structure and composition were confirmed by HPLC, NMR and LCMS. MS (DUIS): MW Calculated 542.21. Found: (+) 543 (M+1).

EXAMPLE l7 4- [4-(9-(4-(tert-Butoxy)oxobutyl)-6, 8 , 8-trimethyloxo-8,9-dihydro-2H-pyrano [3 ,2- g]quinolin-3 -yl)pyridin— 1 -iumyl]butanesulfonate (Compound I- 1 7) N+-(CH2)4303' + —> N OH | KL N+ (CH2)4303' KL C02C(CH3)3 C02C(CH3)3 [0 1 57] tert-Butyl 4-(6-formylhydroxy-2,2,4-trimethyl-quino lin- 1 (2H)-yl)butanoate (0.36 g) was suspended in ethanol (5 mL). 4-[4-(2-Ethoxyoxoethyl)pyridiniumyl)butane- l-sulfonate (0.30 g) was added and the mixture was stirred at room temperature for 30 min. Solution ofpiperidine acetate prepared from piperidine (50 mg) and acetic acid (50 mg) in ethanol (15 mL) was added. This reaction e was stirred at 50°C for 5 hours then stirred ght at room temperature. Solvent was distilled off and the residue was dissolved in a e of water (10 mL) and itrile (10 mL). The resulting on was filtered and purified by preparative HPLC to afford Compound 1-17. Purity, structure and composition were confirmed by HPLC, NMR and LCMS. MS (DUIS): MW Calculated 596.26. Found: (+) 597 (M+1).

EXAMPLE l8 4-[4-(9-(3-Carboxypropyl)-6,8,8-trimethyloxo-8,9-dihydro-2H-pyrano[3 ,2-g]quinolin yl)pyridiniumyl]butanesulfonate (Compound 1-1 8) CO2C(CH3)3 Compound 1-17 was dissolved in DCM (5 ml) and TFA was added. The reaction mixture was stirred overnight at room ature then solvent was distilled off in vacuum. To the residue water (2 ml) and acetonitrile (10 mL) were added and solvent distilled off again to remove the residue TFA. The remaining orange crystalline product was triturated with ether, filtered off and purified by preparative HPLC to afford Compound 1-18. Purity, structure and composition were confirmed by HPLC, NMR and LCMS. MS (DUIS): MW ated 540.19. Found: (-) 539 (M- 1); (+) 541 (M+1).

EXAMPLE 1 9 4-(4-(9-(4-((4-(tert-Butoxy)oxobutyl)(3 -sulfopropyl)amino)oxobutyl)-6, 8 , 8-trimethyl 9-dihydro-2H-pyrano [3 ,2-g]quino lin-3 -yl)pyridin— l -ium- l -yl)butane- l -sulfonate 1 Compound L19 1 / N+-(CH2)4sos' N+‘(CH2)4503' 1. P BOP | | y HO 53 2. N KHVOH HN\/\/803H KL 04393002ch CO2C(CH3)3 0 Compound I-8 (50 mg) was dissolved in DMF (1 mL) and then solvent distilled off in vacuo. This operation was repeated twice. The dried Compound I-8 was re-dissolved in DMF (l .5 mL) at room temperature. (Benzotriazo l- l -yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP, 1.5 eq., 64 mg, 169 umol) was added to the flask then excess of DIPEA (60 uL) was added. The reaction flask was sealed under nitrogen gas. After reaction was complete, the activated dye on in DMF was mixed with 3-[(4-(tert-butoxy) oxobutyl)amino]propane-l-sulfonate (63 mg). More DIPEA (58 uL) was added. Flask was again sealed under en gas and left overnight at room temperature. Reaction progress was monitored by TLC, HPLC and (LCMS). When reaction was complete, water (2 mL) was added. The reaction mixture was stirred for 15 min and then solvent distilled off from the reaction mixture in vacuum at room ature to afford Compound I-l9. This compound was used in next step t any fiarther purification.

EXAMPLE 20 4-(4-(9-(4-((3-Carboxypropyl)(3-sulfopropyl)amino)—4-oxobutyl)-6, 8 ,8-trimethyloxo-8 ,9- dihydro-2H-pyrano [3 ,2-g]quino lin-3 -yl)pyridin- l -ium- l -yl)butane- l -sulfonate (Compound 1-20) -(CH2)4503' -(CH2)4503' /N+ /N+ The dried compound I-19 was dissolved in dichloromethane (5 mL).

Trifluoroacetic acid (0.5 mL) was added and the on was left ng ght at room temperature. The reaction was quenched with water (0.5 mL), concentrated in vacuo and then purified by preparative-HPLC. Purity, structure and composition ofthe product were confirmed by HPLC, NMR and LCMS. MS (DUIS): MW Calculated 747.25. Found: (-) 746 (M-l).

EXAMPLE 21 1 -(5 -Carboxypentyl)(7-(diethylamino)oxo-2H-chromenyl)pyridinium e(Compound 1-21) OCH2 2)5 COZCZHS / N+((2)CH)