Stabilized reagents and compositions for the conversion of aminooxy compounds to alcohols

Abstract

Provided herein are compositions comprising an imine phosphonate compound and, optionally, a buffer. Also provided herein are kits and systems comprising the compositions as well as methods of making and using the compositions.

Description

Attorney Docket No.: 097128-1540435 (046WO1)Reagents and Compositions for the Conversion of Aminooxy Compounds to AlcoholsCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is based on, and claims the benefit of, U. S. Provisional Application No. 63 / 738,391, filed December 23, 2024, the which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Commonly used in nucleic acid chemistry, including sequencing and synthesis, are reversible terminators. These modified nucleotides can be used in next generation sequencing to elongate a primer in a controlled cycle-based workflow, allowing the sequence of nucleotides in the template strand to be determined. Reversible terminators bearing aminooxy groups, e.g., a -ONH2 at the 3’ position, can be used in a sequencing-by-synthesis approach, where the sequence of a template is inferred by stepwise primer elongation. Reversible terminators bearing aminooxy groups can also be used in sequencing by binding technologies. To enable the next cycle of sequencing, any 3’ aminooxy group must be converted to the natural 3’ hydroxy linkage. This aminooxy to hydroxy conversion can be referred to as a cleavage or deblocking step.

[0003] In general, deblocking may be accomplished with reagents capable of efficiently converting the aminooxy groups to alcohols. Conventional deblocking reagents have several issues that limit their utility’ which remain unaddressed. Typically, deblocking is accomplished by exposing the nucleotide sample to reagents capable of generating nitrous acid, i.e., HNO2, which cleaves the aminooxy N=O bond. Nitrous acid cleavage-based reagents often require harsh reaction conditions, e.g. high temperature and / or high acidity, which may damage the nucleotide. Additionally, these harsh reagents often require high concentrations to be effective making them financially and environmentally costly. These significant costs may often be compounded by solubility issues, i.e., the high concentrations required may result in precipitation decreasing efficiency and increasing waste. Accordingly, there exists a need to develop reagents that can achieve efficient cleavage of the N=O bond.BRIEF SUMMARY

[0004] The present disclosure provides a composition comprising an imine phosphonate compound and, optionally, a buffer.

[0005] The present disclosure provides a composition, comprising a buffer and an imine compound of Formula (I), Formula (la), Formula (lb), Formula (Ic), Formula (Id), and Formula (le) or stereoisomers and salts thereof. In some cases, the present disclosure provides a method of producing an imine compound of Formula (I), Formula (la). Formula (lb), Formula (Ic), Formula (Id), or Formula (le), comprising contacting a solution of carbonyl diphosphonate with a primary amine forming a reaction mixture comprising the imine compound; and, optionally, extracting the imine compound from the reaction mixture.

[0006] In some cases, a composition is provided comprising a buffer and an imine compound of Formula (I):or stereoisomers and salts thereof, wherein:n is 0, 1, 2, or 3;m is 0 to 20R1is a bond, H, –CH2–, or –NH–;R2is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polymer, or -NR4R5, wherein R4and R5are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted polymer; andR3is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polymer, -P(O)3‘, or -NR4R5, wherein R4and R5are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted polymer,wherein when R1is a bond and m is 0, n is 1,wherein when R1is -NH- and m is 0, n is 1, andwherein when R1is H, m and n are 0.

[0007] In some cases. Formula (I) has a structure of Formula (la), Formula (lb). Formula (Ic), Formula (Id), or Formula (le):

[0008] Also provided herein are methods of extending polynucleotides and methods of sequencing polynucleotide templates as well as systems for carrying out such methods. In addition, further provided herein is a kit comprising the herein provide imine compounds. For example, the provided kits can include a composition comprising an imine compound of Formula (I), Formula (la), Formula (lb), Formula (Ic), Formula (Id), or Formula (le); a polymerase; and a nucleotide triphosphate comprising an O-N bond.

[0009] The details of one or more embodiments are set forth in the drawings and the description below. Other features, mechanisms, and advantages will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The foregoing embodiments and others will be readily appreciated by the skilled artisan from the following description of illustrative embodiments when read in conjunction with the accompanying drawings.

[0011] FIG. 1 shows a ultraviolet visible (UV-VIS) spectrum demonstrating conversion of carbonyl diphosphonate (CDP) to an imine compound with increasing concentrations of N1,N1-dimethylethane-1,2-diamine (DMEN).

[0012] FIG. 2 shows a ultraviolet visible (UV-VIS) spectrum demonstrating no conversion of carbonyl diphosphonate (CDP) to an imine compound with increasing concentrations of N1,N1,N2,N2-tetramethylethane-1,2-diamine (TMEDA).

[0013] FIG. 3 shows a phosphorus nuclear magnetic resonance (31P NMR) spectra analysis. FIG. 3, Panel A shows the31P NMR spectrum for 50 mM carbonyl diphosphonate (CDP) in 1 M sodium acetate buffer in deuterium oxide. FIG. 3, Panel B shows the31P NMR spectrum for 50 mM CDP with 90 mM N1,N1-dimethylethane-1,2-diamine (DMEN) in 1 M sodium acetate buffer in deuterium oxide.

[0014] FIG. 4 shows proton nuclear magnetic resonance (1H NMR) spectra. FIG. 4, Panel A shows the1H NMR spectrum for 90 mM N1,N1-dimethylethane-1,2-diamine (DMEN) in 1 M sodium acetate buffer in deuterium oxide. FIG. 4, Panel B shows the1H NMR spectrum for 50 mM carbonyl diphosphonate (CDP) with 90 mM DMEN in 1 M sodium acetate buffer in deuterium oxide.

[0015] FIG. 5 shows carbon nuclear magnetic resonance (13C NMR) spectrum for (((2-(dimethylammonio)ethyl)imino)methylene)bis(phosphonate) (DMEN-imine).

[0016] FIG. 6 shows ultra-performance liquid chromatography (UPLC) spectra for AZDye™ 568 hydroxylamine with and without an imine diphosphonate (IDP) formed with N1,N1-dimethylethane-1,2-diamine (DMEN) and reacted with a solution of 2-formylphenylboronic acid (2-FPBA). FIG. 6, Panel A shows 5 pM AZDye™ 568 hydroxylamine 0 pM IDP and 200 pM 2-FPBA. FIG.6, Panel B shows 5 pM AZDye™ 568 hydroxylamine 10 pM IDP and 200 pM 2-FPBA. FIG. 6, Panel C shows 5 µM AZDye™568 hydroxylamine 20 µM IDP and 200 µM 2-FPBA. FIG. 6, Panel D shows 5 µM AZDye™ 568 hydroxylamine 50 µM IDP and 200 µM 2-FPBA.

[0017] FIG. 7 shows a bar graph of increasing acetate concentrations and its effect on oxime condensation reactions in the presence of an imine diphosphonate.

[0018] FIG. 8 shows a graph of fraction deblocked versus time demonstrating increased reaction kinetics for deblocking of polynucleotides in the presence of increasing concentrations of DMEN indicating increased activity of imine diphosphonate (IDP).

[0019] FIG. 9 shows the31P NMR spectra for carbonyl diphosphonate (CDP) and CDP in the presence of (4-(2-aminoethyl)morpholine) (AEM). FIG. 9, Panel A shows the31P NMR spectrum for 50 mM CDP in deuterium oxide. FIG. 9, Panel B shows the31P NMR spectrum for 50 mM CDP and 90 mM AEM in 1 M sodium acetate buffer in a 10% deuterium oxide in water mixture.

[0020] FIG. 10 showsNMR spectra. FIG. 10, Panel A shows theNMR spectrum of 90 mM (4-(2-aminoethyl)morpholine) (AEM) in 1 M sodium acetate buffer in a 10% deuterium oxide in water mixture. FIG. 10, Panel B shows theNMR spectrum of 50 mM carbonyl diphosphonate (CDP) and 90 mM of AEM in 1 M sodium acetate buffer in a 10% deuterium oxide in water mixture.

[0021] FIG. 11 shows ultra-performance liquid chromatography (UPLC) spectra of cleavage activity for imine diphosphonates (IDPs) formed with (4-(2-aminoethyl)morpholine) (AEM) FIG. 11, Panel A shows the UPLC spectrum for 50 mM carbonyl diphosphonate (CDP) in 1 M acetate buffer. FIG. 11, Panel B shows the UPLC spectrum for 50 mM CDP reacted with 90 mM AEM in 1 M sodium acetate buffer.

[0022] FIG. 12 shows the31P NMR spectrum of 50 mM carbonyl diphosphonate (CDP) with 90 mM N1,N1-dimethylpropane-1,3-diamine (DMPN) in 1 M sodium acetate buffer in a 10% deuterium oxide in water mixture

[0023] FIG. 13 showsNMR spectra for the formation of imine diphosphonates (IDPs) with N1,N1-dimethylpropane-1,3-diamine (DMPN). FIG. 13, Panel A shows 90 mM DMPN in 1 M sodium acetate buffer in a 10% deuterium oxide in w ater mixture. FIG. 13, Panel B shows 50 mM carbonyl diphosphonate (CDP) with 90 mM DMPN in 1 M sodium acetate buffer in a 10% deuterium oxide in water mixture.

[0024] FIG. 14 shows ultra-performance liquid chromatography (UPLC) spectra of cleavage activity for imine diphosphonates (IDPs) formed with N1,N1-dimethylpropane-1,3-diamine (DMPN). FIG. 14, Panel A shows 50 mM carbonyl diphosphonate (CDP) in 1 M acetate buffer. FIG. 14, Panel B shows 50 mM CDP with 90 mM DMPN in 1 M sodium acetate buffer.

[0025] FIG. 15 shows31P NMR spectra for carbonyl diphosphonate (CDP), CDP in the presence of 2-aminomethyl-lH-imidazole dihydrochloride, and CDP in the presence of N1,N1-dimethylethane-1,2-diamine (DMEN). FIG. 15, Panel A shows the31P NMR spectrum of 50 mM CDP in deuterium oxide. FIG. 15, Panel B shows the31P NMR spectrum of 90 mM 2-aminomethyl-lH-imidazole dihydrochloride with 50 mM CDP in 1 M sodium acetate buffer. FIG. 15, Panel C shows the31P NMR spectrum of 50 mM CDP with 90 mM DMEN in 1 M sodium acetate in a 10% deuterium oxide in water mixture.

[0026] FIG. 16 showsNMR spectra for carbonyl diphosphonate (CDP). and CDP in the presence of 2-aminomethyl-lH-imidazole dihydrochloride. FIG. 16, Panel A shows theNMR spectrum of 90 mM of 2-aminomethyl-lH-imidazole dichloride in 1 M sodium acetate buffer in a 10% deuterium oxide in water mixture. FIG. 16, Panel B shows the 'H NMR spectrum of 90 mM of 2-aminomethyl-lH-imidazole dihydrochloride with 50 mM CDP in 1 M sodium acetate buffer in a 10% deuterium oxide in water mixture.

[0027] FIG. 17 shows1H NMR spectra for 2-aminomethyl-1H-imidazole dihydrochloride and 2-aminomethyl-lH-imidazole dihydrochloride in the presence of carbonyl diphosphonate (CDP). FIG. 17, Panel A shows aNMR spectrum in the range of 7.9 to 3.0 ppm for 90 mM 2-aminomethyl-1H-imidazole dichloride in 1 M sodium acetate buffer in a 10% deuterium oxide in water mixture. FIG. 17, Panel B show s aNMR spectrum in the range of 7.9 to 3.0 ppm for 90 mM of 2-aminomethyl-1H-imidazole dihydrochloride with 50 mM CDP in 1 M sodium acetate buffer in a 10% deuterium oxide in water mixture.

[0028] FIG. 18 shows ultra-performance liquid chromatography (UPLC) spectra of cleavage activity for carbonyl diphosphonate (CDP) combined with 2-aminomethyl-lH-imidazole dihydrochloride (Amlm) in the presence of O-phenylhydroxylamine. FIG. 18, Panel A shows the UPLC spectrum for phenylhydroxylamine. FIG. 18, Panel B shows the UPLC spectrum for 50 mM CDP in 1 M acetate buffer in the presence of O-phenylhydroxylamine. FIG. 18, Panel C shows the UPLC spectrum for 50 mM CDP with 90 mM Amlm in 1 M sodium acetate buffer that was further mixed with a solution of O-phenylhydroxylamine. FIG. 18, Panel D shows the UPLC spectrum for 50 mM CDP with180 mM Amlm in 1 M sodium acetate buffer that was further mixed with a solution of O-phenylhydroxylamine.

[0029] FIG. 19 shows31P NMR spectra for carbonyl diphosphonate (CDP), CDP in the presence of symmetric N1,N2-dimethylethylenediamine. and CDP in the presence of asymmetric N1,N1-dimethylethylenediamine (DMEN). FIG. 19, Panel A shows the31P NMR spectrum of 50 mM CDP in deuterium oxide. FIG. 19, Panel B shows the31P NMR spectrum of 50 mM CDP with 90 mM of symmetric N1, A-’-dimethy lethy lenediamine in 1 M acetate buffer in a 10% deuterium oxide in water mixture. FIG. 19, Panel C shows the31P NMR spectrum of 50 mM CDP with 90 mM DMEN in 1 M sodium acetate in a 10% deuterium oxide in water mixture.

[0030] FIG. 20 shows no imine diphosphonate formation with aniline and carbonyldiphosphonate is observed by31P NMR. 90 mM aniline was added to 1 M acetate buffer, with or without 50 mM CDP. and the resultant mixture was analyzed by31P NMR. Of note, the peak at ~13 ppm may correspond to CDP hydrate, which is more prevalent at lower pH (5.5). This further supports the conclusion that aniline does not form an adduct with CDP.

[0031] FIG. 21 shows no imine diphosphonate formation with aniline and carbonyldiphosphonate is observed by1H NMR. 90 mM aniline was added to 1 M acetate buffer, with or without 50 mM CDP, and the resultant mixture was analyzed by 'H NMR. These further support the conclusion that aniline does not form an adduct with CDP.DETAILED DESCRIPTION

[0032] Here, we describe specific compositions of matter - imine phosphonates -structures that have not been previously reported, as optimized reagents for deblocking chemistry. Imine phosphonates are highly active, water-stable, and resistant to precipitation compared to CDP.

[0033] Of note, electron withdrawing groups (EWGs), i.e., an atom or group of atoms that pull electron density away from adjacent atoms, are known to activate carbonyls for nucleophilic attack. Typically, this occurs through either resonance and / or inductive effects. This activation is especially pronounced when the EWG is in the a-position, i.e., directly attached to the carbonyl carbon. Phosphonate is a known EWG. Therefore, the carbonyl carbon of carbonyl diphosphonate (CDP) is considered activated, and susceptible to nucleophilic attack.

[0034] CDP has been reported as a deblocking compound in nucleic acid chemistry, including sequencing and synthesis, due to its ability to undergo condensation reactions with reversible terminators bearing 3’ aminooxy groups, i.e., -ONH2 commonly used as blocking moieties. For example, as demonstrated by the general reaction below, CDP undergoes a condensation reaction with hydroxylamine, thereby forming an oxime compound with the loss of water. The oxime compound may then undergo fragmentation, resulting in a free hydroxy compound, and a phosphate. This is useful for nucleic acid chemistry, as a free hydroxy becomes a reactive site for further nucleotide incorporation.

[0035] As described in the examples herein, asymmetric N1,N1-dimethylethane-1,2-diamine (DMEN) mixture with CDP was found to provide more efficient cleavage of aminooxy N=O bonds compared to amine-CDP mixtures previously proposed. Below is the mechanism proposed after review of literature and experimentation.R-OH O R-CNH2Q, ^, P — HO"? ^OH HO-? phosphate©° °Q 0O O@ O@■oxime formation adduct fragmentation

[0036] Carbonyl phosphonates other than CDP have also been reported as deblocking reversible terminators bearing 3‘ aminooxy groups. For example, acetyl phosphonates bearing only a single phosph onate group have been shown to have cleavage activity (see US Patent Application Number 18 / 753,625). The mechanism below abstracts the cleavage of carbonyl phosphonates beyond carbonyl diphosphonate.R ROHo „ R-OO % „ A 'P >- J? fi — > ^'~OH R’ P~OH phosphate°© °0 oxime formation adduct fragmentation

[0037] As can be seen by the general reaction mechanism below, the rate limiting step is the dehydration step, i.e., loss of water, which forms the activated imine compound. Simply stated, general ketone bearing compounds such as CDP are kinetically limited when undergoing direct condensation reactions with amine nucleophiles of typical blocking moi eties.kinetic limitations of directcondensation

[0038] Provided herein, as illustrated by the general reaction mechanism below, is the use of imine phosphonates capable of undergoing oxime formation followed by adduct fragmentation, thereby producing the free hydroxy to serve as a reactive site for nucleotide incorporation. The below mechanism shows oxime formation from imine phosphonates that comprise one or two phosphonate groups, such as acetyl phosphonate or carbonyl diphosphonate.phosphateoxime formation adduct fragmentation imine phosphonate

[0039] The proposed reaction mechanism, shown below, provides an increased rate of oxime formation, by avoiding the kinetic limitations seen with generally used carbonyl phosphonates. In one aspect, the present disclosure relates to activated imine compounds, wherein the rate limiting dehydration step is avoided allowing for a kinetic enhancement of bioconjugation leading to rapid adduct fragmentation. These strategies demonstrate an important development in nucleic acid chemistry.amine catalystsproposed kineticenhancement

[0040] While the above mechanisms show an unstable imine intermediate in the cleavage of aminooxy N=O bond with a carbonyl precursor, it will be understood that the iminecompounds described herein are stable, in equilibrium, and are not a transient intermediate. The benefit of the imine compounds described herein is that they are stable and when combined with aminooxy compounds, such as when combined with nucleotides comprising an aminooxy N=O bond, the compositions of the subject application provide an efficient cleavage of the N=O bond. As such, compositions described herein may be stable, in equilibrium, and may lack any aminooxy substrates.

[0041] These imine phosphonate reagents are understood, based on the experiments reported herein, to undergo deblocking reactions through a similar mechanism relative to that of CDP. The imine undergoes reaction with the oxime, which then fragments to form the deblocked alcohol, cyanophosphonate, and phosphate. Oxime formation is accelerated when using these imines as the starting materials owing to the presence of internal proton donors that can catalyze oxime formation.

[0042] The above mechanism is based on our understanding that 1) compounds that convert hydroxyl amines to alcohols must first form an oxime intermediate (e.g. CDP, monophosphonates, and the like), 2) oxime formation is rate determining, especially at concentrations relevant for efficient cleave reaction (e g. 0.1 mM cleave reagent or higher), and 3) activated imines form oximes more rapidly than the corresponding carbonyl derivative. The increased rate of oxime formation by imine phosphonate (as compared to carbonyl phosphonate analogs) has been demonstrated by the examples and figures provided herein. As such, imines may be more active reagents in general for converting hydroxylamines to alcohols. If carbonylsulfonates show some level of cleave through a similar mechanism to phosphonates, then imine sulfonates are expected to be more active in enabling this same chemical transformation. Carbonyl sulfonates may therefore be converted to imine sulfonates through mixture with suitable primary amines described herein and said imine sulfonates may demonstrate cleavage of aminooxy N=O bonds. The phosphonates of imine phosphonate compounds described herein may be substituted with sulfonates, and imine sulfonates may be used in any of the embodiments described herein.

[0043] The above mechanism of fast cleavage of aminooxy N=O bonds by imine phosphonates predicts that imine phosphonates may exhibit significantly faster cleavage (e.g., 30x faster) than their carbonyl phosphonate analogs which must first undergo the slow step shown above. This is borne out by experimental data described in examples and figures herein. Carbonyl diphosphonate may compete for the same sites as imine diphosphonates and could slow down cleavage by imine phosphonate. As such, formulations of iminephosphonate described herein may be more effective with minimal or no (e.g., undetectable) CDP.

[0044] The presence of CDP in the herein provided imine phosphonate compositions or formulations, contrary to assisting in cleave, may reduce the efficiency of the overall reaction. This is because CDP may occupy aminooxy groups during a slow step that does not complete during the period of incubation, thereby preventing more efficient cleavage by imine phosphonate. Phasing issues may therefore arise from CDP if incubated for short periods of time.

[0045] CDP is known to precipitate from solution, such as at pH 5.5 in acetate buffer. This has been a major issue to date since precipitates can damage instruments and flow cells. Efforts to improve the solubility of deblock active formulations have focused on decreasing carbonyl diphosphonate from solution to prevent precipitation from occurring.

[0046] In terms of mechanism for precipitation, it is hypothesized that CDP can form an equilibrium with the corresponding hydrated form at lower pH, which precipitates from solution. This can be seen when 50 mM CDP solutions are stored in 1 M acetate buffer. The solution begins having a bright yellow color but loses color rapidly at 4 °C or -20 °C storage with the concomitant formation of colorless / white precipitates. The precipitates are thought to be primarily CDP hydrate (or oligomers). It is believed the hydrated form primarily because of the loss of color. CDP itself is yellow, and the loss of this yellow color is indicative of disruption to the carbonyl bond. For this and the above reasons, compositions of imine phosphonate for cleavage of aminooxy N=O bonds have been developed that have no detectible level of CDP.

[0047] Improving solubility of CDP with organic counterions (i.e., protonated organic bases that form a salt with CDP) has been described previously. Amines have been proposed in this context but only as a counterion (protonated base), and no amine solubility enhancers have been proposed that would form an imine phosphonate from a carbonyl phosphonate such as CDP. As such, the use of certain primary amines in mixture with CDP to form imine phosphonate has not been previously reported. The description of amines as counterions for CDP is a different mechanism from the imine phosphonate formation described herein and the use of amines as counterions precludes their use for forming imine phosphonate.

[0048] Further, due to the insolubility of CDP under certain conditions, the loss of color when CDP is in admixture with the primary amines described herein for imine phosphonate formation could indicate CDP had precipitated out of solution and the resultant solution wasnot suitable for cleavage of aminooxy N=O bonds. Reported herein is such a color change indicating the formation of a highly pure imine diphosphonate from CDP. The type of primary amine combined with CDP and its concentration can affect the purity of the resulting imine phosphonate. This high purity also makes it more desirable to isolate or purify the imine phosphonate by means described herein.

[0049] Described further herein is the discovery and mechanism of imine phosphonate formation from mixtures of select primary amines with carbonyl phosphonates such as CDP, and the enhanced aminooxy N=O bonds cleavage efficiency of the imine phosphonates reported herein. Of note, while CDP is referred to in a number of embodiments, other carbonyl phosphonates, such as acetyl phosphonate, demonstrating cleavage of aminooxy N=O bond and are within the scope of the subject application. Similarly, while imine diphosphonates are referred to in a number of embodiments, other imine phosphonates such as compounds comprising a single phosphonate group are within the scope of the subject application. Carbonyl phosphonates, as used herein, refer to carbonyl groups sharing a carbon with one or two phosphonates. Imine phosphonates, as used herein, refer to imine groups sharing a carbon with one or two phosphonates. The group bonded to the N of the imine is the group bonded to the primary amine that reacts with the carbonyl phosphonate to form the imine phosphonate.

[0050] Before the present disclosure is described in detail, it is to be understood that the terminology used herein is for purposes of describing particular examples and illustrative embodiments only and is not intended to be limiting.DEFINITIONS

[0051] Compounds are described using standard nomenclature. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the invention belongs.

[0052] The terms “a” and “an’" do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. The term "or” means "and / or”. The term ■‘and / or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of’ or “one or more” of the listed items is used or present.

[0053] As used herein, the terms “optional” or “optionally” mean that the subsequently described feature or structure may or may not be present, or that the subsequently described event or circumstance may or may not occur, and that the description includes instanceswhere a particular feature or structure is present and instances where the feature or structure is absent, or instances where the event or circumstance occurs and instances where it does not.

[0054] As used herein, '‘comprising’’ is synonymous with '‘including,” ‘'containing,” or ■‘characterized by,” and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, “consisting of’ excludes any element, step, or ingredient not specified in the claim element. As used herein, “consisting essentially of does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term “comprising”, particularly in a description of components of a composition, in a description of a method, or in a description of elements of a device, is understood to encompass those compositions, methods, or devices consisting essentially of and consisting of the recited components or elements, optionally in addition to other components or elements. The invention illustratively described herein suitably may be practiced in the absence of any element, elements, limitation, or limitations which is not specifically disclosed herein.

[0055] As used herein, the term "each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.

[0056] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0057] As used herein, a dashthat is not between two letters or symbols is used to indicate a point of attachment for a substituent. For example, -(C=O)NH2 is attached through the carbon of the keto (C=O) group.

[0058] As used herein, “analog” or “analogue” is used in accordance with its plain ordinary' meaning within Chemistry and Biology and refers to a chemical compound that may be structurally similar to another compound (i.e., a so-called “reference” compound) but differs in composition, e g., in the replacement of one atom by an atom of a different element, or in the presence of a particular functional group, or the replacement of one functional group by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound. Accordingly, an analog may be a similar compound or a compound comparable in function and appearance but not in structure or origin to a reference compound.

[0059] Certain compounds of the present invention may possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute stereochemistry’, as (R)-or (S)-, and individual isomers are encompassed yvithin the scope of the present disclosure. The compounds of the present disclosure do not include those that are know n in art to be too unstable to synthesize and / or isolate. The present disclosure may include compounds in racemic and optically pure forms. Optically active (R)- and (S)-isomers may be prepared using chiral synthons or chiral reagents or resolved using conventional techniques.

[0060] As used herein, the term “isomers” refers to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms.

[0061] As used herein, the term “tautomer” refers to one of two or more structural isomers which exist in equilibrium, and which are readily converted from one isomeric form to another.

[0062] As used herein, the term “salt” refers to acid or base salts of the compounds used in the methods of the present disclosure. Illustrative examples of acceptable salts are mineral acid (hydrochloric acid, hydrobromic acid, phosphoric acid, and the like) salts, organic acid (acetic acid, propionic acid, glutamic acid, citric acid and the like) salts, quaternary ammonium (methyl iodide, ethyl iodide, and the like) salts.

[0063] Certain compounds of the present disclosure can exist in unsolvated forms as well as solvated forms, including hy drated forms. In general, the solvated forms are equivalent to unsolvated forms and are encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. Ingeneral, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention.

[0064] Description of compounds of the present disclosure may be limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding and to give compounds which are not inherently unstable and / or would be known to one of ordinary skill in the art as likely to be unstable under ambient conditions, such as aqueous, neutral, and several known physiological conditions. For example, a heterocycloalkyl or heteroaryl is attached to the remainder of the molecule via a ring heteroatom in compliance with principles of chemical bonding known to those skilled in the art thereby avoiding inherently unstable compounds.

[0065] Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH2O- is equivalent to -OCH2-.

[0066] As used herein, “alkyl” is a branched or straight chain saturated aliphatic hydrocarbon group. In some cases, the alkyl group contains from 1 to about 30 carbon atoms, more generally from 1 to about 20 carbon atoms, or from 1 to about 12 carbon atoms, or from 1 to about 8 carbon atoms. In some cases, the alkyl contains from 1 to about 20 carbon atoms. In some cases, the alkyl is C1-C2, C1-C3, C1-C4, C1-C5, Ci-Ce, C1-C7, Ci-Cs, C1-C9, C1-C10, C1-C11, C1-C12, C1-C13, C1-C14, C1-C15, C1-C16, C1-C17, C1-C18, C1-C19, C1-C20, C1-C21, C1-C22, C1-C23. C1-C24. C1-C25. C1-C26, C1-C27, C1-C28, C1-C29, or Ci-C30. The specified ranges as used herein indicate an alkyl group having each member of the range described as an independent species. For example, the term C1-C20 alkyl as used herein indicates a straight or branched alkyl group having from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms and is intended to mean that each of these is described as an independent species. For example, the term C1-C4 alkyl as used herein indicates a straight or branched alkyl group having from 1, 2, 3, or 4 carbon atoms and is intended to mean that each of these is described as an independent species. Examples of alky l include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, secbutyl. t-butyl. n-pentyl. isopentyl, tert-pentyl, neopentyl, n-hexyl, 2-methylpentane. 3-methylpentane, 2,2-dimethylbutane, and 2,3-dimethylbutane. In some cases, the alky l group is optionally substituted. The term “alky l” also encompasses cycloalkyl or carbocyclicgroups. For example, when a term is used that includes “alk” then “cycloalkyd” or “carbocyclic” can be considered part of the definition, unless unambiguously excluded by the context. For example, and without limitation, the terms alkyl, alkoxy, etc. can all be considered to include the cyclic forms of alkyl, unless unambiguously excluded by context.

[0067] As used herein, “alkenyl” is a linear or branched aliphatic hydrocarbon groups having one or more carbon-carbon double bonds that may occur at a stable point along the chain. In some cases, “alkenyl” is used to indicate those alkenyl groups having 2-20 carbons. The specified ranges as used herein indicate an alkenyl group having each member of the range described as an independent species, as described above for the alkyl moiety.Examples of alkenyl radicals include, but are not limited to ethenyl, propenyl, allyl, propenyl, butenyl and 4-methylbutenyl. The term “alkenyl” also embodies “cis” and “trans” alkenyl geometry, or alternatively, “E” and “Z” alkenyl geometry. In an alternative embodiment, the alkenyl group is optionally substituted. The term “Alkenyl” also encompasses cycloalkyl or carbocyclic groups possessing at least one point of unsaturation.

[0068] As used herein, “alkynyl” is a branched or straight chain aliphatic hydrocarbon group having one or more carbon-carbon triple bonds that may occur at any stable point along the chain. In some cases, “alkynyl” is used to indicate those alkenyl groups having 2-20 carbons. The specified ranges as used herein indicate an alkynyl group having each member of the range described as an independent species, as described above for the alkyl moiety. Examples of alky ny l include, but are not limited to, ethynyl, propynyl, 1-butynyl, 2-butynyl, 3-butynyl, 1 -pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl and 5-hexynyl. In some cases, the alkynyl group is optionally substituted. The term “alkynyl” also encompasses cycloalkyl or carbocyclic groups possessing at least one point of unsaturation.

[0069] As used herein, “alkoxy” is an alkyl group as defined above covalently bound through an oxygen bridge (-O-). Examples of alkoxy include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, 2-butoxy, t-butoxy, n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy, neopentoxy, n-hexoxy, 2-hexoxy, 3-hexoxy, and 3 -methylpentoxy. Similarly, an “alkylthio” or “thioalkyl” group is an alkyl group as defined above with the indicated number of carbon atoms covalently bound through a sulfur bridge (-S-).

[0070] As used herein, “alkanoyl” and “acyl” are used interchangeably. As used herein, "acyl” means, unless otherwise stated, -C(O)R where R is an alkyl group as defined above. For example, R may be a substituted or unsubstituted alkyl, substituted or unsubstitutedcycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. The carbonyl carbon is included in the number of carbons, that is C2alkanoyl is a -(C=O)CHs group.

[0071] As used herein, “aliphatic” refers to a saturated or unsaturated, straight, branched, or cyclic hydrocarbon. “Aliphatic” is intended herein to include, but is not limited to, alkyd, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, and cycloalkynyl moieties. and thus incorporates each of these definitions. In some cases, “aliphatic” is used to indicate those aliphatic groups having 1-30 carbon atoms. The aliphatic group can be for example, alkyl, mono-unsaturated, di-unsaturated, tri-unsaturated, or polyunsaturated, or alky nyl. Unsaturated aliphatic groups can be in a cis or trans configuration. In some cases, the aliphatic group contains from 1 to about 20 carbon atoms, more generally from 1 to about 12 carbon atoms or from 1 to about 8 carbon atoms. In one embodiment, the aliphatic group contains from 1 to about 20 carbon atoms. In certain embodiments, the aliphatic group is C1-C2, C1-C3, C1-C4, C1-C5, Ci-Ce, Ci-C7, C1-C8, C1-C9, C1-C10, C1-C11, C1-C12, C1-C13, C1-C14, C1-C15, C1-C16, C1-C17, C1-C18, C1-C19. C1-C20, C1-C21, C1-C22, C1-C23, C1-C24, C1-C25, C1-C26, C1-C27, C1-C28, C1-C29, or C1-C30. The specified ranges as used herein indicate an aliphatic group having each member of the range described as an independent species. For example, the term C1-C20 aliphatic as used herein indicates a straight or branched alky l, alkenyl, or alkynyl group having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms and is intended to mean that each of these is described as an independent species. For example, the term C1-C4 aliphatic as used herein indicates a straight or branched alkyl, alkenyl, or alkynyl group having from 1, 2, 3, or 4 carbon atoms and is intended to mean that each of these is described as an independent species.

[0072] As used herein, “carbocyclyl”, “carbocyclic”, “carbocycle” or “cycloalkyl” is a saturated or partially unsaturated (i. e., not aromatic) group containing all carbon ring atoms and from 3 to 14 ring carbon atoms (“C3-14 cycloalkyl”) and zero heteroatoms in the nonaromatic ring system. In some cases, a cycloalkyd group has 3 to 10 ring carbon atoms (“C3-10 cycloalky 1”). In some cases, a cycloalky 1 group has 3 to 9 ring carbon atoms (“C3-9 cycloalkyl”). In some cases, a cycloalkyl group has 3 to 8 ring carbon atoms (“C3-8 cycloalkyl”). In some cases, a cycloalkyl group has 3 to 7 ring carbon atoms (“C3-7 cycloalkyd”). In some cases, a cycloalkyd group has 3 to 6 ring carbon atoms (“C3-6 cycloalkyd”). In some cases, a cycloalkyd group has 4 to 6 ring carbon atoms (“C4-6 cycloalkyl”). In some cases, a cycloalkyl group has 5 to 6 ring carbon atoms (“C5-6cycloalky l”). In some cases, a cycloalkyl group has 5 to 10 ring carbon atoms (“C5-10 cycloalkyl”). Exemplary Cs 6 cycloalkyl groups include, without limitation, cyclopropyl (C3), cyclopropenyl (C3), cyclobutyl (C4), cyclobutenyl (C4), cyclopentyl (Cs), cyclopentenyl (Cs), cyclohexyl (Ce), cyclohexenyl (Ce), cyclohexadienyl (Ce), and the like. Exemplary C3-8 cycloalky l groups include, without limitation, the aforementioned C3-6 cycloalkyl groups as well as cycloheptyl (C7), cycloheptenyl (C7), cycloheptadienyl (C7), cycloheptatrienyl (C7), cyclooctyl (Cs), cyclooctenyl (Cs). and the like. Exemplary C3-10 cycloalkyl groups include, without limitation, the aforementioned C3-8 cycloalkyl groups as well as cyclononyl (C$>), cyclononenyl (C9), cyclodecyl (C10), cyclodecenyl (C10), and the like. As the foregoing examples illustrate, in certain embodiments, the cycloalkyl group can be saturated or can contain one or more carbon-carbon double or triple bonds. In some cases, “cycloalkyl” also includes ring systems wherein the cycloalkyl ring, as defined above, is fused with one heterocycle, aryl or heteroar l ring wherein the point of attachment is on the cycloalkyl ring, and in such instances, the number of carbons continue to designate the number of carbons in the carbocyclic ring system. In some cases, each instance of cycloalkyl is optionally substituted with one or more substituents. In some cases, the cycloalkyl group is an unsubstituted C3-14 cycloalkyl.

[0073] As used herein, the term “aryl” means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (for example from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently. A fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring. The term “heteroaryl” refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quatemized. Thus, the term “heteroaryl” includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroary l ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. And a 6,5-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroary l ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl. pyridazinyl, triazinyl,pyrimidiny 1, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, fury l, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl, indolyl. isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1-naphthy 1, 2-naphthyl, 4- biphenyl, 1 -pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazoly 1, 4-imidazolyl, pyrazinyl, 2- oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2- thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl. 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl. 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyL 1 -isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5 -quinoxalinyl, 3-quinolyl, and 6-quinolyl. Example substituents for each of the above noted ary l and heteroaryl ring systems are described below.

[0074] As used herein, the terms “heteroatom” or “ring heteroatom'’ means, unless otherwise stated, atoms other than carbon or hydrogen.

[0075] As used herein, the terms “heteroalkyl.” “heterocycloalkyl,” “heteroalkenyl,” “heterocycloalkenyl,” or “heteroaryl” unless otherwise stated refer to the inclusion of a heteroatom in alkyd, cycloalkyl, alkeny l, cycloalkenyl or ary 1 as described herein.

[0076] As used herein the terms “cycloalkyd,” “heterocycloalky 1,” “cycloalkenyl,” “heterocycloalkenyl,” unless otherwise stated refer to cyclic versions of the alkyl, heteroalkyl, alkenyl, and heteroalkenyl as described herein.

[0077] Each of the above terms (e.g., “alkyl,” “cycloalkyl,” “heteroalkyl,” “heterocycloalkyl,” “alkenyl,” “heteroalkenyl,” “cycloalkenyl,” “heterocycloalkenyl,” “aryl,” and “heteroaryd”) includes both substituted and unsubstituted forms of the indicated group.

[0078] In some cases, each substituted group described in the compounds herein may be substituted with at least one substituent group. More specifically, in some cases, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalky dene, substituted heterocycloalky dene, substituted arylene, and / or substituted heteroarylene described in the compounds herein are substituted with at least one substituent group.

[0079] Substituents for the alkyl and heteroalkyl groups (including those groups often referred to as alkylene, alkenyl, heteroalky dene, heteroalkenyl, alkynyl, cycloalk d, heterocycloalkyd, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to, -OR', =0, =NR', =N-0R', -NR'R", -SR', -OC(O)R', -C(O)R', -CO2R', -CONR'R", -OC(O)NR'R", -NR" C(O)R', -NR'-C(O)NR" R"', -NR" C(O)2R', -NR-C(NR'R" R"')=NR"", -NR- C(NR'R")=NR"', -S(O)R', -S(O)2R', -S(O)2NR'R", -NRSO2R', -NR'NR" R'", -ONR'R", -NR,C=(O)NR" NR"'R"", -CN, -NO2, -NR'SO2R", -NR'C=(O)R", -NR’C(O)-OR", or -NR'OR". R, R', R", R'", and R"" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When a compound of the disclosure includes more than one R group, for example, each of the R groups may be independently selected as are each R', R", R'", and R"" group when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-. 5-, 6-, or 7-membered ring. For example, -NR'R" includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term ‘‘alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as acyl (e.g.. -C(O)CH3, -C(O)CH2OCH3, and the like).

[0080] Substituents for rings (e.g. cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene) may be depicted as substituents on the ring rather than on a specific atom of a ring (commonly referred to as a floating substituent). In such a case, the substituent may be attached to any of the ring atoms (obeying the rules of chemical valency). When a substituent is attached to a ring, but not a specific atom (a floating substituent), and a subscript for the substituent is an integer greater than one, the multiple substituents may be on the same atom, same ring, different atoms, and each substituent may optionally be different. Where a point of attachment of a ring to the remainder of a molecule is not limited to a single atom (a floating substituent), the attachment point may be any atom of the ring while obeying the rules of chemical valency. Where a ring contains one or more ring heteroatoms and the ring, is shown with one more floating substituents (including, but not limited to, points of attachment to the remainder of the molecule), the floating substituents may be bonded to the heteroatoms. Where the ring heteroatoms are shown bound to one or more hydrogens (e.g. a ring nitrogen with two bonds to ring atoms and a third bond to a hydrogen) in the structure or formula with the floating substituent, when the heteroatom is bonded to the floating substituent, the substituent will be understood to replace the hydrogen, while obeying the rules of chemical valency.

[0081] In some cases, a “substituent group,” as used herein, means a group selected from the following moieties: -CN, -OH, -NH2, -COOH. -CONH2. -NO2, -SH. -SCH3, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -NHC(O)NHNH2, -NHC(O)NH2, -NHSO2H, -NHC(O)H, -NHC(O)OH, -NHOH. - O-alkyl, -O-heteroalkyl. -O-cycloalkyl. -O-heterocycloalkyl, -O-aryl, -O-heteroaryL -O-alkenyl, -O-heteroalkenyl, -O-heterocycloalkenyl, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, and / or substituted or unsubstituted heteroaryl.

[0082] As used herein, the term “blocking moiety,” when used in reference to a nucleotide, means a part of the nucleotide that inhibits or prevents the 3 ' oxygen of the nucleotide from forming a covalent linkage to a next correct nucleotide during a nucleic acid polymerization reaction. The blocking moiety of a “reversibly terminated” nucleotide can be removed from the nucleotide analog, or otherwise modified, to allow the 3’ - oxygen of the nucleotide to covalently link to a next correct nucleotide. Such a blocking moiety is referred to herein as a “reversible terminator moiety.” Exemplary reversible terminator moieties are set forth in U. S. Pat. Nos. 7.427,673; 7.414.116; 7,057.026; 7,544,794 or 8,034,923; or PCT publications WO 91 / 06678 or WO 07 / 123744, each of which is incorporated herein by reference. A nucleotide that has a blocking moiety or reversible terminator moiety' can be at the 3’ end of a nucleic acid, such as a primer, or can be a monomer that is not covalently attached to a nucleic acid. A blocking moiety need not hinder or preclude ternary complex formation at the 3’ end of a nucleic acid to which the blocking moiety is attached.

[0083] As used herein, the term “deblock” means to remove or modify a reversible terminator moiety of a nucleotide to render the nucleotide extendable. For example, the nucleotide can be present at the 3 ’ end of a primer such that deblocking renders the primer extendable.

[0084] As used herein, the term “extension,” when used in reference to a nucleic acid, means a process of adding at least one nucleotide to the 3’ end of the nucleic acid. The term “polymerase extension," when used in reference to a nucleic acid, refers to a polymerase catalyzed process of adding at least one nucleotide to the 3’ end of the nucleic acid. A nucleotide or oligonucleotide that is added to a nucleic acid by extension is said to be incorporated into the nucleic acid. A template dependent polymerase, such as Klenow fragment, 9 degrees north, or Phi29, incorporates nucleotides into the 3’ end of a primer hybridized to a template nucleotide. A template independent polymerase, such as TdT,incorporates nucleotides into a single strand of DNA at an 3’ end that is not hybridized to a template. Accordingly, the term "incorporating" can be used to refer to the process of joining a nucleotide or oligonucleotide to the 3' end of a nucleic acid by formation of a phosphodiester bond.

[0085] As used herein, the term “primer” refers to a nucleic acid having a sequence that binds to a nucleic acid at or near a template sequence. Generally, the primer binds in a configuration that allows replication of the template, for example, via polymerase extension of the primer. The primer can be a first portion of a nucleic acid molecule that binds to a second portion of the nucleic acid molecule, the first portion being a primer sequence and the second portion being a primer binding sequence (e.g. a hairpin primer). Alternatively, the primer can be a first nucleic acid molecule that binds to a second nucleic acid molecule having the template sequence. A primer can consist of DNA, RNA or analogs thereof. A primer can have an extendible 3’ end, a 3’ end that is blocked from primer extension or a 3’ end that is capped to hinder or preclude ternary complex formation.

[0086] As used herein, the term “template” means a nucleic acid, or portion thereof, having a sequence of nucleotide bases that act as a pattern for producing a complementary copy of the sequence. A template can be copied via extension of a primer that is hybridized at or adjacent to the template. Extension can be mediated by a polymerase or ligase. A template can consist of DNA, RNA or analogs thereof.

[0087] As used herein, the term “primer-template nucleic acid hybrid” or “primer- template hybrid” refers to a nucleic acid having a double stranded region such that one of the strands is a primer and the other strand is a template. The two strands can be parts of a contiguous nucleic acid molecule (e.g. a hairpin structure) or the two strands can be separable molecules that are not covalently attached to each other.

[0088] As used herein, the term “solid support” refers to a rigid substrate that is insoluble in aqueous liquid. The substrate can be non-porous or porous. The substrate can optionally be capable of taking up a liquid (e.g. due to porosity) but will typically be sufficiently rigid that the substrate does not swell substantially when taking up the liquid and does not contract substantially when the liquid is removed by drying. A nonporous solid support is generally impermeable to liquids or gases. Exemplary solid supports include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TEFLON™, cyclic olefins, polyimides etc ), nylon, ceramics, resins,ZEONOR®, silica or silica-based materials including silicon and modified silicon, carbon, metals, inorganic glasses, optical fiber bundles, and polymers.

[0089] As used herein, the term "‘extendable,” when used in reference to a nucleotide, means that the nucleotide has an oxygen or hydroxyl moiety at the 3’ position, and is capable of forming a covalent linkage to a next correct nucleotide if and when incorporated into a nucleic acid. An extendable nucleotide can be at the 3' position of a primer or it can be a monomeric nucleotide. A nucleotide that is extendable will lack blocking moieties such as reversible terminator moieties.

[0090] As used herein, the term ‘‘nucleotide” can be used to refer to a native nucleotide or analog thereof. Examples include, but are not limited to, nucleotide triphosphates (NTPs) such as ribonucleotide triphosphates (rNTPs), deoxyribonucleotide triphosphates (dNTPs), or non-natural analogs thereof such as dideoxyribonucleotide triphosphates (ddNTPs) or reversibly terminated nucleotide triphosphates (rtNTPs).

[0091] As used herein, the term ‘‘next correct nucleotide” refers to the nucleotide type that will bind and / or incorporate at the 3’ end of a primer to complement a base in a template strand to which the primer is hybridized. The base in the template strand is referred to as the “next base” and is immediately 5’ of the base in the template that is hybridized to the 3’ end of the primer. The next correct nucleotide can be referred to as the “cognate” of the next base and vice versa. Cognate nucleotides that interact with each other in a ternary complex or in a double stranded nucleic acid are said to “pair” with each other. A nucleotide having a base that is not complementary to the next template base is referred to as an “incorrect”, “mismatch” or “non-cognate” nucleotide. The next correct nucleotide can be a detectable (e.g., fluorescent) nucleotide most recently incorporated into the 3’ end of the primer or a detectable (e.g., fluorescent) nucleotide that is stabilized in a ternary complex without being incorporated into the primer.

[0092] As used herein, the term “oligonucleotide” or “polynucleotide” refers to a portion of a nucleic acid that includes at least 2 contiguous nucleotides. An oligonucleotide or polynucleotide can include, for example, at least 5, 8, 10, 15, 20, 25, 50, 100 or more contiguous nucleotides. The terms include, but are not limited to, DNA, RNA, analogs (e.g., derivatives) thereof or any combination thereof, that can be acted upon by a polymerizing enzyme. The term includes single-, double-, or multiple-stranded DNA. RNA and analogs (e.g., derivatives) thereof. Double-stranded nucleic acids advantageously can minimize secondary structures that may hinder nucleic acid synthesis. A double stranded nucleic acidmay possess a nick or a single-stranded gap. A nucleic acid may represent a single, plural, or clonally amplified population of nucleic acid molecules. The polynucleotide may be a primer hybridized to a template nucleic acid.

[0093] As used herein, the term ‘"label” refers to a molecule, or moiety thereof, that provides a detectable characteristic. The detectable characteristic can be, for example, an optical signal such as absorbance of radiation, fluorescence emission, luminescence emission, fluorescence lifetime, fluorescence polarization, or the like; Rayleigh and / or Mie scattering; binding affinity for a ligand or receptor; magnetic properties; electrical properties; charge; mass; radioactivity or the like. Exemplary labels include, without limitation, a fluorophore, luminophore, chromophore, nanoparticle (e.g., gold, silver, carbon nanotubes), heavy atoms, radioactive isotope, mass label, charge label, spin label, receptor, ligand, or the like.

[0094] As used herein, the term “polymerase” can be used to refer to a nucleic acid synthesizing enzyme, including but not limited to. DNA polymerase, RNA polymerase, reverse transcriptase, primase and transferase. Typically, the polymerase has one or more active sites at which nucleotide binding and / or catalysis of nucleotide polymerization may occur. The polymerase may catalyze the polymerization of nucleotides to the 3‘ end of the first strand of the double stranded nucleic acid molecule. For example, a polymerase catalyzes the addition of a next correct nucleotide to the 3’ oxygen group of the first strand of the double stranded nucleic acid molecule via a phosphodiester bond, thereby covalently incorporating the nucleotide to the first strand of the double stranded nucleic acid molecule. Optionally, a polymerase need not be capable of nucleotide incorporation under one or more conditions used in a method set forth herein. For example, a mutant polymerase may be capable of forming a ternary complex but incapable of catalyzing nucleotide incorporation. Further, polymerases can be template dependent or non-template dependent. For example, non-template dependent polymerases such as terminal deoxynucleotidyl transferase (TdT) can be used in DNA synthesis.

[0095] As used herein, the term “ternary complex” refers to an intermolecular association between a polymerase, a double stranded nucleic acid and a nucleotide. Typically, the polymerase facilitates interaction between a next correct nucleotide and a template strand of the primed nucleic acid. A next correct nucleotide can interact with the template strand via Watson-Crick hydrogen bonding. The term “stabilized ternary complex” means a ternary complex having promoted or prolonged existence or a ternary complex for which disruption has been inhibited. Generally, stabilization of the ternary complex prevents covalentincorporation of the nucleotide component of the ternary complex into the primed nucleic acid component of the ternary complex.

[0096] As used herein, the term "type" or "‘species” is used to identify molecules that share the same chemical structure. For example, a mixture of nucleotides can include several dCTP molecules. The dCTP molecules will be understood to be the same type (or species) of nucleotide as each other, but a different type (or species) of nucleotide compared to dATP, dGTP, dTTP etc. Similarly, individual DNA molecules that have the same sequence of nucleotides are the same type (or species) of DNA, whereas DNA molecules with different sequences are different types (or species) of DNA. The term “type” or “species” can also identify' moi eties that share the same chemical structure. For example, the cytosine bases in a template nucleic acid will be understood to have the same type (or species) of base as each other independent of their position in the template sequence.

[0097] As used herein, the term “catalytic metal ion” refers to a metal ion that facilitates phosphodiester bond formation between the 3’-oxygen of a nucleic acid (e g., a primer) and the phosphate of an incoming nucleotide by a polymerase. A “divalent catalytic metal cation” is a catalytic metal ion having a valence of two. Catalytic metal ions can be present at concentrations that stabilize formation of a complex between a polymerase, nucleotide, and primed template nucleic acid, referred to as non-catalytic concentrations of a metal ion insofar as phosphodiester bond formation does not occur. Catalytic concentrations of a metal ion refer to the amount of a metal ion sufficient for polymerases to catalyze the reaction between the 3 '-oxygen of a nucleic acid (e.g., a primer) and the phosphate moiety of an incoming nucleotide. Exemplary catalytic metal ions include Mg2+and Mn2+.

[0098] As used herein, the terms “non-catalytic metal ion” or “inhibitory metal ion” refers to a metal ion that, when in the presence of a polymerase enzyme, inhibits phosphodiester bond formation needed for chemical incorporation of a nucleotide into a primer. An inhibitory metal ion may interact with a polymerase, for example, via competitive binding compared to catalytic metal ions. A "divalent inhibitory metal ion” is an inhibitory metal ion having a valence of two. Examples of divalent inhibitory metal ions include, but are not limited to, Ca2+, Zn2+, Co2+, Ni2+, and Sr2+. The trivalent Eu3+ and Tb3+ ions are inhibitory metal ions having a valence of three.

[0099] As used herein, the term “cycle,” when used in reference to a sequencing process, refer to the portion of a sequencing run that is repeated to indicate the presence of a nucleotide. Typically, a cycle includes several steps such as steps for delivery of reagents,washing away unreacted reagents and detection of signals indicative of changes occurring in response to added reagents.COMPOSITION

[0100] The present disclosure provides a composition, comprising a buffer and an imine compound of Formula (I), Formula (la), Formula (lb), Formula (Ic), Formula (Id), and Formula (le) or stereoisomers and salts thereof.

[0101] In some cases, the compositions described herein are compositions, comprising a buffer and an imine compound of Formula (I):or stereoisomers and salts thereof, wherein:n is 0, 1, 2, or 3;m is 0 to 20R1is a bond, H, –CH2–, or –NH–;R2is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polymer, or -NR4R5, wherein R4and R5are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted polymer; andR3is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polymer, -P(O)?\ or -NR4R5, wherein R4and R5are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyd, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted polymer,wherein when R1is a bond and m is 0, n is i,wherein when R1is -NH- and m is 0, n is 1, andwherein when R1is H, m and n are 0.

[0102] In some cases, Formula (I) has a structure of Formula (la), Formula (lb), Formula (Ic), Formula (Id), or Formula (le):

[0103] In some cases, Formula (I) has a structure of Formula (la), Formula (Ic), or Formula (Id). In some cases. Formula (I) has a structure of Formula (lb) or Formula (le).

[0104] In some cases, m is 0, 1 or 2. In some cases m is 0. In some cases, m is 1. In some cases, m is 2.

[0105] In some cases, n is 1, 2, or 3. In some cases, n is 1. In some cases, n is 2. In some cases, n is 3.

[0104] In some cases, R1is -CH2- or -CH-.

[0106] In some cases, R2is substituted or unsubstituted C1-C20 alkyl. In some cases, R2is substituted or unsubstituted C1-C2 alkyl, C1-C3 alkyl, C1-C4 alky l, C1-C5 alkyl, Ci-Ce alkyl, C1-C7 alkyl, Ci-C8alky l, C1-C9 alkyd, C1-C10 alkyd, C1-C11 alkyd, C1-C12 alkyd, C1-C13 alkyd, Ci-Cu alkyl, C1-C15 alkyl, C1-C16 alkyl, C1-C17 alkyl, Ci-Cis alkyl, C1-C19 alkyl, or C1-C20 alkyd. In some cases, substituted or unsubstituted C2-C3 alkyl, C2-C4 alkyl, C2-C5 alkyd, C2-C6 alkyd, C2-C7 alkyl, C2-C8 alkyl, C2-C9 alkyl, C2-C10 alkyl, C2-C11 alkyl, C2-C12 alkyl, C2-C13 alkyd, C2-C14 alkyd, C2-C15 alkyd, C2-C16 alkyd, C2-C17 alkyl, C2-C18 alkyl, C2-C19 alkyl, or C2-C20 alkyl. In some cases, R2is substituted or unsubstituted C3-C4 alkyl, C3-C5 alkyl, C3-C6 alkyl, C3-C7 alkyl, Cs-Cs alkyl. C3-C9 alkyl, C3-C10 alkyl, C3-C11 alkyl, C3-C12 alkyl, C3-C13 alkyd. C3-C14 alkyd. C3-C15 alkyd. C3-C16 alkyd. C3-C17 alkyl. C3-C18 alkyl. C3-C19 alkyl, or C3-C20 alkyd. In some cases, R2is substituted or unsubstituted C4-C5 alkyl, C4-C6 alkyl, C4-C7 alkyd, C4-C8 alkyd, C4-C9 alkyd, C4-C10 alkyd, C4-C11 alkyd, C4-C12 alkyd, C4-C13 alkyd, C4-C14 alkyd, C4-C15 alkyd, C4-C16 alkyd, C4-C17 alkyd, C4-C18 alkyl, C4-C19 alkyl, or C4-C20 alkyd.

[0107] In some cases, R2is substituted or unsubstituted C1-C20 heteroalkyl. In some cases, R2is substituted or unsubstitued C1-C2 heteroalkyl, C1-C3 heteroalkyl, C1-C4 heteroalkyl, Ci-C5 heteroalkyd, Ci-Ce heteroalkyl, C1-C7 heteroalkyl, Ci-Cs heteroalkyl, C1-C9 heteroalkyd, C1-C10 heteroalkyd, C1-C11 heteroalkyd, C1-C12 heteroalkyl, C1-C13 heteroalkyd, C1-C14 heteroalky 1, C1-C15 heteroalkyl, C1-C16 heteroalkyl, C1-C17 heteroalkyl, Ci-Cis heteroalky 1, C1-C19 heteroalky l. or C1-C20 heteroalkyl. In some cases, R2is substituted or unsubstituted C2-C3 heteroalkyl, C2-C4 heteroalkyl, C2-C5 heteroalkyl, C2-C6 heteroalkyl, C2-C7 heteroalkyl, C2-C8 heteroalkyl, C2-C9 heteroalky 1, C2-C10 heteroalkyl, C2-C11 heteroalkyl, C2-C12 heteroalky 1, C2-C13 heteroalkyl, C2-C14 heteroalky l, C2-C15 heteroalky l, C2-C16 heteroalkyl, C2-C17 heteroalkyl, C2-C18 heteroalkyl, C2-C19 heteroalkyl, or C2-C20 heteroalkyl. In some cases, R2is substituted or unsubstituted C3-C4 heteroalkyl, C3-C5 heteroalkyl, C3-C6 heteroalkyl, C3-C7 heteroalkyl, C3-C8 heteroalkyd, C3-C9 heteroalkyl, C3-C10 heteroalkyd, C3-C11 heteroalkyd, C3-C12 heteroalkyl, C3-C13 heteroalky 1, C3-C14 heteroalky l, C3-C15 heteroalkyl, C3-C16 heteroalkyl, C3-C17 heteroalkyl, C3-C18 heteroalkyl, C3-C19 heteroalky 1, or C3-C20 heteroalky l. In some cases, R2is substituted or unsubstituted C4-C5 heteroalkyl, C4-C6 heteroalkyl, C4-C7 heteroalkyl, C4-C8 heteroalkyl, C4-C9 heteroalkyl, C4-C10 heteroalkyl, C4-C11 heteroalkyd, C4-C12 heteroalkyl, C4-C13 heteroalky 1, C4-C14 heteroalky l, C4-C15heteroalkyl, C4-C16 heteroalky7!, C4-C17 heteroalkyl, C4-C18 heteroalkyl, C4-C19 heteroalkyd, or C4-C20 heteroalk l.

[0108] In some cases, R2is substituted or unsubstituted C3-C10 cycloalkyl. In some cases, R2is substituted or unsubstituted C3-C4 cycloalkyl, C3-C5 cycloalkyl, C3-C6 cycloalkyl, C3-C7 cycloalky7!, C3-C8 cycloalkyl, C3-C9 cycloalkyl, or C3-C10 cycloalky7!. In some cases, R2is substituted or unsubstituted C4-C5 cycloalkyl, C4-C6 cycloalkyl, C4-C7 cycloalky7!, C4-C8 cycloalkyl, C4-C9 cycloalky7!, or C4-C10 cycloalkyl. In some cases. R2is substituted or unsubstituted C5-C6 cycloalkyl, C5-C7 cycloalkyl, Cs-Cs cycloalkyl, C5-C9 cycloalkyl, or Cs-C10 cycloalky7!. In some cases, R2is substituted or unsubstituted C6-C7 cycloalkyl, Ce-Cs cycloalky l, C6-C9 cycloalky7!, or Ce-Cio cycloalkyl.

[0109] In some cases, R2is substituted or unsubstituted C3-C10 heterocycloalky l. In some cases. R2is substituted or unsubstituted C3-C4 heterocycloalky7!, C3-C5 heterocycloalkyl, C3-Cs heterocycloalkyl, C3-C7 heterocycloalkyl, C3-C8 heterocycloalkyl, C3-C9 heterocycloalkyl, or C3-C10 heterocycloalkyl. In some cases, R2is substituted or unsubstituted C4-C5 heterocycloalkyl, C4-C6 heterocycloalkyl, C4-C7 heterocycloalky7!, C4-C8 heterocycloalkyl, C4-C9 heterocycloalky l, or C4-C10 heterocycloalkyl. In some cases, R2is substituted or unsubstituted C5-C6 heterocycloalkyl, C5-C7 heterocycloalkyl. Cs-Cs heterocycloalkyl, C5-C9 heterocycloalkyl, or C5-C10 heterocycloalkyl. In some cases, R2is substituted or unsubstituted C6-C7 heterocycloalkyl, Ce-Cs heterocycloalkyl, C6-C9 heterocycloalkyl, or Ce-Cio heterocycloalkyl.

[0110] In some cases, R2is substituted or unsubstituted C1-C20 alkeny l. In some cases, R2is substituted or unsubstitued C1-C2 alkenyl, C1-C3 alkenyl, C1-C4 alkenyl, C1-C5 alkenyl. Ci-Ce alkenyl, C1-C7 alkenyl, Ci-Cs alkenyl, C1-C9 alkenyl, C1-C10 alkenyl, C1-C11 alkenyl, C1-C12 alkenyl, C1-C13 alkenyl, C1-C14 alkenyl, C1-C15 alkenyl, C1-C16 alkenyl, C1-C17 alkenyl, Ci-Cis alkenyl, C1-C19 alkenyl, or C1-C20 alkenyl. In some cases, R2is substituted or unsubstituted C2-C3 alkenyl, C2-C4 alkenyl, C2-C5 alkenyl, C2-C6 alkenyl. C2-C7 alkenyl, C2-Cs alkenyl, C2-C9 alkenyl, C2-C10 alkenyl, C2-C11 alkenyl, C2-C12 alkenyl, C2-C13 alkenyl, C2-C14 alkenyl, C2-C15 alkenyl, C2-C16 alkenyl, C2-C17 alkenyl, C2-C18 alkenyl, C2-C19 alkenyl, or C2-C20 alkenyl. In some cases, R2is substituted or unsubstituted C3-C4 alkenyl, C3-C5 alkenyl, C3-C6 alkenyl, C3-C7 alkenyl, C3-C8 alkenyl, C3-C9 alkenyl, C3-C10 alkenyl, C3-C11 alkenyl, C3-C12 alkenyl, C3-C13 alkenyl, C3-C14 alkenyl, C3-C15 alkenyl. C3-C16 alkenyl, C3-C17 alkenyl, C3-C18 alkenyl, C3-C19 alkenyl, or C3-C20 alkenyl. In some cases, R2is substituted or unsubstituted C4-C5 alkenyl, C4-C6 alkenyl, C4-C7 alkenyl, C4-C8 alkenyl, C4-C9 alkenyl, C4-C10 alkenyl, C4-C11 alkenyl, C4-C12 alkenyl, C4-C13 alkenyl, C4-C14 alkenyl, C4-C15 alkenyl, C4-C 16 alkenyl. C4-C17 alkenyl, C4-C18 alkenyl, C4-C19 alkenyl, or C4-C20 alkenyl.

[0111] In some cases, R2is substituted or unsubstituted C1-C20 heteroalkenyl. In some cases, R2is substituted or unsubstitued C1-C2 heteroalkenyl, C1-C3 heteroalkenyl, C1-C4 heteroalkenyl, C1-C5 heteroalkenyl, Ci-Ce heteroalkenyl, C1-C7 heteroalkenyl, Ci-Cs heteroalkenyl, C1-C9 heteroalkenyl, C1-C10 heteroalkenyl, C1-C11 heteroalkenyl, C1-C12 heteroalkenyl, C1-C13 heteroalkenyl, C1-C14 heteroalkenyl, Ci-C 15 heteroalkenyl, C1-C16 heteroalkenyl, C1-C17 heteroalkenyl, Ci-Cis heteroalkenyl, Ci-C 19 heteroalkenyl, or C1-C20 heteroalkenyl. In some cases, R2is substituted or unsubstituted C2-C3 heteroalkenyl, C2-C4 heteroalkenyl, C2-C5 heteroalkenyl, C2-C6 heteroalkenyl, C2-C7 heteroalkenyl, C2-C8 heteroalkenyl, C2-C9 heteroalkenyl, C2-C10 heteroalkenyl, C2-C11 heteroalkenyl, C2-C12 heteroalkenyl, C2-C13 heteroalkenyl, C2-C14 heteroalkenyl, C2-C15 heteroalkenyl, C2-C16 heteroalkenyl, C2-C17 heteroalkenyl, C2-C18 heteroalkenyl, C2-C 19 heteroalkenyl, or C2-C20 heteroalkenyl. In some cases, R2is substituted or unsubstituted C3-C4 heteroalkenyl, C3-C5 heteroalkenyl, C3-C6 heteroalkenyl, C3-C7 heteroalkenyl, C3-C8 heteroalkenyl, C3-C9 heteroalkenyl, C3-C10 heteroalkenyl, C3-C11 heteroalkenyl, C3-C 12 heteroalkenyl, C3-C13 heteroalkenyl, C3-C14 heteroalkenyl, C3-C15 heteroalkenyl, C3-C16 heteroalkenyl, C3-C17 heteroalkenyl, C3-C18 heteroalkenyl, C3-C19 heteroalkenyl, or C3-C20 heteroalkenyl. In some cases, R2is substituted or unsubstituted C4-C5 heteroalkenyl, C4-C6 heteroalkenyl, C4-C7 heteroalkenyl, C4-C8 heteroalkenyl, C4-C9 heteroalkenyl, C4-C10 heteroalkenyl, C4-C11 heteroalkenyl, C4-C12 heteroalkenyl, C4-C13 heteroalkenyl, C4-C 14 heteroalkenyl, C4-C15 heteroalkenyl, C4-C16 heteroalkenyl, C4-C 17 heteroalkenyl, C4-C18 heteroalkenyl, C4-C19 heteroalkenyl, or C4-C20 heteroalkenyl.

[0112] In some cases, R2is substituted or unsubstituted C3-C10 heterocycloalkenyl. In some cases. R2is substituted or unsubstituted C3-C4 heterocycloalkenyl, C3-C5 heterocycloalkenyl. C3-C6 heterocycloalkenyl, C3-C7 heterocycloalkenyl, C3-C8 heterocycloalkenyl, C3-C9 heterocycloalkenyl, or C3-C10 heterocycloalkenyl. In some cases, R2is substituted or unsubstituted C4-C5 heterocycloalkenyl, C4-C6 heterocycloalkenyl, C4-C7 heterocycloalkenyl, C4-C8 heterocycloalkenyl, C4-C9 heterocycloalkenyl, or C4-C10 heterocycloalkenyl. In some cases, R2is substituted or unsubstituted Cs-Ce heterocycloalkenyl, C5-C7 heterocycloalkenyl, Cs-Cs heterocycloalkenyl, C5-C9 heterocycloalkenyl, or C5-C10 heterocycloalkenyl. In some cases, R2is substituted or unsubstituted C6-C7 heterocycloalkenyl, Ce-Cs heterocycloalkenyl, C6-C9 heterocycloalkenyl, or Ce-Cio heterocycloalkenyl.

[0113] In some cases, R2is substituted or unsubstituted C5-C10 aryl. In some cases, R2is substituted or unsubstituted C6-C7 aryl, C6-C8 aryl, C6-C9 aryl, or C6-C10 ary l. In some cases, R2is substituted or unsubstituted C7-C8 aryl, C7-C9 aryl, or C7-C10 aryl. In some cases, R2is substituted or unsubstituted C8-C9 aryl, or C8-C10 aryl.

[0114] In some cases, R2is substituted or unsubstituted C6-C10 heteroaryl. In some cases, R2is substituted or unsubstituted C6-C7 heteroaryl, C6-C8 heteroaryl, C6-C9 heteroaryl, or C6-C10 heteroaryl. In some cases, R2is substituted or unsubstituted C7-C8 heteroaryl, C7-C9 heteroaryl, or C7-C10 heteroaryl. In some cases, R2is substituted or unsubstituted C8-C9 heteroaryl, or C8-C10 heteroaryl.

[0115] In some cases, R2is substituted or unsubstituted polymer. The polymer may be a hydrophilic polymer, such as polyethylene glycol. The polymer may comprise repeating units of ethylamine or propylamine.

[0116] In some cases, R2is substituted or unsubstituted C1-C20 alkoxy. In some cases, R2is substituted or unsubstitued C1-C2 alkoxy, C1-C3 alkoxy, C1-C4 alkoxy, C1-C5 alkoxy, C1-C6 alkoxy, C1-C7 alkoxy, C1-C8 alkoxy, C1-C9 alkoxy, C1-C10 alkoxy, C1-C11 alkoxy, C1-C12 alkoxy, C1-C13 alkoxy, C1-C14 alkoxy, C1-C15 alkoxy, C1-C16 alkoxy, C1-C17 alkoxy, C1-C18 alkoxy, C1-C19 alkoxy, or C1-C20 alkoxy. In some cases, R2is substituted or unsubstituted C2-C3 alkoxy, C2-C4 alkoxy, C2-C5 alkoxy, C2-C6 alkoxy, C2-C7 alkoxy, C2-C8 alkoxy, C2-C9 alkoxy, C2-C10 alkoxy, C2-C11 alkoxy, C2-C12 alkoxy, C2-C13 alkoxy, C2-C14 alkoxy, C2-C15 alkoxy, C2-C16 alkoxy, C2-C17 alkoxy, C2-C18 alkoxy, C2-C19 alkoxy, or C2-C20 alkoxy. In some cases. R2is substituted or unsubstituted C3-C4 alkoxy, C3-C5 alkoxy, C3-C6 alkoxy, C3-C7 alkoxy, C3-C8 alkoxy. C3-C9 alkoxy, C3-C10 alkoxy, C3-C11 alkoxy, C3-C12 alkoxy, C3-C13 alkoxy, C3-C14 alkoxy, C3-C15 alkoxy, C3-C16 alkoxy, C3-C17 alkoxy, C3-C18 alkoxy, C3-C19 alkoxy, or C3-C20 alkoxy. In some cases, R2is substituted or unsubstituted C4-C5 alkoxy, C4-C6 alkoxy, C4-C7 alkoxy, C4-C8 alkoxy, C4-C9 alkoxy, C4-C10 alkoxy, C4-C11 alkoxy, C4-C12 alkoxy, C4-C13 alkoxy, C4-C14 alkoxy, C4-C15 alkoxy, C4-C16 alkoxy, C4-C17 alkoxy, C4-C18 alkoxy, C4-C19 alkoxy, or C4-C20 alkoxy.

[0117] In some cases, at least one R2is — NHR5. In some cases, R5is hydrogen. In some cases, R5is substituted alkyl. In some cases, R3is unsubstituted alkyl. In some cases, R3is substituted cycloalkyl. In some cases, R5is unsubstituted cycloalkyl. In some cases, R5is substituted heteroalkyl. In some cases, R5is unsubstituted heteroalkyl. In some cases, R5is substituted heterocycloalkyl. In some cases, R5is unsubstituted heterocycloalkyl. In some cases, R5is a substituted polymer. In some cases, R5is an unsubstituted polymer.

[0118] In some cases, -NHR5is NH2.

[0119] In some cases, at least one R2is an imine phosphonate. In some cases, the imine compound has a structure of Formula (I), Formula (la), Formula (lb), Formula (Ic). Formula (Id), or Formula (le). In some cases, the imine compound has a structure of Formula (I), and at least one R2is an imine phosphonate. In some cases, the imine compound has a structure of Formula (la), and at least one R2is an imine phosphonate. In some cases, the imine compound has a structure of Formula (lb), and at least one R2is an imine phosphonate. In some cases, the imine compound has a structure of Formula (Ic), and at least one R2is an imine phosphonate. In some cases, the imine compound has a structure of Formula (Id), and at least one R2is an imine phosphonate. In some cases, the imine compound has a structure of Formula (le), and at least one R2is an imine phosphonate.

[0120] In some cases, R3is -P(O)3‘. In some cases, the imine compound has a structure of Formula (I), Formula (la), Formula (lb). Formula (Ic). Formula (Id), or Formula (le). In some cases, the imine compound has a structure of Formula (I), and R3is -P(O)s’. In some cases, the imine compound has a structure of Formula (la), and R3is -P(O)s". In some cases, the imine compound has a structure of Formula (lb), and R3is -P(O)3‘. In some cases, the imine compound has a structure of Formula (Ic), and R3is -P(O)3’. In some cases, the imine compound has a structure of Formula (Id), and R3is -P(O)?’. In some cases, the imine compound has a structure of Formula (le), and R3is -P(O)s’.

[0121] In some cases, R3is -S(O)s". In some cases, the imine compound has a structure of Formula (I), Formula (la), Formula (lb), Formula (Ic), Formula (Id), or Formula (le). In some cases, the imine compound has a structure of Formula (I), and R3is -S(O)3". In some cases, the imine compound has a structure of Formula (la), and R3is -S(O)3'. In some cases, the imine compound has a structure of Formula (lb), and R3is -S(O)s". In some cases, the imine compound has a structure of Formula (Ic), and R3is -S(O)3-. In some cases, the imine compound has a structure of Formula (Id), and R3is -S(O)3'. In some cases, the imine compound has a structure of Formula (le), and R3is -S(O)3’.

[0122] In some cases, R2is:

[0123] In some cases, R2is

[0124] In some cases, R2is a substituted polymer. In some cases, the substituted polymer is polyethylene glycol (PEG). In some cases, the substituted polymer comprises a hydrophilic substituent. In some cases, the hydrophilic substituent comprises a polar functional group, and / or a charged group. In some cases, the hydrophilic substituent is selected from the group consisting of a hydroxyl, carboxyl, amine, amide, sulfonic acid, phosphate, ketone, aldehyde, ammonium, sulfonate, phosphonate, and carboxylate.

[0125] In some cases, R2is unsubstituted aryl or unsubstituted heteroaryl wherein m is at least 1. In some cases, R2is substituted aryl or substituted heteroaryl wherein m is at least 1. In some cases, R2is not an aryl or heteroaryl.

[0126] In some cases, R2is a pyridine. N-alkylated tetrazoles, or N-alkylated imidazole.

[0127] In some cases, R2is not aniline, imidazole, methoxy benzoic acid, or benzimidazole.

[0128] In some cases, the imine compound has a structure of Formula (I), and n is 1, 2, or 3; m is 1 or 2; R1is a bond or -CH2, and at least one R2is -NR4R5.

[0129] In some cases, the imine compound has a structure of Formula (I), and n is 1, 2, or 3; m is 1 or 2; R1is a bond or -CH2, and at least one R2is -NH2.

[0130] In some cases, the imine compound has a structure of Formula (I), and n is 1, 2, or 3; m is 1 or 2; R1is a bond or -CH2, and R3is -P(O)3.

[0131] In some cases, n is 1, 2, or 3 and R2is -(NH-CH2-CH2)qR6, wherein q is 1 to 20 and wherein R6is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polymer, or -NR4R5, wherein R4and R5are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted polymer.

[0132] In some cases, n is 1, 2 or 3 and R2is -(NH-CH2-CH2-CH2)qR6, wherein q is 1 to 20 and wherein R6is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polymer, or -NR4R5, wherein R4and R5are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted polymer.

[0133] In some cases, n is 1 or 2 and R2is -NHR5, wherein R5is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted polymer.

[0134] In some cases, n is 1 or 2 and R2is -NR4R5, wherein R4and R5are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted polymer.

[0135] In some cases, at least one of R4or R3is a substituted polymer. In some cases, R4is a substituted polymer. In some cases, R5is a substituted polymer. In some cases, both R4and R5are a substituted polymer.

[0136] In some cases, the substituted polymer is PEG.

[0137] In some cases, the substituted polymer comprises a hydrophilic substituent.

[0138] In some cases, the hydrophilic substituent comprises a polar functional group, and / or a charged group.

[0139] In some cases, the hydrophilic substituent is selected from the group consisting of hydroxyl, carboxyl, amine, amide, sulfonic acid, phosphate, ketone, aldehyde, ammonium, sulfonate, phosphonate, and carboxylate. In some cases, the hydrophilic substituent is a hydroxyl. In some cases, the hydrophilic substituent is a carboxyl. In some cases, the hydrophilic substituent is a amine. In some cases, the hydrophilic substituent is an amide. In some cases, the hydrophilic substituent is a sulfonic acid. In some cases, the hydrophilic substituent is a phosphate. In some cases, the hydrophilic substituent is a ketone. In some cases, the hydrophilic substituent is an aldehyde. In some cases, the hydrophilic substituent is an ammonium. In some cases, the hydrophilic substituent is a sulfonate. In some cases, the hydrophilic substituent is a phosphonate. In some cases, the hydrophilic substituent is a carboxylate.

[0140] In some cases, composition further comprising an amine of the formula below, optionally wherein the amine is in molar excess of the imine phosphonate:(R2)n

[0141] In some cases, the composition comprising a compound of Formula (I) further comprises a carbonyl diphosphonate molecule. In some cases, the composition comprising a compound of Formula (la) further comprises a carbonyl diphosphonate molecule. In some cases, the composition comprising a compound of Formula (lb) further comprises a carbonyl diphosphonate molecule. In some cases, the composition comprising a compound of Formula (Ic) further comprises a carbonyl diphosphonate molecule. In some cases, the composition comprising a compound of Formula (Id) further comprises a carbonyl diphosphonate molecule. In some cases, the composition comprising a compound of Formula (le) further comprises a carbonyl diphosphonate molecule.

[0142] In some cases, a w / w ratio of the imine compound to the carbonyl diphosphonate molecule is >1. In some cases, a molar ratio of the carbonyl diphosphonate molecule to the imine compound is 0.1 or less, such as 0.05 or less, 0.01 or less, or 0.001 or less. In some cases, carbonyl diphosphonate may not be detectible by color change and / or NMR.

[0143] In some cases, the composition comprising a compound of Formula (I) further comprises a polynucleotide. In some cases, the composition comprising a compound of Formula (la) further comprises a polynucleotide. In some cases, the composition comprising a compound of Formula (lb) further comprises a polynucleotide. In some cases, the composition comprising a compound of Formula (Ic) further comprises a polynucleotide. In some cases, the composition comprising a compound of Formula (Id) further comprises a polynucleotide. In some cases, the composition comprising a compound of Formula (le) further comprises a polynucleotide.

[0144] In some cases, the polynucleotide comprises an aminooxy group.

[0145] In some cases, the pH of the composition is between 5.0 and 9.0. In some cases, the pH of the composition is 5.0. In some cases, the pH of the composition is 5.1. In some cases, the pH of the composition is 5.2. In some cases, the pH of the composition is 5.3. In some cases, the pH of the composition is 5.4. In some cases, the pH of the composition is 5.5. In some cases, the pH of the composition is 5.6. In some cases, the pH of the composition is 5.7. In some cases, the pH of the composition is 5.8. In some cases, the pH of the composition is 5.9. In some cases, the pH of the composition is 6.0. In some cases, the pH of the composition is 6.1. In some cases, the pH of the composition is 6.2. In some cases, the pH of the composition is 6.3. In some cases, the pH of the composition is 6.4. In some cases, the pH of the composition is 6.5. In some cases, the pH of the composition is 6.6. In some cases, the pH of the composition is 6.7. In some cases, the pH of the composition is 6.8. In some cases, the pH of the composition is 6.9. In some cases, the pH of the composition is 7.0. In some cases, the pH of the composition is 7.1. In some cases, the pH of the composition is 7.2. In some cases, the pH of the composition is 7.3. In some cases, the pH of the composition is 7.4. In some cases, the pH of the composition is 7.5. In some cases, the pH of the composition is 7.6. In some cases, the pH of the composition is 7.7. In some cases, the pH of the composition is 7.8. In some cases, the pH of the composition is 7.9. In some cases, the pH of the composition is 8.0. In some cases, the pH of the composition is 8.1. In some cases, the pH of the composition is 8.2. In some cases, the pH of the composition is 8.3. In some cases, the pH of the composition is 8.4. In some cases, the pH of the composition is 8.5. In some cases, the pH of the composition is 8.6. In some cases, the pH of the composition is 8.7. In some cases, the pH of the composition is 8.8. In some cases, the pH of the composition is 8.9. In some cases, the pH of the composition is 9.0. Confirmed, though preferred may be 5-8 and 5.5-7.5.

[0146] In some cases, the pH of the composition is between 5.5 to 6.0. In some cases, the pH of the composition is 5.5. In some cases, the pH of the composition is 5.6. In some cases, the pH of the composition is 5.7. In some cases, the pH of the composition is 5.8. In some cases, the pH of the composition is 5.9. In some cases, the pH of the composition is 6.0.

[0147] In some cases, the pH of the composition is between 5.0 to 8.0. In some cases, the pH of the composition is between 5.5 to 8.0. In some cases, the pH of the composition is between 5.5 to 7.5. In some cases, the pH of the composition is between 5.5 to 7.0. In some cases, the pH of the composition is between 6.0 to 7.5.

[0148] In some cases, the buffer of the composition is a phosphate buffer, a bicarbonate buffer, an acetate buffer, or a protein buffer. In some cases, the buffer of the composition is sodium acetate, Tris(hydroxymethyl)aminomethane (Tris), (N-2-hydroxyethylpiperazine-N1- 2-ethanesulfonic acid) (HEPES), 3-(N-Morpholino)propanesulfonic acid (MOPS), 2-Hydroxy-3-morpholinopropanesulfonic acid (MOPSO). piperazine-N, N'-bis(2-ethanesulfonic acid) (PIPES), (2-(N-morpholino)ethanesulfonic acid) (MES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), 2-{[l,3-Dihydroxy-2-(hydroxymethyl)propan-2-yl] amino} ethane- 1 -sulfonic acid (TES), glycylglycine (Gly-Gly), N-cyclohexyl-2-hydroxyl- 3-aminopropanesulfonic acid (CAPSO), N-(2-Acetamido)iminodiacetic acid (ADA), orN, N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), In some cases, the buffer is an acetate buffer, a citrate buffer, a bicarbonate buffer, a phosphate buffer, Tris, HEPES, MOPS, or PIPES. The buffer, such as acetate or citrate, may be a salt with sodium, lithium, magnesium or another suitable cation. The buffer may include a base and / or acid at a molar concentration of between 0.01 molar and 5 molar, such as between 0.02 molar and 2 molar.

[0149] In some cases, the composition further comprises an organic solvent, and / or an inorganic solvent. In some cases, the inorganic solvent is water and is in excess of the organic solvent. In some cases, the organic solvent is a polar organic solvent. In some cases, the organic solvent is a polar organic solvent. In some cases, the organic solvent is Dimethyl sulfoxide (DMSO), Dimethylformamide (DMF), an alcohol, or an aliphatic diol. In some cases, the inorganic solvent is water.

[0150] In some cases, the imine compound has a molarity of at least 0.001 mol / L (1 millimolar), based on a total volume of the composition. In some cases, the imine compound has a molarity of at least at least 0.1 millimolar (mM), such as 0.1 to lOmM or 0.1 to lOOmM. In some cases, the imine compound has a molarity of at least at least 1 mM. such as 1 tolOOmM, 1 to 200mM, 1 to 500mM, 1 to 10 mM. In some cases, the imine compound has a molarity of at least at least 10 mM such as 10 to lOOmM or 10 to 200 mM or 10 to 500mM.

[0151] In some cases, the imine compound has a structure of Formula (I) and n is 1, 2, or 3; m is 1 or 2; R1is a bond or -CH2, and R2is -NHR5.

[0152] In some cases, R4and R3are independently selected from the group consisting of -(NH-CH2-CH2)qR6and -(NH-CH2-CH2-CH2)qR6, wherein q is 1 to 20 and wherein R6is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polymer, or -NR4R5, wherein R4and R3are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted polymer.

[0153] In some cases, R4and R5are independently selected from the group consisting of-(NH-CH2-CH2)qR6and -(NH-CH2-CH2-CH2)qR6, wherein q is 1 and wherein R6is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polymer, or -NR4R5, wherein R4and R5are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted polymer.

[0154] In some cases, R6is hydrogen.

[0155] In some cases, the compound of Formula (I) is:Ax-P PxHO 1 1 OHO O0 0p> / °CL-O0O' / ° CL- O®Xoj □.-o®

[0156] In some cases, the compound of Formula (I) is:qxA 'P xP P< HO I I OH O Oo oQ 0

[0157] In some cases, the compositions described herein are compositions, comprising a mixture of a buffer, a primary amine, and an imine compound of Formula (I):NH2R3(I), or stereoisomers and salts thereof, wherein:n is 0, 1, 2, or 3;m is 0 to 20R1is a bond, H, –CH2–, or –NH–;R2is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polymer, or -NR4R5, wherein R4and R3are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted polymer; andR3is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstitutedheterocycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted and. substituted or unsubstituted heteroaryl, substituted or unsubstituted polymer, -P(O)s’, or -NR4R5, wherein R4and R3are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted polymer,wherein when R1is a bond and m is 0, n is 1,wherein when R1is -NH- and m is 0, n is 1, andwherein when R1is H, m and n are 0.

[0158] In some cases, the compositions described herein are compositions comprising a mixture of a buffer, an imine compound of Formula (I), and a primary amine. The primary amine may be in molar excess of the imine. The primary amine may be in molar excess of the imine by at least 3 fold, at least 10 fold, at least 30 fold, or at least 100 fold. In some cases, the methods of making the compositions described herein comprising providing a buffer and mixing a carbonyl phosphonate with a primary amine in excess of the carbonyl phosphonate. The primary amine may be in molar excess of the carbonyl phosphonate by at least 3 fold, at least 10 fold, at least 30 fold, or at least 100 fold.

[0159] The imine compounds described herein are stable, may be in equilibrium with a carbonyl phosphonate or may be highly pure, and are not an intermediate in a chemical reaction. While imine phosphonates are described herein, the theory of imine formation and cleavage activity of N=O bonds of aminooxy groups also applies to imine sulfonates. As such, the compounds of the subject applications also include imine sulfonate analogs of any of the chemical structures described herein. For example, phosphonates of any of the compounds described herein may be substituted with sulfonates.METHODS OF MAKING

[0160] The present disclosure provides a method of producing an imine compound. In some cases, the methods described herein are methods of producing an imine compound of the composition comprising a buffer and an imine compound of Formula (I), Formula (la), Formula (lb), Formula (Ic), Formula (Id), or Formula (le), comprising contacting a solution of carbonyl diphosphonate with a primary amine forming a reaction mixture comprising the imine compound; and, optionally, extracting the imine compound of Formula (I), Formula (la), Formula (lb), Formula (Ic), Formula (Id), or Formula (le) from the reaction mixture.

[0161] In some cases, the imine compound is an imine phosphonate formed by contacting a carbonyl phosphonate with a suitable primary amine. In some cases, the imine compound is an imine diphosphonate formed by contacting a carbonyl diphosphonate with a suitable primary amine. In some cases, the imine phosphonate composition may be formed without extracting the imine compound, e.g., when the mixture resulting from just the contacting step is used in the subject methods, kits and / or systems.

[0162] In some cases, the methods described herein are methods of producing an imine compound comprises combining a buffer, a primary amine (e.g., a primary amine in excess), and a carbonyl phosphonate to produce a composition comprising a primary amine and an imine compound of Formula (I):or stereoisomers and salts thereof, wherein:n is 0, 1, 2, or 3;m is 0 to 20R1is a bond, H, –CH2–, or –NH–;R2is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polymer, or -NR4R5, wherein R4and R5are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted polymer; andR3is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polymer, -P(O)s‘, or -NR4R5, wherein R4and R5are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted polymer,wherein when R1is a bond and m is 0, n is 1,wherein when R1is -NH- and m is 0, n is 1, andwherein when R1is H, m and n are 0.

[0163] In some cases, the buffer is a phosphate buffer, a bicarbonate buffer, an acetate buffer, or a protein buffer.

[0164] In some cases, the pH of the solution is within the range of 5.0 to 9.0. In some cases, the pH of the composition is 5.0. In some cases, the pH of the composition is 5.1. In some cases, the pH of the composition is 5.2. In some cases, the pH of the composition is 5.3. In some cases, the pH of the composition is 5.4. In some cases, the pH of the composition is 5.5. In some cases, the pH of the composition is 5.6. In some cases, the pH of the composition is 5.7. In some cases, the pH of the composition is 5.8. In some cases, the pH of the composition is 5.9. In some cases, the pH of the composition is 6.0. In some cases, the pH of the composition is 6.1. In some cases, the pH of the composition is 6.2. In some cases, the pH of the composition is 6.3. In some cases, the pH of the composition is 6.4. In some cases, the pH of the composition is 6.5. In some cases, the pH of the composition is 6.6. In some cases, the pH of the composition is 6.7. In some cases, the pH of the composition is 6.8. In some cases, the pH of the composition is 6.9. In some cases, the pH of the composition is 7.0. In some cases, the pH of the composition is 7.1. In some cases, the pH of the composition is 7.2. In some cases, the pH of the composition is 7.3. In some cases, the pH of the composition is 7.4. In some cases, the pH of the composition is 7.5. In some cases, the pH of the composition is 7.6. In some cases, the pH of the composition is 7.7. In some cases, the pH of the composition is 7.8. In some cases, the pH of the composition is 7.9. In some cases, the pH of the composition is 8.0. In some cases, the pH of the composition is 8.1. In some cases, the pH of the composition is 8.2. In some cases, the pH of the composition is 8.3. In some cases,the pH of the composition is 8.4. In some cases, the pH of the composition is 8.5. In some cases, the pH of the composition is 8.6. In some cases, the pH of the composition is 8.7. In some cases, the pH of the composition is 8.8. In some cases, the pH of the composition is 8.9. In some cases, the pH of the composition is 9.0.

[0165] In some cases, the pH of the composition is between 5.0 to 8.0. In some cases, the pH of the composition is between 5.5 to 8.0. In some cases, the pH of the composition is between 5.5 to 7.5. In some cases, the pH of the composition is between 5.5 to 7.0. In some cases, the pH of the composition is between 6.0 to 7.5.

[0166] In some cases, the buffer is sodium acetate,Tris(hydroxymethyl)aminomethane (Tris), (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) (HEPES). 3-(N-Morpholino)propanesulfonic acid (MOPS), 2-Hydroxy-3-morpholinopropanesulfonic acid (MOPSO), piperazine-N, N'-bis(2-ethanesulfonic acid) (PIPES), (2-(N-morpholino)ethanesulfonic acid) (MES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), 2-{[l,3-Dihydroxy-2-(hydroxymethyl)propan-2-yl] amino} ethane- 1 -sulfonic acid (TES), glycylglycine (Gly-Gly), N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid (CAPSO), N-(2-Acetamido)iminodiacetic acid (ADA), orN, N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), In some cases, the buffer is an acetate buffer, a citrate buffer, a bicarbonate buffer, a phosphate buffer, Tris, HEPES, MOPS, or PIPES. The buffer, such as acetate or citrate, may be a salt with sodium, lithium, magnesium or another suitable cation. The buffer may include a base and / or acid at a molar concentration of between 0.01 molar and 5 molar, such as between 0.02 molar and 2 molar.

[0167] In some cases, the solution further comprises an organic solvent, and / or an inorganic solvent. In some cases, the inorganic solvent is water and is in excess of the organic solvent.

[0168] In some cases, the organic solvent is a polar organic solvent. In some cases, the organic solvent is a polar organic solvent. In some cases, the organic solvent is Dimethyl sulfoxide (DMSO), Dimethylformamide (DMF), an alcohol, or an aliphatic diol.

[0169] In some cases, the inorganic solvent is water.

[0170] In some cases, the method of producing an imine compound of the composition comprising a buffer and an imine compound of Formula (I). Formula (la), Formula (lb), Formula (Ic), Formula (Id), or Formula (le), further comprising introducing a solid support tothe solution of carbonyl diphosphonate before contacting the solution of carbonyl diphosphonate with the primary amine.

[0171] In some cases, the solid support is an ion exchange resin, a magnetic bead, or a glass bead.

[0172] In some cases, the solid support is an ion exchange resin.

[0173] In some cases, the ion exchange resin is a cation exchange resin or an anion exchange resin. In some cases, the ion exchange resin is a strong acid cation (SAC) resin. In some cases, the ion exchange resin is a weak acid cation (WAC) resin. In some cases, the ion exchange resin is a strong base anion (SBA) resin. In some cases, the ion exchange resin is a weak base anion (WBA) resin.

[0174] In some cases, the primary amine is selected from a list consisting of:NHH3C-(CH2)^NH2H3C-(CH2VNH2H3C— (CH2> TNH2 H3C— (CH2)j-NH2 H3c— (CH2XTNH2H3C-(CH2)ITNH2 H3C— (CH2)rrNH2 H3C — (CH2)77NH2 H3C — (CH2)i3-NH2 H3c— (CH2yiTNH2H3C— (CH2)iyNH2 H3C— (CH2)T^NH2H3C — (CH2)17NH2 H3c— (CH2)i8-NH2H3C — (CH2)i9" NH2NH2

[0175] In some cases, the primary amine is selected from a list consisting of:

[0176] In some cases, the primary amine may be selected from N. N- dimethylethylenediamine (DMEN), 1,2-ethylenediamine (en). and 2-(2- Aminoethylamino)ethanol, Tris(2-aminoethyl)amine (Tren), or 3 -(Dimethylamino)- 1- propylamine (DMPN), or salts thereof. In some cases the primary amine is not ethylenediamine, for example, the primary amine may be an ethylamine or propylamine other than ethylenediamine. In some cases, the primary amine is not 2-amino-5-methoxy benzoic acid, 2-(Aminomethyl)benzimidazole, analine, isopropylamine or an amino acid.

[0177] In some cases, the primary amine does not comprise conjugation within a bond of the primary amine. In some cases, the primary amine is linear and does not have a branched or ring structure. In some cases, an NH2 of the primary amine does not contain a group that sterically hinders efficient formation of imine phosphonate. In some cases, the primary amine does not form an adduct with the carbonyl phosphonate. In some cases, the primary amine is soluble in the buffer, such as at a molarity of at least 1 mM, at least 10 mM, or at least 100 mM. Solubility of amine in buffer can be easily tested. Efficient and highly pure imine formation can be easily checked by looking for color change (e.g., in the visible or UV spectrum) and / or NMR spectra (e.g., hydrogen, carbon and / or phophorous NMR), and adduct formation can be ruled out by NMR spectra.

[0178] In some cases, the amine has a molarity of at least 0.001 mol / L (1 millimolar), based on a total volume of the composition. In some cases, the amine has a molarity of at least at least 0.1 millimolar (mM), such as 0.1 to 1 OmM or 0.1 to 1 OOmM. In some cases, the amine has a molarity of at least at least 1 mM, such as 1 to 1 OOmM, 1 to 200mM, 1 to 500mM, 1 to 10 mM. In some cases, the amine has a molarity of at least at least 10 mM such as 10 to lOOmM or 10 to 200 mM or 10 to 500mM or 10 to 1000 mM.

[0179] In some cases, the imine is extracted by rinsing with a reagent, crystallization, chromatography, lyophilization and / or filtration. In some cases, the chromatography is column chromatography or ion exchange chromatography. In some cases, the reagent is a primary amine.METHODS

[0180] Provided herein are methods of extending polynucleotides. The methods include contacting polynucleotides comprising aminooxy groups with a first composition to produce polynucleotides comprising extendible 3’ OH groups, wherein the first composition comprises an imine phosphonate compound as provided herein, wherein each polynucleotide has an aminooxy group at a 3' end of the polynucleotide, and wherein the composition cleaves an N=O bond of the aminooxy group to produce the 3’ OH group thereby producing extendible polynucleotides with 3’ OH groups. The polynucleotides comprising the 3’ OH groups are then extended in a nucleic acid polymerization reaction by contacting the polynucleotides comprising the 3 ’ OH groups with a polymerase and one or more nucleotides; and repeating the contacting and extending steps in a plurality of cycles.Optionally, the contacting occurs over a period of time and the period of time is less than 5 minutes, less than 3 minutes, less than 2 minutes, less than 1 minute, less than 30 seconds, orless than 10 seconds, such as between 5 seconds and 1 minute. In some methods, the nulcleotides incorporated into the polynucleotides in the extending step have 3‘ aminooxy groups. Alternatively, the nucleotides incorporated into the polynucleotides in the extending step may have a 3’ aminooxy precursor group that is converted to an aminooxy group after incorporation and before the cleavage in the next cycle.

[0181] The imine phosphonate compound can be an imine diphosphonate. Optionally, the imine phosphonate compound is aliphatic. Optionally, the imine phosphonate compound comprises a substituted alkyl, unsubstituted alkyl, substituted alkenyl, or unsubstituted alkenyl. The imine phosphonate compound can comprise one or more ethylamine groups or one or more propylamine groups. Optionally, the imine phosphonate compound comprises an amine separated from the imine by 2 or 3 carbons. Optionally, the imine phosphonate compound is not aromatic. Optionally, the imine phosphonate compound does not contain ethylenediamine, isopropylamine, aniline, or an amino acid.

[0182] The first composition can include other components, for example, a carbonyl diphosphonate. In the first composition, the w / w ratio of the imine compound to the carbonyl diphosphonate molecule can be greater than 1. Optionally, first composition comprises the carbonyl diphosphonate at a molar ratio to imine phosphonate of less than 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02 or less than 0.01. If the first composition comprises a carbonyl diphosphonate, the presence of the carbonyl diphosphonate can reduce the number of extendible nucleotides produced.

[0183] The herein provided methods can result in at least 99.9% of the polynucleotides comprising the aminooxy groups are converted to extendable polynucleotides comprising the 3’ OH groups. For example, at least 99% of the extendable polynucleotides can be produced by cleavage of the O-N bond by imine phosphonate.

[0184] In the provided methods, the polynucleotides can be coupled, directly or indirectly, to a surface. Optionally, the surface is a bead, pad or well. Optionally, the surface is within a flow cell. The polynucleotides can be clonally identical and repeated in the same concatemer, hybridized to the same concatemer, attached to a bead, attached to a pad, or attached to a well. Optionally, the polynucleotides are coupled to the surface indirectly and wherein the polynucleotides are primers that are hybridized to a template nucleotide that is coupled to the surface. Optionally, the polymerase is a template dependent polymerases. Optionally, the nucleotides do not comprise a fluorescent label.

[0185] In the provided methods, the extended polynucleotides can be a primer hybridized to a template nucleotide. Further, the plurality of cycles in the provided methods can include a step of detecting a next correct nucleotide at a 3’ position of the primer. Optionally, the next correct nucleotide is detected by detecting a ternary complex comprising a polymerase, the polynucleotides hybridized to a template nucleotide, and a fluorescent nucleotide, wherein the fluorescent nucleotide is not incorporated into the primer. The ternary complex can be formed with polynucleotides comprising the aminooxy groups. Alternatively, the ternary complex can be formed with polynucleotides comprising the 3’ OH groups in the presence of a non-catalytic cation. The non-catalytic cation can be, for example, strontium or calcium.

[0186] In the provided methods, the next correct nucleotide is a fluorescent nucleotide incorporated into the polynucleotide during the extension step. Optionally, the incorporated fluorescent nucleotide comprises a fluorophore coupled to the base of the nucleotide through an aminooxy group comprising an N=O bond that is cleaved in step a) of a next cycle of the plurality' of cycles.

[0187] Some embodiments include methods of DNA synthesis, such as chemical or enzymatic DNA synthesis. Enzymatic DNA synthesis may be with a template-independent polymerase, such as Terminal deoxynucleotidyl transferase (TdT), such as is described in US Patent Publication Nos. 2019 / 0300923 and 2019 / 0112627. In the provided methods, the polymerase can be a template independent polymerase, such as in a DNA synthesis reaction with sequencing of a template polynucleotide. Some embodiments include methods of DNA synthesis, such as chemical or enzymatic DNA synthesis. Enzymatic DNA synthesis may be with a template-independent polymerase, such as Terminal deoxynucleotidyl transferase (TdT), such as is described in US Patent Publication Nos. 2019 / 0300923 and 2019 / 0112627.

[0188] The nucleotides used in the provided method during extension step can be of a single base type and comprise a 3' N=O bond. Optionally, the nucleotides used during the extension step are of a single base and differ between some of the cycles of the plurality of cycles. Thus, for example, in a cycle of the plurality of cycles the one or more nucleotides can be an A, while in the next cycle the one or more nucleotides are a G, then a C and then a T. The one or more nucleotides can be used in combination and in any order. Thus, for example, the method can comprise contacting the polynucleotides with AGCT, AT, AG, AC, GC, GT, CT, A, T, C, G, or any combination thereof, in any one of the cycles in the plurality of cycles.

[0189] Also provided herein are methods of sequencing a polynucleotide templates. The methods include providing polynucleotides hybridized to polynucleotide templates coupled to a surface, wherein each polynucleotide is a primer; contacting the polynucleotides with a first composition comprising an imine phosphonate to cleave an N=O bond of an aminooxy group of the 3’ nucleotide of each polynucleotide; b) extending the polynucleotides in a nucleic acid polymerization reaction by contacting the polynucleotides with a polymerase and one or more nucleotides; c) detecting, before or after step b) of extending, the next correct nucleotide of the polynucleotide template; and d) repeating steps a), b) and c) in a plurality of cycles. The detecting can include detecting a ternary complex comprising a polymerase, the polynucleotide, and a fluorescent nucleotide, wherein the fluorescent nucleotide is not incorporated to extend the extendible polynucleotide in step b). Optionally, the N=O bond cleaved in step a) is at the 3’ position of the 3’ nucleotide of the polynucleotide and wherein the nucleotides of step b) each comprise an N=O bond at their 3’ end. The ternary complex can be formed with polynucleotides comprising the aminooxy groups and wherein step c) of detecting is after step b) of extending. Optionally, the ternary complex is formed with polynucleotides comprising the 3’ OH groups in the presence of anon-catalytic cation.Optionally, the non-catalytic cation is strontium or calcium and wherein step c) of detecting is between step a) of contacting and step b) of extending. Optionally, the next correct nucleotide is a fluorescent nucleotide incorporated in step b) and wherein step c) of detection is after step b) of extending. Optionally, the incorporated fluorescent nucleotide comprises a fluorophore coupled to the base of the nucleotide through an aminooxy bond and comprises an aminooxy group on the 3’ end, wherein an N=O bond of both aminooxy groups are cleaved in step a) of the next cycle of the plurality of cycles. Optionally, the incorporated fluorescent nucleotide comprises a fluorophore coupled to the base of the nucleotide through an N=O bond and does not comprise an N=O bond at the 3’ end. Optionally, the nucleotides of step b) are of a single base type and wherein the method further comprises repeating each base type every four cycles of the plurality' of cycles. Optionally, the contacting occurs over a period of time and the period of time is less than 5 minutes, less than 3 minutes, less than 2 minutes, less than 1 minute, less than 30 seconds, or less than 10 seconds, such as between 5 seconds and 1 minute.

[0190] Any of a variety of sequencing techniques can be used in a method set forth herein, for example, sequencing-by-synthesis (SBS) or sequencing-by-binding (SBB). SBS generally involves the enzymatic extension of a nascent primer through the iterative addition of nucleotides against a template strand to which the primer is hybridized. Briefly, SBS canbe initiated by contacting target nucleic acids with one or more labeled nucleotides, DNA polymerase, etc. A primer that is extended using the target nucleic acid as template will incorporate a labeled nucleotide that can be detected. Optionally, the labeled nucleotides can further include a reversible terminator that terminates further primer extension once a nucleotide has been added to a primer. For example, a nucleotide analog having a reversible terminator moiety can be added to a primer such that subsequent extension cannot occur until a deblocking agent is delivered to remove the moiety. Thus, for embodiments that use reversible termination, a deblocking reagent can be delivered to the vessel (before or after detection occurs). Washes can be carried out between the various delivery' steps. In some forms of SBS, nucleotides are not reversibly terminated and are instead added one base ty pe at a time, with fluorophores off the base being detected and then cleaved between additions. The cycle can be performed n times to extend the primer by n nucleotides, thereby detecting a sequence of length n. Exemplary' SBS procedures, reagents and detection components that can be readily adapted for use with a method, system or apparatus of the present disclosure are described, for example, in Bentley et al., Nature 456:53-59 (2008), WO 04 / 018497; WO 91 / 06678; WO 07 / 123744; U. S. Pat. No. 7,057,026; 7,329,492; 7.211,414; 7.315,019 or 7,405,281, and US Pat. App. Pub. No. 2008 / 0108082 Al, each of which is incorporated herein by reference.

[0191] Sequencing By Binding (SBB) techniques can be used in the provided methods, for example, as described in US Pat. App. Pub. Nos. 2017 / 0022553 Al; 2018 / 0044727 Al; 2018 / 0187245 Al; or 2018 / 0208983 Al, each of which is incorporated herein by reference. Generally, SBB methods for determining the sequence of a template nucleic acid molecule can be based on formation of a stabilized ternary' complex (between polymerase, primed nucleic acid and cognate nucleotide) under specified conditions. The method can include an examination phase followed by a nucleotide incorporation phase. One or more sequencing phases can be carried out using a mixed-phase fluid (e.g. a fluid foam, fluid slurry or fluid emulsion).

[0192] The examination phase of an SBB process can be carried out in a flow' cell, the flow' cell containing at least one template nucleic acid molecule primed with a primer by delivering to the flow cell reagents to form a first reaction mixture. The reaction mixture can include the primed template nucleic acid, a polymerase and at least one nucleotide type. The interaction between the primed template, polymerase and nucleotide can be detected in a variety' of schemes. For example, the nucleotides can contain a detectable label. Each nucleotide can have a distinguishable label with respect to other nucleotides. Alternatively.some or all of the different nucleotide types can have the same label and the nucleotide ty pes can be distinguished based on separate deliveries of different nucleotide types to the flow cell. Detection can be carried out by scanning the flow cell using an apparatus or method set forth herein.

[0193] The extension phase can be carried out by creating conditions where a nucleotide can be added to the primer on each template nucleic acid molecule. Alternatively, one or more reagents can be added to the examination phase reaction to create extension conditions. For example, catalytic divalent cations can be added to an examination mixture that was deficient in the cations, and / or polymerase inhibitors can be removed or disabled, and / or extension competent nucleotides can be added, and / or a deblocking reagent can be added to render primer(s) extension competent, and / or extension competent polymerase can be added.

[0194] Other sequencing processes can be used, such as pyrosequencing. Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as nucleotides are incorporated into a nascent primer hybridized to a template nucleic acid strand (Ronaghi, et al., Analytical Biochemistry 242 (1), 84-9 (1996); Ronaghi, Genome Res. 11 (1), 3-11 (2001); Ronaghi et al. Science 281 (5375), 363 (1998); U. S. Pat. Nos. 6,210,891; 6.258,568 and 6,274,320, each of which is incorporated herein by reference). In pyrosequencing, released PPi can be detected by being converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the resulting ATP can be detected via luciferase-produced photons.

[0195] The nucleotides used in the provided methods can be of a single base type or can be used in combination and, thus, be of multiple base types. The nucleotides used in each cycle of the methods can be of the same or different base types. Optionally, the nucleotides used in the provided methods are of a single base and differ between some of the cycles of the plurality of cycles. Thus, for example, in a cycle of the plurality of cycles the one or more nucleotides can be an A, while in the next cycle the one or more nucleotides are a G, then a C and then a T. The one or more nucleotides can be used in combination and in any order. Thus, for example, the method can comprise contacting the polynucleotides with AGCT, AT, AG, AC, GC, GT, CT, A, T, C, G, or any combination thereof, in any one of the cycles in the plurality of cycles.

[0196] Optionally, synthesis of a ssDNA nucleic acid of a desired sequence may include: elongating initial nucleic acid fragments or attached to a support and having free 3'-hydroxyls by: (a) reacting said fragments with a modified nucleotide and a template-independent DNA polymerase, wherein said modified nucleoside triphosphate comprises a 3’ N=O group thatprevents multiple additions of nucleotides, (b) cleaving the N=O group with a deblocking reagent or solution described herein, and (c) repeating steps (a) and (b).

[0197] Optionally, synthesis of an ssDNA nucleic acid of a desired sequence may include elongating initial nucleic acid fragments or attached to a support and having free 3'-hydroxyls by: (a) reacting said fragments with a modified nucleoside triphosphate and a templateindependent DNA polymerase, wherein said modified nucleotide comprises a linker comprising an N=O group is joined to the base of the nucleotide and coupling the nucleotide to the polymerase, (b) cleaving the N=O group with a deblocking composition comprising imine phosphonate as described herein, and (c) repeating steps (a) and (b). The coupling of the nucleotide to the polymerase acts as a reversable termination, as further elongation may not be possible until after cleavage of the linker. The linker comprising an N=O group may be joined to the base of the nucleotide, such as at an atom of the base that is not involved in base pairing. In other embodiments, the linker is attached to an aldehyde specifically generated within the polymerase, as described in Carrico et al. (Nat. Chem. Biol. 3, (2007) 321-322). This aldehyde may then be specifically labeled with the aminooxy moiety of a linker. In some embodiments, the linker may be attached to the polymerase via non-covalent binding of a moiety of the linker to a moiety fused to the polymerase. Examples of such attachment strategies include fusing a polymerase to streptavidin that can bind a biotin moiety of a linker or fusing a polymerase to anti-digoxigenin that can bind a digoxigenin moiety of a linker. As described in the above embodiments, the aminooxy group cleaved by the deblocking agent may be on the 3’ end and / or base of a nucleotide. As such, any of the kits, methods and systems described herein with a 3’ aminooxy group may, alternatively or in addition to the 3’ aminooxy group, have an aminooxy group off the base.SYSTEMS

[0198] Also provided herein are systems comprising a composition comprising an imine phosphonate and a flow cell comprising a surface, wherein the system is configured to deliver the composition and reagents to the surface of the flow cell to extend polynucleotides coupled to the surface of the flow cell. Optionally, the system further comprises a detection system configured to detect fluorescent nucleotides incorporated into the polynucleotides or in a ternary complex with the polynucleotides. The system can include any of the imine phosphonate compounds described herein. Further, the system can be configured to carry out the methods described throughout. Thus, a system of the present disclosure can be configured for detecting nucleic acids, for example, using methods set forth herein.Optionally, the system includes components and reagents for performing one or more steps set forth herein for extending polynucleotides and / or sequence polynucleotide templates.

[0199] A system of the present disclosure can include a vessel, solid support or other apparatus for carrying out a nucleic acid detection method. For example, the system can include an array, flow cell, multi-well plate or other convenient apparatus. The apparatus can be removable, thereby allowing it to be placed into or removed from the system. As such, a system can be configured to process a plurality of apparatus (e.g. vessels or solid supports) sequentially or in parallel. The system can include a fluidic component having reservoirs for containing one or more of the reagents set forth herein (e g. polymerase, primer, template nucleic acid, nucleotide(s) for ternary complex formation, nucleotides for primer extension, deblocking reagents, or mixtures of such components). The fluidic system can be configured to deliver reagents to a vessel or solid support, for example, via channels or droplet transfer apparatus (e.g. electro wetting apparatus). Any of a variety of detection apparatus can be configured to detect the vessel or solid support where reagents interact. Examples include luminescence detectors, surface plasmon resonance detectors and others know n in the art. Exemplary systems having fluidic and detection components that can be readily modified for use in a system herein include, but are not limited to, those set forth in US Pat. App. Pub. No.2018 / 0280975 Al, U. S. Pat. Nos. 8,241,573; 7,329,860 or 8,039,817; or US Pat. App. Pub. Nos. 2009 / 0272914 Al or 2012 / 0270305 Al, each of which is incorporated herein by reference.KITS

[0200] Provided herein are kits that include one or more compositions comprising one or more of an imine phosphonate compound as provided herein. The kits can also include one or more polymerases and / or one or more nucleotides comprising an N=O bond. Optionally, the nucleotides are nucleotide triphosphates. Optionally, the nucleotides comprise a 3’ N=O bond. Optionally, the nucleotides further comprise a fluorescent nucleotide comprising an extendible 3’ OH group. Optionally, the nucleotides comprise the N=O bond off the base between the nucleotide and a fluorophore. Optionally, the nucleotides comprise the 3’ N=O bond and the N=O bond off the base between the nucleotide and a fluorophore. Optionally, the polymerase is a template independent polymerase.

[0201] In the provided kits the kit can include any of the herein provided compounds. Thus, the imine phosphonate in the kit can be an imine diphosphonate. Optionally, the imine phosphonate is aliphatic. Optionally, the imine phosphonate comprises one or moreethylamine groups one or more propylamine groups. Optionally, the imine phosphonate comprises an amine spaced from the imine by 2 or 3 carbon bonds. Optionally, the imine phosphonate lacks any aryl group. Optionally, the composition, polymerase and nucleotides are in separate compartments of the kit. Optionally, the kit comprises a plastic cartridge comprising the composition, polymerase and nucleotides.

[0202] Optionally, a kit can include additional reagents for earn ing out one or more of the methods provided herein. Reagents included in a kit, such as nucleotide(s), polymerase(s) or both, can include an exogenous label, for example, as set forth herein in the context of various methods.

[0203] Accordingly, any of the components or articles used in performing the methods set forth herein can be usefully packaged into a kit. For example, the kits can be packed to include some, many or all of the components or articles used in performing the methods set forth herein. Exemplary components include, for example, labeled nucleotides (e.g. extendible labeled nucleotides), polymerases (labeled or unlabeled), nucleotides having terminator moieties (e.g. unlabeled, reversibly terminated nucleotides), deblocking reagents, and the like as set forth herein and in references cited herein. Any of such reagents can include, for example, some, many or all of the buffers, components and / or articles used for performing one or more of the subsequent steps for analysis of a primer-template nucleic acid hybrid. A kit need not include a primer or template nucleic acid. Rather, a user of the kit can provide one or more primer-template nucleic acid hybrids which are to be combined with components of the kit by the user.

[0204] One or more ancillary reagents also can be included in a kit. Such ancillary reagents can include any of the reagents exemplified above and / or other types of reagents useful in performing the methods set forth herein. Instructions can further be included in a kit. The instructions can include, for example, procedures for making any components or articles used in the methods set forth herein, performing one or more steps of any embodiment of the methods set forth herein and / or instructions for performing any of the subsequent analysis steps employing a primer-template nucleic acid hybrid.

[0205] In some aspects, a kit can include an apparatus set forth herein, such as a flow cell or solid support. Optionally, a kit includes a cartridge having reservoirs to contain the reagents and further having fluidic components for transferring reagents from the reservoirs to a detection instrument. For example, the fluidic components can be configured to transfer reagents to a flow cell where stabilized ternary complexes are detected. An exemplary fluidiccartridge that can be included in a kit (or system) of the present disclosure is described in US Pat. App. Pub. No. 2018 / 0280975 Al, which is incorporated herein by reference.

[0206] One or more embodiments of the disclosure and the invention may be further understood by the following non-limiting examples.EXAMPLESExample 1: Ultraviolet-visible (UV-Vis) spectroscopy characterization of imine forming formulations.

[0207] A solution of tetrasodium carbonyl diphosphonate (CDP) showed a yellow color in solution. As has been reported elsewhere, this coloration is expected for a tetrasodium carbonyl diphosphonate solution. The color has been reported to arise from an electronic transition of the carbonyl group at ~ 413 nm. Surprisingly, it was observed that the addition of asymmetric N1,N1-dimethylethane-1,2-diamine (DMEN) to CDP resulted in in a loss of yellow coloration. Ultraviolet- visible (UV-Vis) spectroscopy was used to further analyze the change in color and absorbance transition.

[0208] For the UV-Vis experiments, 50 mM of CDP was dissolved in 1 M sodium acetate buffer (5 times the CDP concentration). To the CDP in sodium acetate solution, 30 mM, 90 mM, or 150 mM DMEN or Nl. Nl. N2. N2-tetramethylethane-l.2-diamine (TMEDA) were added. Control conditions were also tested where no DMEN or TMEDA were added. Of note, symmetric N. N'-dimethylethane-l.2-diamine (symmetric DMEDA) showed no color change, similar to TMEDA and consistent with the mechanism described herein that a particular subset of primary amines are necessary for imine phosphonate formation from CDP.

[0209] FIG. 1 shows UV-Vis spectral data where a loss of the -420 nm absorbance (-70% drop) was observed with increasing concentrations of DMEN. At the same time, a new peak at -320 nm was formed and an isosbestic point was readily observed during the transition. In FIG. 1, the top peak at -420 nm absorbance is the control, where no DMEN or TMEDA was added, the second peak, i.e., the peak below the top peak, is for where 30 mM DMEN was added, the third peak is for where 90 mM DMEN was added and the bottom peak at -420 absorbance is for the condition where 150 mM DMEN was added. The loss of coloration observed and the loss of -420 nm absorbance measured may indicate that the carbonyl group of the CDP was consumed upon DMEN addition.

[0210] In contrast, FIG. 2 shows UV-Vis spectral data where TMEDA was used shows no loss of the -420 nm absorbance peak, nor the formation of a new peak at -320 nm, nor an isosbestic point. These data suggests that the primary amine characteristic of DMEN may be important for imine formation.Example 2: Spectroscopic Analysis of N^N'-dimethylethane-l^-diamine DMEN and carbonyl diphosphonate (CDP) Imine Formation (31P NMR)

[0211] To further analyze the imine formation from the CDP and DMEN solution, two31P NMR experiments were conducted. First, 50 mM CDP in 1 M sodium acetate was dissolved in D2O. Second, 50 mM CDP and 90 mM DMEN in 1 M sodium acetate were dissolved in D2O.31P NMR spectra was measured for both experimental conditions.

[0212] FIG. 3, Panel A shows the31P NMR spectrum for the CDP solution where the arrow represents the singlet formed by the symmetric di-phosphonate moiety of CDP. In contrast, FIG. 3, Panel B shows the31P NMR spectrum for the CDP + DMEN solution where the arrows point to a pair of doublets. The data suggests a reaction between the CDP to form a single conjugated compound. Furthermore, data is consistent with imine formation when CDP and DMEN are mixed in acetate buffer. The formation of a doublet suggests the desymmetrization of the two phosphorus atoms which couple to each other.

[0213] An ion exchange experiment with DMEN was also performed to verify the results. Ion exchange was performed to remove sodium cations, yielding a white solid. A31P NMR experiment of the product after cation exchange between CDP and DMEN showed the characteristic doublet of doublets consistent with the formation of the imine compound. Example 3: Spectroscopic Analysis of N^N’-dimethylethane-l^-diamine DMEN and carbonyl diphosphonate (CDP) Imine Formation (’H NMR)

[0214] To further inquire about the interaction of the CDP and DMEN in solution, twoXH NMR experiments were conducted. First 90 mM DMEN in IM sodium acetate was dissolved in D2O. Second, 50 mM CDP and 90 mM DMEN in 1 M solution acetate were dissolved in D2O. NMR spectra was measured for both experimental conditions.

[0215] FIG. 4, Panel A shows theNMR spectrum for the DMEN solution where the methyl protons of the DMEN molecule appeared as a singlet at -3.3 ppm and the -CEE protons of the DMEN molecule appeared as a singlet at -2.3 ppm. The acetate and solvent were properly labeled at -4.3 and -1.8 ppm, respectively. In contrast, FIG. 4, Panel B shows the NMR spectrum for the CDP + DMEN solution, where new resonance peaks wereobserved. In particular, the methyl groups of the DMEN moiety were split into a doublet around the -3.3 ppm resonance. The -CH2 protons were also split and shifted downfield to ~4.3 ppm and ~3.4 ppm resonance, respectively. The downfield shift of the -CH2 protons suggests the formation of an imine. Notably, the relative integrations of the peaks was -1:1:3, consistent with the assignment as the imine.Example 4: Analysis of Isolated N^N'-dimethylethane-lj -diamine DMEN Imine Diphosphonate after Ion Exchange by13C NMR to assess Imine Formation

[0216] An ion exchange experiment with DMEN was performed to verify the results as described in Example 1. Ion exchange was performed to remove sodium cations, yielding a white solid. As shown in FIG.5, a13C NMR spectrum of the product after cation exchange between CDP and DMEN revealed the formation the carbonyl as a triplet around ~ 197 ppm (pointed to by an arrow) versus the expected -244 ppm for CDP. Such an upfield shift in resonance is expected for the formation of an imine. Indeed, a similar -40 ppm upfield shift is observed for analogous ketones and their corresponding imines.Examples 5: Cleave Kinetics Increase with Higher Concentration of Imine Diphosphonate (IDP)

[0217] AZDye™ 568 Hydroxylamine (FP-1092) (VectorLabs, Newark, CA) was dissolved in dimethyl sulfoxide (DMSO) to form a 10 mM stock solution. This solution was subsequently diluted to 250 pM in Millipore Milli-Q® water (EDM Millipore, Burlington MA). Five (5) pL of this solution was then added to an ultra performance liquid chromatography (UPLC) vial. Next, 250 pL of various formulations with potential cleaving activity were added to the solution and the solution was mixed with a pipette. At the prescribed time (e.g. 15 or 60 seconds), 100 pL of 1 mM methoxyamine in 1 M acetate buffer w as added to the solution to quench any remaining imine diphosphonate (IDP) in solution and the solution mixed. Finally, 150 pL of 1 mM 2-formylphenylboronic acid (2-FPBA) in Millipore Milli-Q® water (diluted from a 50 mM stock in DMSO) was added to the mixture.2-FPBA forms a 1:1 adduct with any unreacted hydroxylamine in solution and induces a retention time shift on UPLC. Any hydroxylamine that is converted to the corresponding alcohol will not form an adduct, which facilitates measuring the conversion of the AZDye™ 568 hydroxylamine to AZDye™ 568 alcohol, which otherwise have nearly identical retention times.

[0218] As show n in FIG.6, Panels A through D. the mixture was analyzed directly on UPLC, with absorption wavelength for analysis set at 568 nm. The relative integrations of thealcohol and hydroxylamine peaks provided information on % conversion. As an initial test, imine diphosphonate was titrated into a 1 M acetate buffer solution. Concentration dependence was observed in reaction kinetics as captured in FIG.6, Panels A through D. Specifically, 37%, 67%, and 92% conversion was measured after 1 minute at 10 pM (FIG. 6, Panel B), 20 pM (FIG. 6, Panel C), and 50 pM (FIG.6, Panel D) reagent concentration, where the control had 0 pM of the reagent added (FIG. 6, Panel A).

[0219] It has been reported that oxime condensations may be accelerated by the presence of salt. To further investigate the affect salt had on NfNkdimelhylethane-l.2-diamme (DMEN) mediated cleavage, reactions of carbonyl diphosphonate (CDP) with increasing acetate concentrations (i.e., 20 mM, 100 mM and 1000 mM) were monitored for the conversion of AZDye™ 568 hydroxylamine to AZDye™ 568 alcohol over time (i.e., 15, 60, 120, 300 seconds). It was found that increasing acetate concentration provided a 2X increase in reaction rate over the range 20 nM to 1 M acetate.

[0220] Next, FIG. 7 shows how increasing acetate concentrations (i.e., 0, 20, 100, and 1000 mM), in CDP mediated reactions with 30 mM of DMEN were monitored over 15 seconds for conversion of AZDye™ 568 hydroxylamine to AZDye™ 568 alcohol. It was found all DMEN containing reactions, independent of acetate concentration, resulted in a 40X increase in rate in conversion of the AZDye™ 568 hydroxylamine to AZDye™ 568 alcohol.Example 6: Analysis of N1,N1-dimethylethane-1,2-diamine (DMEN) and CDP in deblock kinetics assay

[0221] A “deblock kinetics assay” may be defined as an extension assay that measures the cleave kinetics of DNA on a solid support. In the “deblock kinetics assay” a fluorescent primer was hybridized to a solid support, and a hydroxylamine-containing reversible terminator was incorporated. Following, 100 mM of carbonyl diphosphonate (CDP) in 1 M sodium acetate buffer (pH 6.5) at 40 °C with increasing concentrations of DMEN, i.e., 0.3 mM, 15.3 mM, 30.3 mM, and 45.3 mM, were added and screened. Once the reaction finished, incorporation of a new base was attempted. The DNA primer was then analyzed using capillary electrophoresis. The original primer ran as a +0, DNA with the reversible terminator as +1, and the primer with efficient deblock and extension as +2. Once run, analysis of the relative intensities of the +2 and +1 peaks gave information on DNA cleave efficiency. To obtain kinetic information, the reaction was quenched at key timepoints through the addition of hydroxylamine-containing buffers.

[0222] As show n in FIG.8, the fraction of the deblocked DNA was monitored over a period of 200 seconds. Concentration dependence was observed with a 10X increase in reaction rate at higher concentrations (e.g.. 0.3 mM DMEN vs. 15 mM or 30 mM DMEN) as indicated by the arrow in FIG.8. In addition, the effect of the buffer on deblock kinetics w as also investigated. To test the buffer effects, a solution of 400 pM CDP, 45 mM DMEN at 40 °C, and one of the buffers A-D from Table 1 w ere added. The tested range of buffers having a pH of ~7 was tolerated, demonstrating a surprising and unexpected reactivity profile of IDP as compared to CDP alone.Table 1. Conditions for buffer effect analysis.Buffer Composition pH Jinax Kabs A 0.79 M Na / 1 M Acetate 6.5 0.95 0.104 s’1B 1 M Na / 1.1 M Acetate 7.5 0.96 0.065 s’1C 0.2 M Phosphate / 0.085 M Acetate 7.4 0.96 0.029 s'1D 0.2 M Tris HC1 / 0.06 M Acetate 7.5 0.82 0.052 s’1Example 7: Spectroscopic Analysis of 4-(2-aminoethyl)morpholine (AEM) and carbonyl diphosphonate (CDP) Imine Formation (31P NMR)

[0223] To further analyze the imine formation, a31P NMR experiment of 50 mM CDP with 90 mM AEM in IM sodium acetate dissolved in D2O was conducted. Consistent with what was observed when CDP w as combined with DMEN as seen in FIG.3, Panel B, FIG.9 shows the characteristic doublet of doublets at ~2.6 ppm and ~ -3.5 ppm. The formation of a doublet suggests the desymmetrization of the two phosphorus atoms which couple to each other. Notably, integration of the peaks demonstrates an 88% conversion of CDP to imine.Example 8: Spectroscopic Analysis of 4-(2-aminoethyl)morpholine (AEM) and carbonyl diphosphonate (CDP) Forms Imine Diphosphonate (’ H NMR)

[0224] To further analyze the imine formation, 'H NMR experiments were conducted. First, 90 mM AEM in IM sodium acetate was dissolved in D2O. Second, 50 mM CDP and 90 mM of AEM in IM sodium acetate were dissolved in D2O. 'H NMR spectra was measured for both experimental conditions.

[0225] FIG. 10, Panel A shows theXH NMR spectrum for the AEM solution where the methylene protons adjacent to the oxygen in the morpholine ring are at ~3.6 ppm, the methylene protons adjacent to the morpholine nitrogen ~ 2.50 ppm. and the methylene protons at ~ 3.0 and ~ 2.6 ppm with those closest to the morpholine ring being the most upfield. In contrast, FIG. 10, Panel B shows theJH NMR spectrum for the CDP + AEM solution, where new resonance peaks are observed. In particular, the morpholine methylene protons were shifted downfield to ~4.1 ppm and ~3.0 ppm. And the methylene protons adjacent to the morpholine ring were shifted downfield to ~3.2 ppm. Notably, the methylene adjacent to the imine had a substantial 0.7 ppm shift downfield to ~3.7 ppm indicating imine formation.Example 9: Confirmation of cleavage activity of imine diphosphonate formed from 4-(2-aminoethyljmorpholine (AEM) and carbonyl diphosphonate (CDP)

[0226] The disclosed assay quantifies total active diphosphonate content in solution by monitoring the conversion of pheylhydroxylamine to phenol. The two species are resolved by reverse-phase UPLC with absorption wavelength for analysis set at 271 nm. First, a 200 mM solution of phenylhydroxylamine was prepared in a 1:1 mixture of DMSO and aqueous buffer. The aqueous buffer consists of 1 M sodium acetate (final concentrations of 0.79 M sodium acetate and 0.21 M acetic acid) and 30 mM N\N imethylethane-l,2-diamine (DMEN).

[0227] Second, 2 uL of the phenylhydroxylamine stock was further diluted with 16 uL of 1 M sodium acetate buffer. To this mixture was then added 4 uL of the diphosphonate solution. After mixing the solution for about 1 to 5 minutes, the solution was subjected to UPLC analysis. The relative size of the peaks provides information on the concentration of cleave active species in solution. In general, higher conversion to phenol is consistent with a greater concentration of cleave active species in solution.

[0228] FIG. 11, Panel A, shows the UPLC chromatogram for 50 mM CDP in 1 M acetate buffer. FIG. 11, Panel B, shows the UPLC chromatogram for 50 mM CDP with 90 mM AEM in 1 M acetate buffer. Integration of the peaks demonstrates both 50 mM CDP and 50 mM CDP with 90 mM AEM (which exists predominantly as the imine diphosphonate) provide comparable total “cleave active’' species in solution. This indicates that imine diphosphonates prepared from AEM are competent in performing the cleave reaction.Example 10: Spectroscopic Analysis of Imine Formation (31P NMR) from N^N1-dimethylpropane-1.3-diamine (DMPN) and carbonyl diphosphonate (CDP)

[0229] To further analyze the imine formation, a31P NMR experiment of 50 mM CDP with 90 mM DMPN in 1 M sodium acetate was dissolved in D2O was conducted. Consistent with what was observed with when CDP was combined with DMEN, FIG. 12 shows the characteristic doublet of doublets at ~ 0 ppm and ~ -3.8 ppm. Notably, integration of the peaks demonstrates a 88% conversion of CDP to imine.Example 11: Spectroscopic Analysis of Imine Formation (' H NMR) from N^N1-dimethylpropane-l,3-diamine (DMPN) and carbonyl diphosphonate (CDP)

[0230] To further analyze the imine formation, 'H NMR experiments were conducted. First, 90 mM DMPN in 1 M sodium acetate was dissolved in a D2O / H2O mixture. Second, 50 mM CDP with 90 mM of DMPN in IM sodium acetate were dissolved in aD2O / H2O mixture. 'H NMR spectra was measured for both experimental conditions.

[0231] FIG. 13, Panel A shows theNMR spectrum for the DMPN solution wherein resonance peaks for the methylene protons adjacent to the primary nitrogen produce a triplet at ~3.0 ppm, and resonance peaks for the protons adjacent to the tertiary nitrogen produce a triplet at -2.7 ppm. Finally, the resonance peaks for the methylene protons in the center of the propyl chain produces a quintet at ~1.8 ppm and the N-methyl protons produce a singlet at -2.7 ppm. Conversely. FIG. 13, Panel B shows the 'H NMR spectrum for the CDP + DMPN solution, where new resonance peaks are observed. In particular, the triplet at -4.0 ppm corresponding to the methylene protons adjacent to the imine nitrogen, the singlet at -2.6 corresponding to the N-methyl protons, and the triplet at -2.1 corresponding to the methylene protons adjacent to the tertiary amine. The downfield shift of the methylene protons suggests the formation of an imine.Example 12: Confirmation of imine diphosphonate (IDP) cleave activity formed from N'N'-dimethylpropane-l,3-diamine (DMPN) and carbonyl diphosphonate (CDP)

[0232] To further investigate the activity of DMPN + CDP, the cleavage activity of the imine diphosphonate produced therefrom was analyzed by UPLC. First, 50 mM of CDP was incubated with 90 mM of DMPN in 1 M acetate buffer. Then, to the solution was incubated with excess phenylhydroxylamine. During incubation a portion of the phenylhydroxylamine is converted to phenol.

[0233] FIG. 14, Panel A, shows the UPLC chromatogram for 50 mM CDP in 1 M acetate buffer. FIG. 14, Panel B, shows the UPLC chromatogram for 50 mM CDP with 90 mM DMPN in 1 M acetate buffer. Comparison of the peak integration between Panel A and PanelB demonstrates the total “active CDP” remains constant, consistent with the proposal that imine diphosphonate with DMPN are active reagents for the conversion of hydroxylamines to alcohols.Example 13: Spectroscopic Analysis of 2-Aminomethyl-lH-Imidazole and carbonyl diphosphonate (CDP) demonstrates No Imine Formation (31P NMR)

[0234] To compare imine formation from the CDP and DMEN solution to a 2-Aminomethyl-IH-Imidazole and CDP solution, three31P NMR experiments were conducted. First, 50 mM CDP in 1 M sodium acetate was dissolved in D2O. Second, 50 mM CDP and 90 mM 2-Aminomethyl-lH-Imidazole in 1 M sodium acetate were dissolved in D2O. Lastly, 50 mM CDP and 90 mM N ^N1-dimethyl ethane- 1,2-diamine (DMEN) in 1 M sodium acetate were dissolved in D20.31P NMR spectra were measured for all three experimental conditions.

[0235] FIG. 15, Panel A shows the31P NMR spectrum for the CDP solution where the singlet formed by the symmetric di-phosphonate moiety of CDP is present at ~ 0.0 ppm. FIG.15, panel B shows the31P NMR spectrum 50 mM CDP and 90 mM 2-Aminomethyl-lH-Imidazole where two new singlet peaks at ~9.0 and 7.0 ppm are formed, at ratio of ~2:1 respectively. However, no doublet of doublet diagnostic of imine formation is observed. Conversely, FIG. 15. Panel C shows the doublet of doublets diagnostic of imine formation from 50 mM CDP and 90 mM DMEN. Therefore, no imine diphosphonate was formed by CDP and 2- Aminomethyl- IH-Imidazole.

[0236] Of note, CDP combined with 2-aminomethyl-lH-benzimidazole (2-AMBI) was also tested. While CDP and 2-AMBI are soluble compounds on their own at, for example, 50 mM and 90 mM, respectively. Upon combining CDP and 2-AMBI, large quantities of insoluble precipitates formed indicating 2-AMBI may undergo a cyclization reaction to form an insoluble product. The CDP and 2-AMBI byproduct was not analyzed further.Example 14: Spectroscopic Analysis of 2-Aminomethyl-lH-Imidazole and carbonyl diphosphonate (CDP) demonstrates No Imine Formation (XH NMR)

[0237] To confirm no imine formation from CDP and 2- Aminomethyl- IH-Imidazole, 'H NMR experiments were conducted. First, 90 mM 2-Aminomethyl- IH-Imidazole in 1 M sodium acetate were dissolved in D2O. Second, 50 mM CDP and 90 mM 2-Aminomethyl-IH-Imidazole in 1 M sodium acetate were dissolved in D2O. 'H NMR spectra was measured for both experimental conditions.

[0238] FIG. 16, Panel A shows the 'H NMR spectrum for the 2-Aminomethyl-lH-Imidazole solution wherein the resonance peaks for the methylene protons between the primary amine and imidazole ring form a single at ~ 4.1 ppm and the imidazole olefinic protons form a singlet at ~ 7.1 ppm. FIG. 16, Panel B show s theTH NMR spectrum for the CDP + 2-Aminomethyl-lH-Imidazole solution wherein the resonance peaks are not shifted dow nfield as is diagnostic of imine formation, demonstrating no imine is formed.Example 15: Spectroscopic Analysis of 2-Aminomethyl-lH-Imidazole and carbonyl diphosphonate (CDP) demonstrates Imidazole Adducts ('ll NMR)

[0239] As described in Example 13, it was observed that the combination of 2-aminomethyl-lH-benzimidazole (2-AMBI) and (CDP) in a solution of 1 M acetate buffer resulted in precipitate formation. However, neither 2-AMBI nor (CDP) alone in a 1 M acetate buffer produced a precipitate suggesting the combination of 2-AMBI with (CDP) resulted in the formation of an insoluble adduct. Specifically, a possible mechanism of adduct formation is the secondary amine in the imidazole ring undergoing an intramolecular cyclization with the imine forming and insoluble adduct. To assess if adduct formation prevents imine formation leading to the results of Examples 13 and 14, the 1H NMR spectrum of the CDP and 2-Aminomethyl-lH-Imidazole was subjected to further analysis.

[0240] FIG. 17, Panel A shows that when 2-Aminomethyl-lH-Imidazole alone in solution produces only resonance peaks at ~ 7.1 ppm and ~4.1 ppm representing the imidazole olefinic protons and the methylene protons respectively. FIG. 17, Panel B shows when 2-Aminomethyl-IH-Imidazole and (CDP) are in solution together multiple singlet peaks between 7.4 and 6.9 ppm are produced. Thus, the 2-Aminomethyl-lH-Imidazole ring undergoes a similar intramolecular cyclization as was described for 2-AMBI preventing imine diphosphonate formation.Examples 16: Combination of Carbonyldiphosphonate (CDP) with 2-Aminomethyl-lH-Imidazole (Amlm) Results in a Decrease in Cleave Active Species as Demonstrated by Cleave Kinetics Inconsistent with Formation of Imine Diphosphonate (IDP)

[0241] To further investigate the formation of an IDP produced from the combination of carbonyl diphosphonate (CDP) and aminomethylimidazole (Amlm), four solutions were analyzed by UPLC. First, CDP w as incubated with Amlm. Then, the combined reagents were incubated with excess phenylhydroxylamine, which converts a portion of the reagent to phenol.

[0242] FIG. 18, Panel A, shows the UPLC chromatogram for phyenylhydroxylamine in 1 M acetate buffer. FIG. 18, Panel B, shows the UPLC chromatogram for 50 mM CDP in 1 M acetate buffer. FIG. 18, Panel C, shows the UPLC chromatogram for 50 mM CDP with 90 mM Amlm in 1 M acetate buffer. FIG. 18, Panel D, UPLC chromatogram for 50 mM CDP with 180 mM Amlm in 1 M acetate buffer. It was observed that presence of Amlm decreases active CDP concentration, as indicated by the decreased fraction of phenylhydroxylamine converted into phenol. This suggests that the adduct between CDP and Amlm is inactive for performing the desired deblock reaction.Example 17: Spectroscopic Analysis of N^N^Dimethylethylenediamine and carbonyl diphosphonate (CDP) demonstrates No Imine Formation (31P NMR)

[0243] To determine whether an imine forms from a CDP and N', N2-Dimethylethylenediamine solution, three31P NMR experiments were conducted. First, 50 mM CDP in 1 M sodium acetate buffer prepared in D2O. Second, 50 mM CDP and 90 mM N1, N2-Dimethylethylenediamine in 1 M sodium acetate buffer prepared in D2O. Lastly, 50 mM CDP and 90 mM N ' N1-di methyl ethane- 1,2-diamine (DMEN) in 1 M sodium acetate buffer prepared in D20.31P NMR spectra were measured for all three experimental conditions.

[0244] FIG. 19, Panel A shows the31P NMR spectrum for the CDP solution where the singlet formed by the symmetric di-phosphonate moiety of CDP is present at ~ 0.0 ppm. FIG.19, Panel B shows the31P NMR spectrum 50 mM CDP and 90 mM N N2-Dimethylethylenediamine where no new resonance peaks are seen, only the singlet corresponding to the symmetric di-phosphonate moiety of CDP is present at ~ 0.0 ppm. The absence of any additional peaks suggests no imine or adduct formation. Conversely, FIG. 19, Panel C shows the doublet of doublets that is diagnostic of imine formation from 50 mM CDP and 90 mM DMEN.

[0245] Further, 'H NMR data (not shown) was obtained for 90 mM N’, N2-Dimethylethylenediamine alone in solution and 90 mM N'. N2-Dimeth\ lethylenediamine in combination with 50 mM CDP. Results of bothNMR spectra were identical suggesting no imine and / or adduct formation as observed in the31P NMR spectrum of FIG. 19, Panel B.

[0246] Further,31P NMR analyses were performed for 2-amino-5 -methylphenyl phosphonic acid (AMP A) when combined with CDP and 2-amino-5 -methoxy benzoic acid (AMBA) when combined with CDP under the same conditions as described herein. Noimine formation was observed by31P NMR analyses for AMPA and CDP or AMBA or CDP (data not shown).Example 18: Color Change and Spectroscopic Analysis of Aniline and Carbonyl diphosphonate (CDP) Demonstrates No Imine Formation (31P NMR)

[0247] The mechanism of imine phosphonate formation provided herein predicts that imine phosphonate would not form from aniline and carbonyldiphosphonate. Consistent with this, no noticeable loss of yellow coloration was observed when mixing 50 mM CDP with 90 mM aniline in 1 M acetate buffer. In contrast, a transition from yellow to colorless is observed when CDP is combinedwith other amines, such as DMEN, described herein.

[0248] FIG.20 shows no imine diphosphonate formation with aniline and carbonyldiphosphonate is observed by31P NMR. Briefly, 90 mM aniline was added to 1 M acetate buffer, with or without 50 mM CDP, and the resultant mixture was analyzed by31P NMR. Of note, the peak at ~13 ppm may correspond to CDP hydrate, which is more prevalent at lower pH (5.5). This further supports the conclusion that aniline does not form an adduct with CDP.

[0249] FIG.21 shows no imine diphosphonate formation with aniline andcarbony ldiphosphonate is observed by 'll NMR. Briefly, 90 mM aniline was added to 1 M acetate buffer, with or without 50 mM CDP, and the resultant mixture was analyzed by 'H NMR. These data further support the conclusion that aniline does not form an adduct with CDP.Example 19: Additional Experiments on Imine Diphosphonate Formation and Tolerance

[0250] Imine diphosphonate was found to be formed with CDP in combination with primary amines, including a number of ethylamines and propylamines. As described in Example 17, primary amines where NH2 was bonded directly to an aryl group, such as 2-amino-5-methylphenyl phosphonic acid and 2-amino-5 -methoxy benzoic acid, did not form imine phosphonates. As noted in Example 18, anilines did not form imine phosphonates in aqueous solution. It is believed to be because of the strong deactivation of the amine in aniline by resonance with the nearby aromatic group. 2-aminomethylbenzimidazole suffers from a similar issue as 2-aminomethyl-IH imidazole in terms of adduct formation with CDP by the imidazole nitrogen. N-alkylation may reduce this adduct formation.

[0251] In general, imine phosphonates were found to be stable to various buffers, pH, and salt concentrations. pH ranges between 5 and 9 were tested. Unlike CDP, imine phosphonate was found to maintain deblocking activity above pH 7. Citrate, acetate, phosphate and Tris buffers were tested at different pH and deblocking activity was observed by the deblock kinetics assay described earlier in Example 6.Example 20: Sequencing with Imine Phosphonate Deblocking of 3’ Aminooxy Reversibly Terminated Nucleotides Demonstrates High Efficiency

[0252] Sequencing by binding was performed under conditions similar to the Pacific Biosciences Onso Sequencing System (Pacific Biosciences, Menlo Park, CA) with the exception that imine diphosphonate was used for cleavage of 3’ aminooxy reversibly terminated nucleotides incorporated at the 3‘ end of the extended primer. The imine diphosphonate was formed from carbonyldiphosphonate and an excess of DMEN. The formulation was colorless, indicating no detectible level of carbonyldiphosphonate. Less than 17% of negative phasing (lagging) was observed after 150 cycles, indicating that in an average cycle over 99.8% of 3 ' aminooxy groups were cleaved at the N=O bond by the cleavage composition comprising imine diphosphonate.REFERENCES

[0253] Quimby and coworkers, J. Org. Chem. 1967, 32. 4111.

[0254] Dawson and coworkers, Angew. Chem. Int. Ed. 2006, 45. 7581.

[0255] Kool and coworkers, Org. Lett. 2015, 17, 274.

[0256] Kool and coworkers, Chem. Sci. 2018, 9, 5252.

[0257] Varghese, O. P. and coworkers. Sci Rep. 2018, 8, 2193.

[0258] All references throughout this application, for example patent documents, including issued or granted patents or equivalents and patent application publications, and non-patent literature documents or other source material are hereby incorporated by reference herein in their entireties, as though individually incorporated by reference.

[0259] All patents and publications mentioned in the specification are indicative of the levels of skill of those skilled in the art to which the invention pertains. References cited herein are incorporated by reference herein in their entirety to indicate the state of the art, in some cases as of their filing date, and it is intended that this information can be employedherein, if needed, to exclude (for example, to disclaim) specific embodiments that are in the prior art.

[0260] When a group of substituents is disclosed herein, it is understood that all individual members of those groups and all subgroups and classes that can be formed using the substituents are disclosed separately. When a Markush group or other grouping is used herein, all individual members of the group and all combinations and subcombinations possible of the group are intended to be individually included in the disclosure. As used herein, ”and / or“ means that one, all, or any combination of items in a list separated by ■‘and / o ’ are included in the list; for example “1, 2 and / or 3” is equivalent to ‘'1, 2, 3, 1 and 2, 1 and 3, 2 and 3, or 1, 2 and 3”.

[0261] Every' formulation or combination of components described or exemplified can be used to practice the invention, unless otherwise stated. Specific names of materials are intended to be exemplary, as it is know n that one of ordinary skill in the art can name the same material differently. It will be appreciated that methods, device elements, starting materials, and synthetic methods other than those specifically exemplified can be employed in the practice of the invention without resort to undue experimentation. All art-known functional equivalents, of any such methods, device elements, starting materials, and synthetic methods are intended to be included in this invention. Whenever a range is given in the specification, for example, a temperature range, a time range, or a composition range, all intermediate ranges and subranges, as well as all individual values included in the ranges given are intended to be included in the disclosure.

[0262] The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any7equivalents of the features show n and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may' be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

Claims

Attorney Docket No.: 097128-1540435 (046WO1) WHAT IS CLAIMED IS:

1. A composition, comprising a buffer and an imine compound of Formula (I):or stereoisomers and salts thereof, wherein:n is 0, 1, 2, or 3;m is 0 to 20R1is a bond, H, -CH2-. or -NH-;R2is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polymer, or -NR4R5, wherein R4and R3are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted polymer; andR3is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polymer, -P(O)?\ or -NR4R5, wherein R4and R3are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted polymer,wherein when R1is a bond and m is 0, n is 1,wherein when R1is -NH- and m is 0, n is 1, andwherein when R1is H, m and n are 0.

2. The composition of claim 1, wherein Formula (I) has a structure of Formula (la), Formula (lb), Formula (Ic), Formula (Id), or Formula (le):

3. The composition of claim 1 or claim 2, wherein m is 0. 1 or 2.

4. The composition of any one of claims 1 to 3, wherein n is 1, 2, or 3.

5. The composition of any one of claims 1 to 4, wherein R2is substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C1-C20 heteroalkyl, substituted or unsubstituted C3-C10 cycloalkyl. substituted or unsubstituted C3-C 10 heterocycloalkyl, substituted or unsubstituted C1-C20 alkenyl, substituted or unsubstituted C1-C20 heteroalkenyl, substituted or unsubstituted C3-C10 heterocycloalkenyl, substituted or unsubstituted C6-C10 aryl, substituted or unsubstituted C6-C10 heteroaryl, or substituted or unsubstituted polymer.

6. The composition of any one of claims 1 to 4, wherein at least one R2is — NHR5.

7. The composition of claim 6, wherein -NHR5is NH2.

8. The composition of any one of claims 1 to 4, wherein at least one R2is an imine phosphonate.

9. The composition of any one of claims 1 to 7, wherein R3is -P(O 3’.

10. The composition of any one of claims 1 to 9, wherein R2is:

12. The composition of any one of claims 1 to 5, wherein R2is a substituted polymer.

13. The composition of claim 12, wherein the substituted polymer is polyethylene glycol (PEG).

14. The composition of claim 12, wherein the substituted polymer comprises a hydrophilic substituent.

15. The composition of claim 14, wherein the hydrophilic substituent comprises a polar functional group, and / or a charged group.

16. The composition of claim 14 or claim 15, wherein the hydrophilic substituent is selected from the group consisting of a hydroxyl, carboxyl, amine, amide, sulfonic acid, phosphate, ketone, aldehyde, ammonium, sulfonate, phosphonate, and carboxylate.

17. The composition of any one of claims 1 to 5, wherein R2is unsubstituted aryl or unsubstituted heteroaryl wherein m is at least 1.

18. The composition of any one of claims 1 to 5, wherein R2is substituted aryl or substituted heteroaryl wherein m is at least 1.

19. The composition of any one of claims 1 to 16, wherein R2is not an aryl or heteroaryl.

20. The composition of any one of claims 1 to 5 or 13 to 15, wherein R2is not aniline, imidazole, methoxy benzoic acid, or benzimidazole.

21. The composition of claim 1, whereinn is 1, 2. or 3;m is 1 or 2;R1is a bond or -CH2-; andat least one R2is -NR4R5.

22. The composition of claim 21, wherein the at least one R2is -NH2.

23. The composition of claim 21 or claim 22, whereinR3is -P(O)3.

24. The composition of any one of claims 1 to 5, wherein n is 1, 2, or 3 and R2is -(NH-CH2-CH2)qR6, wherein q is 1 to 20 and wherein R6is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted polymer, or -NR4R5. wherein R4and R5are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyd, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted polymer.

25. The composition of any one of claims 1 to 5, wherein n is 1, 2 or 3 and R2is -(NH-CH2-CH2-CH2)qR6, wherein q is 1 to 20 and wherein R6is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyd, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted ary l, substituted or unsubstituted heteroary l, substituted or unsubstituted polymer, or -NR4R5. wherein R4and R5are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted polymer.

26. The composition of any one of claims 1 to 5, wherein n is 1 or 2 and R2is -NHR5, wherein R3is hydrogen, substituted or unsubstituted alkyd, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalky l, substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted polymer.

27. The composition of any one of claims 1 to 5, wherein n is 1 or 2 and R2is -NR4R3, wherein R4and R5are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted polymer.

28. The composition of claim 26 or claim 27. wherein at least one of R4or R5is a substituted polymer.

29. The composition of claim 28, wherein the substituted polymer is PEG.

30. The composition of claim 28. wherein the substituted polymer comprises a hydrophilic substituent.

31. The composition of any one of claims 1 to 30, further comprising an amine of formula:(R2)n \yR1^NH232. The composition of claim 31, wherein the amine is in molar excess of the imine phosphonate.

33. The composition of any one of claims 1 to 32, further comprising a carbonyl diphosphonate molecule.

34. The composition of claim 33, wherein a molar ratio of the carbonyl diphosphonate molecule to the imine compound is 0.1 or less.

35. The composition of any one of claims 1 to 34, further comprising a polynucleotide.

36. The composition of claim 35, wherein the polynucleotide comprises an aminooxy group.

37. The composition any of one of claims 1 to 36, wherein a pH of the composition is between 5.0 and 9.0.

38. The composition of claim 37, wherein a pH of the composition is between 5.5 to 7.0.

39. The composition of any one of claims 1 to 38, wherein the imine compound has a molarity of at least 0.001 mol / L, based on a total volume of the composition.

40. A method of producing an imine compound of any one of claims 1 to 39, comprising:contacting a solution of carbonyl phosphonate with a primary amine forming a reaction mixture comprising the imine compound; andoptionally extracting the imine compound from the reaction mixture.

41. The method of claim 40, wherein the primary amine is in excess of the carbonyl phosphonate.

42. The method of claim 40 or claim 41, wherein the pH of the solution is within the range of 5.0 to 9.0.

43. The method of any one of claims 40 to 42, wherein the buffer is an acetate buffer, a citrate buffer, a bicarbonate buffer, a phosphate buffer, Tris, HEPES, MOPS, or PIPES.

44. The method of any one of claims 40 to 43, wherein the solution further comprises an organic solvent, and / or an inorganic solvent.

45. The method of claim 44, wherein the organic solvent is a polar organic solvent.

46. The method of claim 44, wherein the inorganic solvent is water.

47. The method of any one of claims 40 to 46, further comprising introducing a solid support to the solution of carbonyl phosphonate before contacting the solution of carbonyl phosphonate with the primary amine.

48. The method of claim 47, wherein the solid support is an ion exchange resin, a magnetic bead, or a glass bead.

49. The method of claim 48, wherein the solid support is an ion exchange resin.

50. The method of claim 49, wherein the ion exchange resin is a cation exchange resin or an anion exchange resin.

51. A method of any one of claims 40 to 50, wherein the primary amine is selected from a list consisting of:NHH3C-(CH2)5-NH2H3C-(CH2VNH2 H3C— (CH2)T-NH2H3C— (CH2)g— NH2H3C— (CH2>9-NH2H3C— (CH2)io-NH2H3C — (CH2)ypNH2H3C — (CH2)fjNH2H3C — (CH2)73-NH2H3C— (CH2)T4-NH2H3c— (CH2hyNH2H3C— (CH2)^NH2H3c— (CH2>i7NH2H3c— (CH2)i8-NH2', or salts thereof.

52. The method of any one of claims 40 to 50, wherein the primary amine is preferably selected from a list consisting of:

53. The method of any one of claims 40 to 52, wherein during the step of contacting, the primary amine has a molarity in the range of 0.001 mol / L, based on a total volume of the reaction mixture.

54. The method of any one of claims 40 to 53, wherein the imine is extracted by rinsing with a reagent, crystallization, chromatography, lyophilization and / or filtration.

55. The method of claim 54, wherein the chromatography is column chromatography or ion exchange chromatography.

56. The method of claim 54, wherein the reagent is a primary amine.

57. The composition of claim 21, wherein R2is -NHR5.

58. The composition of claim 21 or claim 57, wherein R4and R5are independently selected from the group consisting of-(NH-CH2-CH2)qR6and -(NH-CH2- CH2-CH2)qR6, wherein q is 1 to 20 and wherein R6is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted heteroalkenyl, substituted or unsubstituted heterocycloalkenyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstitutedpolymer, or -NR4R5, wherein R4and R5are independently selected from the group consisting of hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, and substituted or unsubstituted polymer.

59. The composition of claim 57, wherein q is 1.

60. The composition of claim 57 or claim 58, wherein R6is hydrogen.

61. A method of extending polynucleotides, comprising:a) contacting polynucleotides comprising aminooxy groups with a first composition to produce polynucleotides comprising extendible 3’ OH groups:wherein the first composition is the composition of any one of claims 1 to 39 and 57 to 60,wherein each polynucleotide has an aminooxy group at a 3' end of the polynucleotide, andwherein the composition cleaves an N=O bond of the aminooxy group to produce the 3’ OH group thereby producing extendible poly nucleotides with 3’ OH groups;b) extending the polynucleotides comprising the 3‘ OH groups in a nucleic acid polymerization reaction by contacting the polynucleotides comprising the 3’ OH groups with a polymerase and one or more nucleotides; andc) repeating steps a) and b) in a plurality of cycles.

62. A method of extending polynucleotides, comprising:a) contacting polynucleotides comprising aminooxy groups with a first composition comprising an imine phosphonate compound to produce polynucleotides comprising extendible 3’ OH groups,wherein each polynucleotide has an aminooxy group at a 3’ end of the polynucleotide, andwherein the composition cleaves an N=O bond of the aminooxy group to produce the 3’ OH group thereby producing extendible poly nucleotides with 3’ OH groups;b) extending the polynucleotides comprising the 3’ OH groups in a nucleic acid polymerization reaction by contacting the polynucleotides comprising the 3’ OH groups with a polymerase and one or more nucleotides; andc) repeating steps a) and b) in a plurality of cycles.

63. The method of claim 62, wherein the carbonyl phosphonate is a carbony l diphosphonate and wherein the imine phosphonate compound is an imine diphosphonate.

64. The method of claim 62 or claim 63, wherein the imine phosphonate compound comprises a substituted alkyl, unsubstituted alkyl, substituted alkenyl, or unsubstituted alkenyl.

65. The method of any one of claims 62 to 64, wherein the imine phosphonate compound comprises one or more ethylamine groups.

66. The method of any one of claims 62 to 64, wherein the imine phosphonate compound comprises one or more propylamine groups.

67. The method of any one of claims 62 to 66, wherein the imine phosphonate compound comprises an amine separated from the imine by 2 or 3 carbons.

68. The method of any one of claims 62 to 67, wherein the imine phosphonate compound is not aromatic.

69. The method of any one of claims 62 to 68, wherein the imine phosphonate compound does not contain ethylenediamine, isopropylamine, aniline, or an amino acid.

70. The method of any one of claims 61-69, wherein at least 99.9% of the polynucleotides comprising the aminooxy groups are converted to extendable polynucleotides comprising the 3’ OH groups.

71. The method of any one of claims 61 to 69, wherein at least 99% of the extendable polynucleotides are produced by cleavage of the O-N bond by imine phosphonate.

72. The method of any one of claims 61 to 71, wherein the first composition further comprises a carbonyl diphosphonate.

73. The method of claim 72, wherein a w / w ratio of the imine compound to the carbonyl diphosphonate molecule is greater than 1.

74. The method of claim 72, wherein the first composition comprises the carbonyl diphosphonate at a molar ratio to imine phosphonate of less than 0.1.

75. The method of claim 74, wherein the molar ratio is less than 0.

01.

76. The method of claim 72, wherein the presence of the carbonyl diphosphonate reduces the number of extendible nucleotides produced.

77. The method of any one of claims 61 to 76, wherein the contacting of step a) occurs over a period of time and the period of time is less than 1 minute.

78. The method of any one of claims 61 to 77, wherein the polynucleotides are coupled, directly or indirectly, to a surface.

79. The method of claim 78, wherein the surface is a bead, pad or well.

80. The method of claim 78, wherein the surface is within a flow cell.

81. The method of claim 78, wherein the polynucleotides are clonally identical and are repeated in the same concatemer, hybridized to the same concatemer, attached to a bead, attached to a pad, or attached to a well.

82. The method of any one of claims 78 to 81, wherein the polynucleotides are coupled to the surface indirectly and wherein the polynucleotides are primers that are hybridized to a template nucleotide that is coupled to the surface.

83. The method of any one of claims 61-82, wherein the polymerase is a template dependent polymerases.

84. The method of any one of claims 61-83, wherein the nucleotides do not comprise a fluorescent label.

85. The method of any one of claims 61-84, wherein the extended polynucleotides are a primer hybridized to a template nucleotide.

86. The method of claim 85, wherein the plurality of cycles further comprises a step of detecting a next correct nucleotide at a 3’ position of the primer.

87. The method of claim 86, wherein the next correct nucleotide is detected by detecting a ternary complex comprising a polymerase, the polynucleotides hybridized to a template nucleotide, and a fluorescent nucleotide, wherein the fluorescent nucleotide is not incorporated into the primer.

88. The method of claim 87, wherein the ternary complex is formed with polynucleotides comprising the aminooxy groups.89 The method of claim 87, wherein the ternary complex is formed with polynucleotides comprising the 3 ’ OH groups in the presence of a non-catalytic cation.

90. The method of claim 89, wherein the non-catalytic cation is strontium or calcium.

91. The method of claim 86, wherein the next correct nucleotide is a fluorescent nucleotide incorporated in step b).

92. The method of claim 91, wherein the incorporated fluorescent nucleotide comprises a fluorophore coupled to the base of the nucleotide through an aminooxy group comprising an N=O bond that is cleaved in step a) of a next cycle of the plurality of cycles.

93. The method of any one of claim 61-81, wherein the polymerase is a template independent polymerase.

94. The method of claim 93, wherein the nucleotides of step b) are of a single base type and comprise a 3’ N=O bond.

95. The method of claim 94, wherein the nucleotides of step b) are of a single base and differ between some of the cycles of the plurality of cycles.

96. The method of any one of claims 62-95, wherein the first composition is the composition of any one of claims 1 to 39 and 57 to 60.

97. A kit comprising:a) the composition comprising the composition of any one claims 1 to 39 and 57 to 60;b) a polymerase; andc) nucleotides comprising an N=O bond.

98. A kit comprising:a) a composition comprising an imine phosphonate;b) a polymerase; andc) nucleotides comprising an N=O bond.

99. The kit of claim 97 or 98, wherein the nucleotides are nucleotide triphosphates.

100. The kit of any one of claims 97-99, wherein the nucleotides comprise a 3’ N=O bond.

101. The kit of claim 100, further comprising a fluorescent nucleotide comprising an extendible 3’ OH group.

102. The kit of any one of claims 97-99, wherein the nucleotides comprise the N=O bond off the base between the nucleotide and a fluorophore.

103. The kit of claim 100, wherein the nucleotides comprise the 3’ N=O bond and the N=O bond off the base between the nucleotide and a fluorophore.

104. The kit of any one of claims 97-103, wherein the polymerase is a template independent polymerase.

105. The kit of any one of claims 98-104, wherein the imine phosphonate is an imine diphosphonate.

106. The kit of any one of claims 98-104, wherein the imine phosphonate is aliphatic.

107. The kit of any one of claims 98-104, wherein the imine phosphonate comprises one or more ethylamine groups.

108. The kit of any one of claims 98-104, wherein the imine phosphonate comprises one or more propylamine groups.

109. The kit of any one of claims 98-104, wherein the imine phosphonate comprises an amine spaced from the imine by 2 or 3 carbon bonds.

110. The kit of any one of claims 98-104, wherein the imine phosphonate lacks any aryl group.

111. The kit of any one of claims 98-104, wherein the composition is the composition of any one of claims 1 to 60.

112. The kit of any one of claims 97-111, wherein the composition, polymerase and nucleotides are in separate compartments of the kit.

113. The kit of any one of claims 97-112, wherein the kit comprises a plastic cartridge comprising the composition, polymerase and nucleotides.

114. A method of sequencing a polynucleotide templates, comprising: providing polynucleotides hybridized to polynucleotide templates coupled to a surface, wherein each polynucleotide is a primer;a) contacting the polynucleotides with a first composition, wherein the first composition is the composition of any one of claims 1 to 39 and 57 to 60. to cleave an N=O bond of an aminooxy group of the 3’ nucleotide of each polynucleotide;b) extending the polynucleotides in a nucleic acid polymerization reaction by contacting the polynucleotides with a polymerase and one or more nucleotides;c) detecting, before or after step b) of extending, the next correct nucleotide of the polynucleotide template; andd) repeating steps a), b) and c) in a plurality of cycles.

115. A method of method of sequencing polynucleotide templates, comprising:providing polynucleotides hybridized to polynucleotide templates coupled to a surface, wherein each polynucleotide is a primer;a) contacting the polynucleotides with a first composition comprising an imine phosphonate to cleave an N=O bond of an aminooxy group of the 3’ nucleotide of each polynucleotide;b) extending the polynucleotides in a nucleic acid polymerization reaction by contacting the polynucleotides with a polymerase and one or more nucleotides;c) detecting, before or after step b) of extending, the next correct nucleotide of the polynucleotide template; andd) repeating steps a), b) and c) in a plurality of cycles.

116. The method of claim 115. wherein the first composition is the composition of any one of claims 1 to 39 and 57 to 60.

117. The method of any one of claims 114-116, wherein detecting comprises detecting a ternary complex comprising a polymerase, the polynucleotide, and afluorescent nucleotide, wherein the fluorescent nucleotide is not incorporated to extend the extendible polynucleotide in step b).

118. The method of claim 117, wherein the N=O bond cleaved in step a) is at the 3’ position of the 3‘ nucleotide of the polynucleotide and wherein the nucleotides of step b) each comprise an N=O bond at their 3’ end.

119. The method of claim 117 or 118, wherein the ternary complex is formed with polynucleotides comprising the aminooxy groups and wherein step c) of detecting is after step b).

120. The method of claim 117 or 118, wherein the ternary complex is formed with polynucleotides comprising the 3’ OH groups in the presence of a non-catalytic cation.

121. The method of claim 120, wherein the non-catalytic cation is strontium or calcium and wherein step c) of detecting is between step a) of contacting and step b) of extending.

122. The method of any one of claims 114-116, wherein the next correct nucleotide is a fluorescent nucleotide incorporated in step b) and wherein step c) of detection is after step b) of extending.

123. The method of claim 122, wherein the incorporated fluorescent nucleotide comprises a fluorophore coupled to the base of the nucleotide through an aminooxy bond and comprises an aminooxy group on the 3‘ end, wherein an N=O bond of both aminooxy groups are cleaved in step a) of the next cycle of the plurality of cycles.

124. The method of claim 122, wherein the incorporated fluorescent nucleotide comprises a fluorophore coupled to the base of the nucleotide through an N=O bond and does not comprise an N=O bond at the 3’ end.

125. The method of any one of claims 124, wherein the nucleotides of step b) are of a single base type and wherein the method further comprises repeating each base type every four cycles of the plurality of cycles.

126. A system comprising:a) the composition of any one of claims of claims 1 to 39 and 57 to 60; andb) a flow cell comprising a surface,wherein the system is configured to deliver the composition and reagents to the surface of the flow cell to extend polynucleotides coupled to the surface of the flow cell.

127. A system comprising:a) a composition comprising an imine phosphonate;b) a flow cell comprising a surface,wherein the system is configured to deliver the composition and reagents to the surface of the flow cell to extend polynucleotides coupled to the surface of the flow cell.

128. The system of claim 126 or 127, further comprising a detection system configured to detect fluorescent nucleotides incorporated into the polynucleotides or in a ternary complex with the polynucleotides.

129. The system of claim 127 or 128, wherein the system comprises a composition of any one of claims 1 to 39 and 57 to 60.

129. The system of any one of claims 126 to 129, wherein the system is configured to perform the methods of any of claims 61 to 96 or 114 to 125.

Images