Diastereomeric ligation reagents for nucleotide probes

Abstract

A multifunctional reagent having a non-nucleotide monomer unit, and first and second coupling groups attached to the non-nucleotide monomer unit, wherein the non-nucleotide monomer unit comprises an enantiomeric center and has a ligand. The reagent allows for the preparation of multifunctional nucleotide / non-nucleotide polymers having any desired sequence of nucleotide and non-nucleotide monomer units, each of which carries a desired ligand. These polymers can, for example, be used as probes that exhibit increased sensitivity and / or are capable of detecting a genus of nucleotides, where each species of the genus has a common target nucleotide sequence bridged by a different non-target sequence.

Description

Technical Field

[0001] The present invention relates to diastereomeric non-nucleotide reagents and mixtures thereof that conveniently allow attachment of single or multiple moieties (e.g., labels, intercalators, metal particles, reactive species, or composite particles) to any specific preselected position on a nucleotide probe or oligonucleotide. Background of the Invention

[0003] In clinical research and diagnostics, a known technique for determining the presence of a specific RNA or DNA nucleotide sequence ("target nucleotide sequence" or simply "target sequence") is to perform a nucleic acid hybridization assay. In such an assay, a nucleotide polymer probe (usually an oligonucleotide) is selected that has a nucleotide sequence that is complementary to at least a portion of the target sequence. Typically, the probe is labeled, that is, it has an atom or group attached to it, the presence of which can be readily detected. When the labeled probe is exposed to a test sample suspected of containing the target nucleotide sequence, the target will hybridize to any of the labeled probes under hybridization conditions. The presence of the target sequence in the sample can be determined qualitatively or quantitatively, typically by separating hybridized and unhybridized probes and then determining the amount of hybridized labeled probe by determining the presence of the label in the probe hybrid or by determining the amount of the label in the non-hybridized probe. There are a variety of methods for labeling oligonucleotides. Such methods include: radiolabeling; attachment of a biotin moiety to the C-5 position of the pyrimidine ring of a uridine analog, followed by enzymatic incorporation into the oligonucleotide; terminal labeling, which is more suitable for label attachment as the final step in solid-phase oligonucleotide synthesis; and derivatives of nucleotides linked to phosphate groups, whose nucleophilic moieties can be labeled after their incorporation into the oligonucleotide, to name a few. Each of the above methods has disadvantages such as environmental and health safety (e.g., radioactivity); sensitivity issues; specificity issues; or automation issues. The present disclosure relates to the use of enantiomeric enrichment reagents compatible with automated oligonucleotide synthesis to improve the sensitivity and specificity of nucleic acid hybridization assays. Summary of the Invention

[0004] The present disclosure relates to enantiospecific non-nucleotide reagents having non-nucleotide monomer units that can be introduced into nucleic acids. Non-nucleotide reagents, also referred to as linking reagents, can be placed at any position within the nucleotide sequence backbone. Obviously, linking reagents can be synthesized to have protective groups commonly used in nucleic acid synthesis, and various nucleic acid synthesis methods can be used to introduce them into nucleic acids. Linking reagents can allow the connection of other chemical moieties, such as, but not limited to, detectable markers, intercalators, chelating agents, metal particles, active substances, composite particles, drugs, hormones, proteins, peptides, haptens, free radical generators, nuclear dissolving agents, proteolytic agents, catalysts, receptor binding substances, other biologically significant binding substances, reagents that change nucleic acid transport across biological barriers, and reagents that change nucleotide polymer solubility.

[0005] In one embodiment, a diastereomeric linking reagent comprising a compound based on the R enantiomer phosphoramidite having the formula: D—X 1 —R 2 (X 3 —M) n —Z. In these embodiments, “Z” can be a 2 The reactive phosphorus group at the end, the OH group of the first nucleotide or connected to R through a cleavable ester bond 2 DNA synthesis support at the end. 2 " can be with X 1 and an atomic chain of 2-20 atoms connected by Z. 2 Stable to DNA synthesis and deprotection conditions. 1 " can be O, S, NH or -N=N-. "D" can be a protective group that can be modified or removed, which allows X to be modified or removed. 1 Coupled to the phosphorus group of the second nucleotide. Each "X 3 ” can be independently connected at the first end to R 2 And connected to the linker-arm of M at the second end. X 3 Stable to DNA synthesis and deprotection conditions. Each "M" can independently be a label that is stable to DNA synthesis and deprotection conditions, or a changeable or removable protecting group that allows X to be modified or removed. 3 coupled to a label."n" is a positive integer.

[0006] In another embodiment, a diastereomeric linking reagent comprising a compound based on the S enantiomer phosphoramidite having the formula: D—X1 —R 2 (X 3 —M) n —Z. In these embodiments, “Z” can be a 2 The reactive phosphorus group at the end, the OH group of the first nucleotide or connected to R through a cleavable ester bond 2 DNA synthesis vector at the end. 2 " can be with X 1 and an atomic chain of 2-20 atoms connected by Z. 2 Stable to DNA synthesis and deprotection conditions. 1 " can be O, S, NH or -N=N-. "D" can be a protective group that can be modified or removed, which allows X to be modified or removed. 1 Coupled to the phosphorus group of the second nucleotide. Each "X 3 ” can be independently connected at the first end to R 2 And connected to the linker-arm of M at the second end. X 3 Stable to DNA synthesis and deprotection conditions. Each "M" can independently be a label that is stable to DNA synthesis and deprotection conditions, or a changeable or removable protecting group that allows X to be modified or removed. 3 is coupled to a label."n" is a positive integer.

[0007] In one embodiment, is a diastereomeric linking reagent comprising a compound based on the R enantiomer phosphoramidite having the formula: Z—X 1 —R 2 (X 3 —M) n —D. In these embodiments, “Z” may be a 2 The reactive phosphorus group at the end, the OH group of the first nucleotide or connected to R through a cleavable ester bond 2 DNA synthesis vector at the end. 2 " can be with X 1 and an atomic chain of 2-20 atoms connected by Z. 2 Stable to DNA synthesis and deprotection conditions. 1 " can be O, S, NH or -N=N-. "D" can be a protective group that can be modified or removed, which allows X to be modified or removed. 1 Coupled to the phosphorus group of the second nucleotide. Each "X 3 ” can be independently connected at the first end to R 2 And connected to the linker-arm of M at the second end. X 3Stable to DNA synthesis and deprotection conditions. Each "M" can independently be a label that is stable to DNA synthesis and deprotection conditions, or a changeable or removable protecting group that allows X to be modified or removed. 3 is coupled to a label."n" is a positive integer.

[0008] In one embodiment, is a diastereomeric linking reagent comprising a compound based on the S enantiomer phosphoramidite having the formula: Z—X 1 —R 2 (X 3 —M) n —D. In these embodiments, “Z” may be a 2 The reactive phosphorus group at the end, the OH group of the first nucleotide or connected to R through a cleavable ester bond 2 DNA synthesis vector at the end. 2 " can be with X 1 and an atomic chain of 2-20 atoms connected by Z. 2 Stable to DNA synthesis and deprotection conditions. 1 " can be O, S, NH or -N=N-. "D" can be a protective group that can be modified or removed, which allows X to be modified or removed. 1 Coupled to the phosphorus group of the second nucleotide. Each "X 3 ” can be independently connected at the first end to R 2 And connected to the linker-arm of M at the second end. X 3 Stable to DNA synthesis and deprotection conditions. Each "M" can independently be a label that is stable to DNA synthesis and deprotection conditions, or a changeable or removable protecting group that allows X to be modified or removed. 3 is coupled to a label."n" is a positive integer.

[0009] In general, for any of the above linking reagents, Z may have the formula:

[0010]

[0011] When this happens, "X 2 " can be halogen or substituted amino. "R 3 " can be an alkyl, alkoxy or phenoxy group. "X 4 " can be halogen, amino or O - . “R 5 " can be alkyl, alkoxy, aryloxy or H, however, only when X 4 Yes O - When R 5It may be H. In one embodiment, Z may have the formula:

[0012]

[0013] When this happens, X 2 Can be Cl or secondary amino, and R 3 It can be chlorophenoxy, methoxy, ethoxy or β-cyanoethoxy. Alternatively, X 2 It can be diisopropylamino, dimethyl or morpholino.

[0014] Generally, for any of the above linking reagents, Z may have the formula:

[0015]

[0016] When this happens, X 4 Can be Cl, secondary amino or O - , and R 5 It can be methoxy, ethoxy, monochlorophenoxy, β-cyanoethoxy or H. However, only when X 4 Yes O - When R 5 It can be H.

[0017] For any of the above linking reagents, particularly when Z is a reactive phosphorus-containing group, X 1 It can be O.

[0018] For any of the above linking reagents, R 2 X may be a hydrocarbon chain optionally substituted with one or more heteroatoms, each of which independently may be oxygen, nitrogen or sulfur. 3 can independently be NH, O, S, -NNH-, or a chain of 1-25 atoms in length terminated by NH, O, S or -NNH-. 3 When R is an atom chain, it may be a hydrocarbon chain optionally substituted with one or more heteroatoms independently selected from oxygen, nitrogen or sulfur. 2 In another embodiment, R 2 The hydrocarbon chain may be an acyclic hydrocarbon chain. The hydrocarbon chain may be 2 to 10 carbon atoms in length. Alternatively, the hydrocarbon chain may be 2 to 3 carbon atoms in length. In addition, n may be 1.

[0019] For any of the above linking reagents, R 2 It can be an acyclic hydrocarbon chain. Typically, the hydrocarbon chain has a length of 2 to 10 carbon atoms, or a length of 2 to 3 carbon atoms. In addition, n can be 1.

[0020] For any of the above linking reagents, particularly when Z is a reactive phosphorus-containing group, X 3 Can be connected to R through carbon 2 and is linked to M through nitrogen. Additionally, M may independently be trifluoroacetyl or 9-trifluorenylmethoxycarbonyl.

[0021] For any of the above linking reagents, D can be trityl or dimethoxytrityl.

[0022] For any of the above linking reagents, M can be a label that is stable to DNA synthesis and deprotection conditions.

[0023] For any of the above linking reagents, particularly when Z is a reactive phosphorus-containing group, M may be a protecting group that can be removed to allow X to 3 coupled to the label.

[0024] In one embodiment is a diastereomeric linking reagent comprising an R enantiomer phosphoramidite-based compound having the formula:

[0025]

[0026] In these embodiments, "Z" can be a reactive phosphorus-containing group, an OH group of the first nucleotide, or a cleavable ester attached to a DNA synthesis support. "D" can be a modifiable or removable protecting group that, when modified or removed, allows the non-nucleotide linking reagent to couple to the second nucleotide. "i" can be 0, 1, 2, or 3, and j can be 0, 1, 2, or 3, provided that i+j is at least 1. "L 1 " can be the first joint arm. "L 2 " can be H or a non-linked alkyl chain or a non-linked mixed alkyl chain or a second linker arm. When L 1 and L 2 When they are not identical, the carbon to which they are attached is the R enantiomer. Alternatively, when L 1 and L 2 When different, the carbon to which they are attached is the S enantiomer. Typically, the first linker-arm and the second linker-arm each independently have the following formula: (CH2) k —NH—(CO—(CH2) q NH) r—M. “M” can independently be H, fluorenylmethoxycarbonyl, trifluoroacetyl, or a label that is stable to DNA synthesis and deprotection conditions. “k” can be an integer between 0 and 4, inclusive. “q” can be an integer between 1 and 11, inclusive. Each “r” can independently be 0, 1, or 2, provided that each k+1+(2+q)r in the first linker arm and the second linker arm is independently an integer between 1 and 25, inclusive.

[0027] In another embodiment is a diastereomeric linking reagent comprising an S-enantiomer phosphoramidite-based compound having the formula:

[0028]

[0029] In these embodiments, "Z" can be a reactive phosphorus-containing group, an OH group of the first nucleotide, or a cleavable ester attached to a DNA synthesis support. "D" can be a modifiable or removable protecting group that, when modified or removed, allows the non-nucleotide linking reagent to couple to the second nucleotide. "i" can be 0, 1, 2, or 3, and j can be 0, 1, 2, or 3, provided that i+j is at least 1. "L 1 " can be the first joint arm. "L 2 " can be H or a non-linked alkyl chain or a non-linked mixed alkyl chain or a second linker arm. When L 1 and L 2 When they are not identical, the carbon to which they are attached is the R enantiomer. Alternatively, when L 1 and L 2 When different, the carbon to which they are attached is the S enantiomer. Typically, the first linker arm and the second linker arm each independently have the formula: (CH2) k —NH—(CO—(CH2) q NH) r —M. “M” can independently be H, fluorenylmethoxycarbonyl, trifluoroacetyl, or a label that is stable to DNA synthesis and deprotection conditions. “k” can be an integer between 0 and 4, inclusive. “q” can be an integer between 1 and 11, inclusive. Each “r” can independently be 0, 1, or 2, provided that each k+1+(2+q)r in the first linker arm and the second linker arm is independently an integer between 1 and 25, inclusive.

[0030] In another embodiment is a diastereomeric linking reagent comprising an R enantiomer phosphoramidite-based compound having the formula:

[0031]

[0032] In these embodiments, "Z" can be a reactive phosphorus-containing group, an OH group of the first nucleotide, or a cleavable ester attached to a DNA synthesis support. "D" can be a modifiable or removable protecting group that, when modified or removed, allows the non-nucleotide linking reagent to couple to the second nucleotide. "i" can be 0, 1, 2, or 3, and j can be 0, 1, 2, or 3, provided that i+j is at least 1. "L 1 " can be the first joint arm. "L 2 " can be H or a non-linked alkyl chain or a non-linked mixed alkyl chain or a second linker arm. When L 1 and L 2 When they are not identical, the carbon to which they are attached is the R enantiomer. Alternatively, when L 1 and L 2 When different, the carbon to which they are attached is the S enantiomer. Typically, the first linker arm and the second linker arm each independently have the formula: (CH2) k —NH—(CO—(CH2) q NH) r —M. “M” can independently be H, fluorenylmethoxycarbonyl, trifluoroacetyl, or a label that is stable to DNA synthesis and deprotection conditions. “k” can be an integer between 0 and 4, inclusive. “q” can be an integer between 1 and 11, inclusive. Each “r” can independently be 0, 1, or 2, provided that each k+1+(2+q)r in the first linker arm and the second linker arm is independently an integer between 1 and 25, inclusive.

[0033] In another embodiment is a diastereomeric linking reagent comprising an S-enantiomer phosphoramidite-based compound having the formula:

[0034]

[0035] In these embodiments, "Z" can be a reactive phosphorus-containing group, an OH group of the first nucleotide, or a cleavable ester attached to a DNA synthesis support. "D" can be a modifiable or removable protecting group that, when modified or removed, allows the non-nucleotide linking reagent to couple to the second nucleotide. "i" can be 0, 1, 2, or 3, and j can be 0, 1, 2, or 3, provided that i+j is at least 1. "L 1 " can be the first joint arm. "L 2 " can be H or a non-linked alkyl chain or a non-linked mixed alkyl chain or a second linker arm. When L 1 and L 2 When they are not identical, the carbon to which they are attached is the R enantiomer. Alternatively, when L 1 and L 2When different, the carbon to which they are attached is the S enantiomer. Typically, the first linker arm and the second linker arm each independently have the formula: (CH2) k —NH—(CO—(CH2) q NH) r —M. “M” can independently be H, fluorenylmethoxycarbonyl, trifluoroacetyl, or a label that is stable to DNA synthesis and deprotection conditions. “k” can be an integer between 0 and 4, inclusive. “q” can be an integer between 1 and 11, inclusive. Each “r” can independently be 0, 1, or 2, provided that each k+1+(2+q)r in the first linker arm and the second linker arm is independently an integer between 1 and 25, inclusive.

[0036] Generally, for any of the above linking reagents, Z may have the following formula:

[0037]

[0038] When this happens, "X 2 " can be halogen or substituted amino. "R 3 " can be an alkyl, alkoxy or phenoxy group. "X 4 " can be halogen, amino or O - . “R 5 " can be alkyl, alkoxy, aryloxy or H, however, only when X 4 Yes O - When R 5 It may be H. In one embodiment, Z may have the formula:

[0039]

[0040] When this happens, X 2 Can be Cl or secondary amino, and R 3 It can be chlorophenoxy, methoxy, ethoxy or β-cyanoethoxy. Alternatively, X 2 It may be diisopropylamino, dimethylamino or morpholino.

[0041] Generally, for any of the above linking reagents, Z may have the formula:

[0042]

[0043] When this happens, X 4 Can be Cl, secondary amino or O - , and R 5 It can be methoxy, ethoxy, monochlorophenoxy, β-cyanoethoxy or H. However, only when X 4Yes O - When R 5 It can be H.

[0044] For any of the above linking reagents, j can be 0. For any of the above linking reagents, L 2 Can be H. For any of the above linking reagents, L 2 can be H and j can be 0. For any of the above linking reagents, i can be 1. For any of the above linking reagents, j can be 0. For any of the above linking reagents, i can be 1 and j can be 1. For any of the above linking reagents, r can be 0. For any of the above linking reagents, r can be 0 and R 5 For any of the above linking agents, q may be an integer between 1 and 6, inclusive. For any of the above linking agents, q may be an integer between 1 and 6, L 2 can be H and j can be 0. For any of the above linking reagents, k can independently be an integer between 1 and 3, inclusive. For any of the above linking reagents, i can be 0. For any of the above linking reagents, L 2 may be H and i may be 0. For any of the above linking reagents, j may be 1.

[0045] Generally, any of the linking agents described above can be incorporated into an oligonucleotide.The oligonucleotide may also include a detectable label.

[0046] Methods for preparing the above-mentioned linking reagents are also contemplated. Typically, the linking reagents are prepared by synthesizing the linker and purifying the isomers. Alternatively, the linking reagents are prepared by synthesizing the linker using an isomer-specific reagent, thereby eliminating the need for purifying the isomers.

[0047] Typically, the linking reagents described above can be introduced into oligonucleotides. In one embodiment, the linking reagents are introduced by (a) coupling the reactive phosphorus group of the linking reagent to a first nucleotide or chain or nucleotides under DNA synthesis conditions, and (b) removing the protecting group D to allow coupling of the activated phosphorus group of the second nucleotide or second linking reagent.

[0048] Also contemplated are kits comprising any of the above-described ligation reagents and instructions for use. Alternatively, the kit may include oligonucleotides having the above-described ligation reagents introduced into the sequence and instructions for use. The kit may also include various buffers or reagents that can be used in conjunction with the ligation reagents or oligonucleotides.

[0049] Also contemplated are assays that may include any of the above-described linking agents. Alternatively, the assay may include any of the above-described oligonucleotides into which a linking agent has been introduced. The assay may also include various buffers or reagents that may be used in the assay.

[0050] The following brief summary provides a basic understanding of some aspects of the claimed subject matter. This summary is not an exhaustive overview and is not intended to identify key / critical elements of the claimed subject matter or to delineate the scope of the claimed subject matter. Its purpose is to present some concepts in a simplified form to introduce the more detailed description presented below. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Embodiments of the present invention will now be described with reference to the accompanying drawings, in which:

[0052] Figure 1a The preparation of 2-(3-N-trifluoroacetamidopropyl)-1,3-propanediol (3) is shown;

[0053] Figure 1b The addition of a dimethoxytrityl (DMT) protecting group to (3) to prepare 1-O-DMT-2-(3-N-trifluoroacetamidopropyl)-1,3-propanediol (4) is shown;

[0054] Figure 1c Phosphorylation of (4) is shown to prepare the 2-(3-aminopropyl)-1,3-propanediol phosphoramidite linker reagent (5).

[0055] Figure 2a The preparation of 2,2-bis-(trifluoroacetamidopropyl)-1,3-propanediol (8) is shown;

[0056] Figure 2b The addition of a DMT protecting group to (8) to prepare (9) is shown;

[0057] Figure 2c Phosphorylation of (9) is shown to prepare a 2,2-bis(ε-aminopropyl)-1,3-propanediol phosphoramidite reagent (10) having two linker arm moieties.

[0058] Figure 3a The preparation of intermediate (12) of O-DMT and N-trifluoroacetyl protected 3-amino-1,2-propanediol is shown;

[0059] Figure 3b Phosphorylation of (12) to prepare the 3-amino-1,2-propanediol phosphoramidite linker reagent (13) is shown.

[0060] Figure 4aThe preparation of 1,2-isopropylidin-6-azido-1,2-hexanediol (16) is shown;

[0061] Figure 4b The reduction of (16) and protection of the amino group with Fmoc to give (18) are depicted;

[0062] Figure 4c The addition of the DMT protecting group is shown to give (19);

[0063] Figure 4d Phosphorylation of (19) to prepare the 6-amino-1,2-hexanediol phosphoramidite linker reagent (20) is shown.

[0064] Figure 5a Three reagents (21), (22), and (23) with aminoalkylcarboxyl-extended linker arms are depicted;

[0065] Figure 5b is a linker reagent (24), which is a further extended analog of compound (23).

[0066] Figure 6a shows the coupling of Fmoc-glycine with 3-amino-1,2-propanediol to give (25);

[0067] Figure 6b DMT protection and phosphorylation of (25) to yield linker reagent (21) is shown.

[0068] Figure 7a Depicted are the Fmoc protection of aminoalkylcarboxylic acids and activation with trimethylacetyl chloride to give (27) and (28);

[0069] Figure 7b Shows the Figure 7a The activated intermediates of (29) and (30) were reacted with 3-amino-1,2-propanediol.

[0070] Figure 8a Depicted is the reaction of the DMT-protected derivative of (30) with ammonium hydroxide to remove Fmoc;

[0071] Figure 8b Continued Figure 8a Depicted are (32) after Fmoc removal from (31) and further extension of the amino compound (32) via coupling with the pivalic anhydride-activated intermediate of (28) to afford the extended analog (33).

[0072] Figure 9a Shows Figure 7aintermediate (28), and its reaction with trimethylacetyl chloride to form activated intermediate (35), and its subsequent reaction with (S)-3-amino-1,2-propanediol to form enantiomeric intermediate (36).

[0073] Figure 9b Shows Figure 9a intermediate (36), and its reaction with 4,4'-dimethoxytrityl chloride to generate enantiomeric intermediate (37).

[0074] Figure 9c Shows Figure 9b The intermediate (37) is reacted with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite to obtain the diastereomeric reagent (38).

[0075] Figure 10 Diastereomeric reagents (39) containing (R)-3-amino-1,2-propanediol are shown.

[0076] Figure 11 Diastereomeric reagents (40-44) comprising (S)-3-amino-1,2-propanediol but having shorter and longer alkyl chain lengths than reagent (38) are shown, and diastereomeric reagents (44-47) comprising (R)-3-amino-1,2-propanediol but having shorter and longer alkyl chain lengths than reagent (39) are shown.

[0077] Figure 12 The diastereomeric reagent N-Fmoc-O 1 -DMT-O 2 -cyanoethoxydiisopropylaminophosphinyl-(S)-3-amino-1,2-propanediol (48) and N-Fmoc-O 1 -DMT-O 2 -Cyanoethoxydiisopropylaminophosphinyl-(R)-3-amino-1,2-propanediol (49).

[0078] Figure 13 The diastereomeric reagent N-Fmoc-O 1 -DMT-O 3 -cyanoethoxydiisopropylaminophosphinyl-(S)-2-(aminomethyl)-1,3-propanediol (50) and N-Fmoc-O 1 -DMT-O 3 -Cyanoethoxydiisopropylaminophosphinyl-(R)-2-(aminomethyl)-1,3-propanediol (51).

[0079] Figure 14 The diastereomeric reagent N-Fmoc-O 1 -DMT-O 3-cyanoethoxydiisopropylaminophosphinyl-(S)-2-(4-aminobutyl)-1,3-propanediol (52) and N-Fmoc-O 1 -DMT-O 3 -Cyanoethoxydiisopropylaminophosphinyl-(R)-2-(4-aminobutyl)-1,3-propanediol (53).

[0080] Figure 15 The diastereomeric reagent N-(fluorenylmethoxycarbonylamino)propionylamino-O 1 -DMT-O 3 -cyanoethoxydiisopropylphosphinyl-(S)-2-amino-1,3-propanediol (54) and N-(fluorenylmethoxycarbonylamino)propionylamino-O 1 -DMT-O 3 -Cyanoethoxydiisopropylphosphinyl-(R)-2-amino-1,3-propanediol (55).

[0081] Figure 16 A partial nucleic acid structure (56) containing a 5'-to-3'(R)-3-amino-1,2-propanediol moiety and a partial nucleic acid structure (57) containing a 5'-to-3'(R)-3-amino-1,2-propanediol moiety are shown. Each (S) isomer has an opposite configuration at the chiral carbon center. B1 and B2 replace any nucleotide base. Wavy lines indicate the positions of nucleotide / non-nucleotide polymer stretches.

[0082] Figure 17 Diastereomeric reagents (58) and (59) containing (S)-2'-amino-1,2-propanediol and (R)-2'-amino-1,2-propanediol intermediates, respectively, are shown.

[0083] Figure 18a and Figure 18b Diastereomeric reagents (60), (61), (62), and (63) comprising (S)-4-aminobutane-1,3-diol and (R)-4-aminobutane-1,3-diol intermediates are shown. DETAILED DESCRIPTION

[0084] definition

[0085] As used herein, unless otherwise indicated, abbreviations for any protecting groups, amino acids, and other compounds are according to their common usage, recognized abbreviations, or the IUPAC-IUB Commission on Biochemical Nomenclature (see Biochem. 11:942-944 (1972)).

[0086] Nucleotide polymer: A chain of nucleotides linked by phosphodiester bonds, or their analogs.

[0087] Oligonucleotides : Nucleotide polymers, usually about 10 to about 100 nucleotides in length, but can be greater than 100 nucleotides in length. They are usually considered to be synthesized from nucleotide monomers, but can also be obtained enzymatically.

[0088] polynucleotides : A nucleotide polymer, typically about 100 nucleotides or longer in length. However, the nucleotide polymer can also be shorter, ranging from about 15 to about 100 nucleotides in length (e.g., for microRNAs (miRNAs), small interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), or transfer RNAs (tRNAs). These are typically of biological origin or obtained enzymatically.

[0089] Nucleotide polymer probe, or probe : nucleotide polymer, its nucleotide sequence is complementary to the target nucleotide sequence included in the second nucleotide polymer (normally polynucleotide).Usually the probe selected is completely complementary to the corresponding base in the target sequence.Yet in some cases, one or more Nucleotide in the probe are not complementary to the corresponding base in the target sequence and can be enough, or even desirable.Usually, the probe is connected to at least one label.

[0090] Non-nucleotide monomer units : refers to monomeric units that do not significantly participate in polymer hybridization. For example, the monomeric units must not participate in any significant hydrogen bonding with nucleotides, and monomeric units having as components one of the five nucleotide bases or their analogs are excluded;

[0091] markers : an atom, group of atoms or compound that can be covalently or non-covalently attached to a molecule for direct or indirect detection and / or quantification of the molecule. Direct label detection may be due to their radioactive or stable isotopic properties, chemiluminescence, phosphorescence, fluorescence, optics, electron density or magnetism. Indirect label detection may be due to a complex of at least a second molecule and a label, wherein the at least second molecule is attached to a direct detection label (e.g., a detectable label attached to an avidin or antibody that complexes biotin or a hapten label, respectively, to the molecule).

[0092] Enantiomers : One of a pair of stereoisomers (I) is the mirror image of its counterpart (II). It is chiral and is usually indicated by the prefix (R)- or (S)-. This term can be used to refer to a specific chiral site in a molecule with multiple chiral centers.

[0093]

[0094] Essentially pure enantiomer : An enantiomeric composition or enantiomeric center that is almost entirely one enantiomer. Almost entirely, the ratio of one enantiomer to the other enantiomer is between about 90:10 and about 100:0.

[0095] Diastereomers : Stereoisomeric compounds that are not mirror images of one another and in which two or more stereocenters have different configurations at one or more equivalent stereocenters.

[0096] Epimer : Diastereomers that differ from each other at only one stereocenter.

[0097] Essentially racemic : A composition having approximately equal numbers of enantiomers or enantiomeric centers of a chiral molecule. The ratio of the two enantiomers is approximately between 45:55 and 55:45.

[0098] Acridinium esters : Any of a series of related molecules comprising an acridinium moiety, an ester moiety, and a leaving moiety. Schematic example (III) is shown below. A and B with numbers represent various substitution sites. (If B is hydrogen (H), then III is acridic acid.) These ionic molecules have counterions (not shown).

[0099]

[0100] Acridinium ester compounds : Any of a series of related molecules comprising an acridinium moiety, an unstable moiety, and a leaving moiety. Schematic example (IV) is shown below. A and B with numbers represent various substitution sites. These ionic molecules have counterions (not shown).

[0101]

[0102] Ligation reagents

[0103] Non-nucleotide reagents with non-nucleotide monomer units have been described. An example is a non-nucleotide reagent consisting of protected phosphoramidite 1,2-propylene glycol and 1,3-propylene glycol linkers, in which alkyl chains of various lengths terminate with protected amine functional groups. Compared to natural nucleic acids, reagents with 1,2-propylene glycol linkers have a limited internucleotidyl distance, while reagents with 1,3-propylene glycol linkers have the same number of atoms between phosphates as natural nucleotides. When inserted between two adjacent nucleotides, reagents with 1,2-propylene glycol linkers can be particularly advantageous, while when inserted for nucleotide substitution, reagents with 1,3-propylene glycol linkers can be particularly advantageous. These reagents can be introduced into oligonucleotides by standard oligonucleotide synthesis methods to form non-nucleotide monomer units at the 5'-end of an oligonucleotide, the 3'-end of an oligonucleotide, between two nucleotides, between a nucleotide and a non-nucleotide monomer unit, or between two non-nucleotide monomer units of the same or different structures. More than one disclosed non-nucleotide reagent can be introduced into an oligonucleotide to form a modified oligonucleotide having a plurality of non-nucleotide monomer units adjacent to each other or separated by one or more nucleotide or non-nucleotide monomer units. The amines of the non-nucleotide monomer units are then covalently bound to fluorescein via an isothiocyanate labeling reagent, or to biotin or an acridinium ester via a suitable N-hydroxysuccinimide labeling reagent.

[0104] Others have reported a similar protected phosphoramidite 1,2-propanediol linker with a shorter alkylamine functionality (N-Fmoc-O 1 -DMT-O 2 -cyanoethoxydiisopropylaminophosphinyl-3-amino-1,2-propanediol) and multiple non-nucleotide monomer units of this non-nucleotide reagent are introduced into the 5'-terminus of the oligonucleotide. In addition to the chiral carbon of the propylene glycol moiety, the diisopropylphosphoramidite phosphorus atom is chiral and is a mixture of phosphorus isomers; therefore, the non-nucleotide reagent is a diastereomer. The amines on these non-nucleotide monomer units are labeled with biotin. This reagent, and therefore the monomer units it introduces, are composed of a mixture of stereoisomers of the propylene glycol moiety, rather than a substantially pure product of one isomer or the other. The literature does not discuss the different properties that may arise when racemates or individual enantiomers are exposed to a chiral environment. For example, the enantiomeric carbon center of one diastereomer can direct the alkylamine portion of the linker monomer unit in one direction relative to the nucleic acid duplex, while the other enantiomer of the propylene glycol moiety can direct the group in an approximately orthogonal direction.

[0105] Also disclosed are related 1,2-propanediol non-nucleotide reagents for derivatizing and labeling the 3'-terminus of oligonucleotides during solid phase synthesis. Similarly disclosed are related 1,3-propanediol but non-nucleotide reagents with longer alkyl amines for derivatizing and labeling the 3'-terminus, internal position, or 5'-terminus of oligonucleotides during solid phase synthesis. In addition to the chiral carbon of the propylene glycol moiety, the diisopropylphosphoramidite phosphorus atom is chiral and is a mixture of phosphorus isomers; therefore, the non-nucleotide reagents are diastereomers. Each of these related reagents and their monomeric units also consists of a mixture of stereoisomers of the propylene glycol moiety rather than a pure product of one isomer or the other; the literature does not discuss the different properties that may arise when the racemate or individual enantiomers are exposed to a chiral environment. Unlike N-Fmoc-O 1 -DMT-O 2 As with the reagents for -cyanoethoxydiisopropylaminophosphinyl-3-amino-1,2-propanediol, the amines of the two carbon enantiomers of the 1,2-propanediol and 1,3-propanediol moieties will be oriented approximately orthogonally relative to the nucleic acid duplex.

[0106] Another example is a protected phosphoramidite serinol linker with an alkylamine functional group (N-Fmoc-β-α-O 1 -DMT-O 3 -cyanoethoxydiisopropylaminophosphinyl-serinol), and multiple non-nucleotide monomer units of the non-nucleotide reagent or related solid support reagent are introduced at any internal position or 5'-end or 3'-end of the oligonucleotide. In addition to the chiral carbon of the propylene glycol portion, the diisopropylphosphoramidite phosphorus atom is chiral and is a mixture of phosphorus isomers; therefore, the non-nucleotide, non-solid support reagent is a diastereomer. More than one disclosed non-nucleotide reagent can be introduced into an oligonucleotide to form a modified oligonucleotide having multiple non-nucleotide monomer units adjacent to each other or separated by one or more nucleotides. These reagents and therefore the monomer units introduced therein are composed of a mixture of stereoisomers, rather than a substantially pure product of one isomer or the other, but the prior art does not discuss the different properties that may arise when a racemate or a single enantiomer is exposed to a chiral environment. Like the above reagents, the amine of one carbon enantiomer of the propylene glycol portion of the serinol linker reagent will be oriented approximately orthogonally relative to the nucleic acid double-stranded complex.

[0107] Detectable markers

[0108] Any of a variety of detectable labels, including but not limited to isotopes, luminescence, phosphorescence, fluorescence, optical, electron density, or magnetic moieties, can be attached to a linker. One class of sensitive labels can be composed of acridinium esters, which are chemiluminescent molecules. Like other chemiluminescent molecules, acridinium esters can be used as probes for detecting and / or quantifying other molecules under appropriate conditions. Acridinium esters consist of an acridinium moiety, an unstable ester moiety, a leaving group moiety, and a counterion moiety. Phenol and substituted phenols are exemplary leaving groups. The ring positions of the acridinium and leaving groups can be substituted differently. Closely related molecules, acridinium ester compounds, include acridinium sulfonylcarboxamides, acridinium thioesters, and acridinium amides, which differ from acridinium esters in their unstable moieties. One of the substituents on the acridinium ring or the leaving group ring can be a moiety that allows for specific reaction with a moiety on another molecule to form a conjugate; in this case, the acridinium compound is an acridinium ester label or an acridinium ester compound label.

[0109] When oligonucleotide hybridization is to the nucleic acid of complete matching and hybridization is to the nucleic acid with mispairing near the joint site, the acridinium ester connected with the internucleotide linker introduced in these oligonucleotides demonstrates different sensitivity to modification.For example, when oligonucleotide hybridization is to the nucleic acid of complete matching and hybridization is to the nucleic acid with mispairing near the joint site, the acridinium ester connected with the internucleotide linker introduced in these oligonucleotides demonstrates different sensitivity to hydrolysis.Sensitivity difference refers to the difference in the hydrolysis rate of the phenyl ester part of acridinium ester based on nucleic acid hybridization.For example, the hydrolysis degree of the acridinium ester connected with the oligonucleotide of complete matching nucleic acid hybridization is lower than the acridinium ester connected with the oligonucleotide of nucleic acid hybridization containing one or more mispairing.Before starting chemiluminescent detection, pH value is increased to alkaline range (but lower than the pH value causing nucleic acid dehybridization (dehybridization)) and solution temperature keeps certain time both and affects hydrolysis difference.After this time, the amount of remaining unhydrolyzed acridinium ester is determined by chemical reaction with oxidant (such as hydrogen peroxide) and alkaline solution (such as sodium hydroxide), then records luminescent output. These properties make acridinium ester-labeled oligonucleotides excellent probes for differential detection of closely related sequences found in certain bacterial species or variants.

[0110] Similarly, for example, when an oligonucleotide is hybridized to a nucleic acid that is fully matched and hybridized to a nucleic acid with a mispairing near a joint site, the acridinium ester connected to the internucleotide linker introducing these oligonucleotides shows different sensitivities to adduct formation at the C9 position of the acridinium ring. The different sensitivities to adduct formation refer to differences in adduct formation at the C9 position of an acridinium ester based on nucleic acid hybridization. For example, compared to an acridinium ester connected to an oligonucleotide hybridizing to a nucleic acid containing one or more mispairings, the acridinium ester connected to an oligonucleotide hybridizing to a fully matched nucleic acid is less likely to form an adduct at the C9 position. Before starting chemiluminescent detection, different adduct formations are achieved by increasing the concentration of the chemical forming the adduct in the solution for a certain period of time. The chemical forming the adduct includes sulfite, mercaptoethanesulfonic acid, propylthiol, and thiophosphate.

[0111] Isomers

[0112] When four chemical substituents are bonded to the same atomic center, their three-dimensional configuration is a tetrahedron. If all substituents are identical, for example four hydrogens are bonded to carbon to form methane, then the tetrahedron is symmetrical, wherein all substituents-atomic center-substituent bonds form an angle of about 109.5 degrees. If at least one of the substituents is different from other substituents, then the tetrahedral configuration becomes distorted and the bond forms an angle range greater than and less than about 109.5 degrees. No matter how perfect the symmetry is, any two substituents bonded to the atoms in the tetrahedral configuration are substantially away from each other. In the case where all four substituents are bonded to the same atomic center but are different from each other, these substituents are also substantially away from each other. However, in this case, the substituents can be combined with the atomic center so that their orientations in space are different, forming stereoisomers. In addition, these stereoisomers are mirror images of each other, and their central atoms are designated as chiral centers. The two different chiral isomers are called enantiomers and are designated Rectus ("R") or Sinister ("S") according to the Cahn-Ingold-Prelog priority rules for assigning substituent orientations in space. Molecules with at least two chiral centers that are not mirror images of each chiral center are diastereomers.

[0113] The propylene glycol portion of the non-nucleotide reagent and its monomer units contain at least one chiral atom. In addition to the chiral carbon of the propylene glycol portion, the diisopropylphosphoramidite phosphorus atom is chiral and is a mixture of phosphorus isomers; therefore, the non-nucleotide reagent is a diastereomer. As known to those skilled in the art, enantiomeric pairs have identical chemical and physical properties in an achiral environment, but have different properties in a chiral environment, including biological systems. For example, a single S p or R pPhosphorothioate oligonucleotides have the same reactivity for labeling with iodoacetamide but have opposite stabilities to hydrolysis by the two nucleases.

[0114] In one embodiment, the linker phosphoramidite is described by Formula V as shown below.

[0115]

[0116] In this formula, Z is a reactive phosphorus-containing group, such as an O-cyanoethoxydiisopropylphosphinyl moiety, OH, or a cleavable ester attached to a DNA synthesis support. The reactive phosphorus-containing group is capable of coupling to the OH group of the first nucleotide or being activated to couple to the OH group of the first nucleotide. In the case where the reactive phosphorus-containing group is an O-cyanoethoxydiisopropylphosphinyl moiety, the O-cyanoethoxydiisopropylphosphinyl moiety is represented by R P and R S The product is a mixture of isomers (R and S enantiomers at the phosphorus center) if not purified. P and R S The isomers will react with approximately equal efficiency with the OH group of the first nucleotide to form a phosphite triester intermediate in the growing oligonucleotide chain, which becomes achiral upon oxidation and elimination of the cyanoethyl protecting group. D is a protecting group that can be removed to allow coupling of a non-nucleotide linking reagent to the second nucleotide; the protecting group can be selected from a trityl, dimethoxytrityl, or other moiety. L 1 It has the formula (CH2) k —NH—(CO—(CH2) q NH) r —The first joint arm of M. L 2 is hydrogen (H), a non-connected alkyl chain, a non-connected mixed alkyl chain or a compound having the formula (CH2) k —NH—(CO—(CH2) q NH) r —The second joint arm of M. When L 1 and L 2When they are different from each other, the carbon atom (X element) to which they are attached is chiral. The chiral center of the molecule can have an R or S stereochemical configuration, and the composition can be composed of different non-racemic ratios of R and S isomers. The number of CH2 (methylene) parts in Formula V can vary between 0 and 3, but must have at least one CH2 part (i.e., i+j>1). Each M is independently hydrogen, fluorenylmethoxycarbonyl, trifluoroacetyl or a label that is stable to DNA synthesis and deprotection conditions. The fluorenylmethoxycarbonyl and trifluoroacetyl parts are groups that protect the amine from undesirable reactions with chemicals before they are removed and reacted as expected. Each k is independently an integer between 0 and 4 CH2 parts, each q is independently an integer between 1 and 11 CH2 parts, and each r is independently 0 to 2 (CO—(CH2) q NH) groups, as long as each k+1+(2+q)r in the first linker arm and the second linker arm is independently an integer between 1 and 25.

[0117] In another embodiment, the linker phosphoramidite is described by Formula VI as shown below.

[0118] DX 1 -R 2 (X 3 -M) n -Z

[0119] In this formula, Z is a reactive phosphorus-containing group, such as an O-cyanoethoxydiisopropylphosphinyl moiety, OH, or a cleavable ester attached to a DNA synthesis support. The reactive phosphorus-containing group is capable of coupling to the OH group of the first nucleotide or being activated to couple to the OH group of the first nucleotide. In the case where the reactive phosphorus-containing group is an O-cyanoethoxydiisopropylphosphinyl moiety, the O-cyanoethoxydiisopropylphosphinyl moiety is represented by R P and R S A mixture of isomers (R and S enantiomers at the phosphorus center) is expected to be a mixture of isomers (R and S enantiomers at the phosphorus center) if not purified. P and R S The isomers will react with approximately equal efficiency with the OH group of the first nucleotide to form a phosphite triester intermediate in the growing oligonucleotide chain, which becomes achiral upon oxidation and elimination of the cyanoethyl protecting group. D is a protecting group that can be removed to allow coupling of a non-nucleotide linking reagent to the second nucleotide; the protecting group can be selected from a trityl, dimethoxytrityl, or other moiety. X 1 It can be O, S, NH or -N=N-. 2 is a chain of atoms. X 3M is a joint arm. M is independently hydrogen, fluorenylmethoxycarbonyl, trifluoroacetyl or a label stable to DNA synthesis and deprotection conditions. Fluorenylmethoxycarbonyl and trifluoroacetyl moieties are groups that protect amines before they are removed and the expected reaction does not occur with chemicals. Without wishing to be bound by any theory, it is believed that the enantiomeric carbon center of a diastereomeric non-nucleotide reagent introduced into nucleic acid polymers can guide the alkylamine moiety of the joint monomer unit to a direction along the nucleic acid double-stranded complex, and another carbon enantiomer can guide the alkylamine group to an approximately orthogonal direction, including away from complex. Alternatively, the enantiomeric carbon center of a diastereomeric non-nucleotide reagent introduced into nucleic acid polymers can guide the alkylamine moiety of the joint monomer unit toward the minor groove (minor groove) of the nucleic acid double-stranded complex, and another carbon enantiomer can guide the alkylamine group toward the major groove (major groove). Further alternatively, the enantiomeric carbon center of one diastereomeric non-nucleotide agent introduced into the nucleic acid polymer can direct the alkylamine moiety of the linker monomer unit in one direction along the nucleic acid duplex, while the other carbon enantiomer can direct the group toward the major groove or the minor groove or out of either groove.

[0120] Linker phosphoramidites of formula V (supra) where i+j=odd number (e.g., 1, 3, 5, 7, etc.), and L 1 is the first joint arm, L 2 is hydrogen or other low priority moiety - when introduced into a nucleotide / non-nucleotide polymer, the asymmetry will be maintained regardless of which carbon is the X element. In addition, the nature of the asymmetry changes depending on whether i>j or j>i. For example, but not intended to be limiting, when i+j=1, it is possible that two different nucleotide / non-nucleotide polymers (partially illustrated by structures (56) and (57)) have the R configuration at the X element ( Figure 16). In one embodiment, a reagent (39) having an R configuration at the X element is allowed to react with the 5'-hydroxyl group of a growing polymer attached to a solid support via its 3'-hydroxyl group; the polymer proceeds to attach additional linking reagents or nucleoside phosphoramidites to produce structure (56). In another embodiment, a reagent (39) is allowed to react with the 3'-hydroxyl group of a growing polymer attached to a solid support via its 5'-hydroxyl group; the polymer proceeds to attach additional linking reagents or nucleoside phosphoramidites to produce structure (57). In another embodiment, a reagent (59) having an R configuration at the X element is allowed to react with the 5'-hydroxyl group of a growing polymer attached to a solid support via its 3'-hydroxyl group; the polymer proceeds to attach additional linking reagents or nucleoside phosphoramidites to produce structure (57). In another embodiment, a reagent (59) having an R configuration at the X element is allowed to react with the 3'-hydroxyl group of a growing polymer attached to a solid support via its 5'-hydroxyl group; the polymer proceeds to attach additional linking reagents or nucleoside phosphoramidites to produce structure (56). In another embodiment, two different nucleotide / non-nucleotide polymers are possible, having an S configuration at the X element, initiated by the relevant reagents. Similar to the comparison of linker enantiomers (supra), introduction of reagents (39) or (59) to form structures (56) and (57)—even though both (56) and (57) are in the R configuration at element X—results in the alkylamine groups (and the label, if attached) pointing in substantially different directions. This is also true for the structures formed by introduction of reagents (38) and (58) into nucleotide / non-nucleotide polymers.

[0121] Linker phosphoramidites of formula V (supra) wherein i=j and L 1 is the first joint arm, L 2is hydrogen or other lower priority moieties - will maintain its symmetry when introduced into the nucleotide / non-nucleotide polymer, that is, will have the same number of CH2 moieties between the X element and the phosphate attached to the 5'-hydroxyl group or the 3'-hydroxyl group of the nucleotide / non-nucleotide polymer. However, in these cases, incorporating a linking agent of a given chirality to the 5'-hydroxyl group of a growing polymer attached to a solid support via its 3'-hydroxyl group, or to the 3'-hydroxyl group of a growing polymer attached to a solid support via its 5'-hydroxyl group, may or may not result in a nucleotide / non-nucleotide polymer having a different chirality at the X element. Due to the symmetrical connectivity of the linker moiety once incorporated into a nucleotide / non-nucleotide polymer, the chiral configuration at the X element depends on a number of priority rule possibilities, including the type of nucleobases (e.g., low priority nucleobases are thymine, uracil, cytosine, guanine, and adenine) that are connected to the carbohydrate moiety, the type of the carbohydrate moiety (e.g., low priority carbohydrates are 2-fluororibose, 2-methoxyribose, ribose, and 2-deoxyribose) and the number of nucleotides (or non-nucleotide linkers) on the phosphate of the 5'-hydroxyl or 3'-hydroxyl of the nucleotide / non-nucleotide polymer. If the linker moiety is at the terminal position of the nucleotide / non-nucleotide polymer, these rules are simplified to the phosphate that is connected to the nucleotide polymer having a higher priority than the terminal hydroxyl of the non-nucleotide linker. If there are two adjacent linker moieties, these rules are simplified to the ribose of the connected nucleotide polymer having a higher priority than the non-nucleotide linker. Regardless of the absolute configuration of the X element, which can be determined on a case-by-case basis, the alkylamine groups (and labels, if connected) of the different possible configurations of the nucleotide / non-nucleotide polymer point to substantially different directions.

[0122] Linker phosphoramidites of formula V (supra) where i+j=even number (e.g., 2, 4, 6, 8, etc.) and i≠j and L 1 is the first joint arm, L 2 is hydrogen or other low priority moieties (e.g., methyl or alkyl moieties) - when introduced into a nucleotide / non-nucleotide polymer, the asymmetry will be maintained regardless of which carbon is the X element. That is, there will be a different number of CH2 moieties between the X element and the phosphate attached to the 5'-hydroxyl or 3'-hydroxyl of the nucleotide / non-nucleotide polymer. As for when i+j=odd, the nature of the asymmetry changes, depending on i>j or j>i. For example, but not intended to be limiting, when i=0 and j=2 or i=2 and j=0, two different nucleotide / non-nucleotide polymers with S configuration at the X element are possible. In one embodiment, it is allowed Figure 18aThe reagent (60) shown in , which has an S configuration at the X element, is allowed to react with the 5'-hydroxyl group of a growing polymer attached to a solid support via its 3'-hydroxyl group; the polymer continues to attach additional linking reagents or nucleoside phosphoramidites to produce a structure in which the non-nucleotide linker maintains an S configuration at the X element. In another embodiment, the reagent (60) is allowed to react with the 3'-hydroxyl group of a growing polymer attached to a solid support via its 5'-hydroxyl group; the polymer continues to attach additional linking reagents or nucleoside phosphoramidites to produce a structure in which the non-nucleotide linker maintains an S configuration at the X element. In yet another embodiment, the reagent (60) is allowed to react with the 3'-hydroxyl group of a growing polymer attached to a solid support via its 5'-hydroxyl group; the polymer continues to attach additional linking reagents or nucleoside phosphoramidites to produce a structure in which the non-nucleotide linker maintains an S configuration at the X element. Figure 18b The reagent (62) shown in FIG—having an S configuration at the X element—reacts with the 5′-hydroxyl group of a growing polymer attached to a solid support via its 3′-hydroxyl group; the polymer is subsequently attached to additional linking reagents or nucleoside phosphoramidites to produce a structure in which the non-nucleotide linker maintains an S configuration at the X element. In another embodiment, the reagent (62) is allowed to react with the 3′-hydroxyl group of a growing polymer attached to a solid support via its 5′-hydroxyl group; the polymer is subsequently attached to additional linking reagents or nucleoside phosphoramidites to produce a structure in which the non-nucleotide linker maintains an S configuration at the X element. In yet another embodiment, two different nucleotide / non-nucleotide polymers are possible, having an R configuration at the X element, initiated by the relevant reagents (61, 63). Similar to the comparison of linker enantiomers (supra), the introduction of reagents (60) or (62) to form a nucleotide / non-nucleotide linker results in the alkylamine group (and label, if attached) pointing in substantially different directions. The same comparison holds true for the structures formed by introducing reagents (61) and (63) into nucleotide / non-nucleotide polymers.

[0123] What is unknown is that the non-nucleotide reagent that will be made up of the racemic mixture of propylene glycol chiral site or the single isomer of propylene glycol chiral site is introduced into nucleic acid polymer will have what kind of impact on the performance of oligonucleotide probe with markers such as acridinium ester.It is believed that using pure isomer will provide higher sensitivity and specificity in the mensuration based on nucleic acid hybridization.Therefore, less material can be used while still keeping the same or better mensuration performance.In addition, using pure isomer can improve the detection of complete matching sequence with respect to mispairing (especially single base mispairing, insertion, deletion, inversion and transposition).Because the amine part of joint and thereby the detectable label of a kind of carbon enantiomer will be oriented with respect to duplex with the mode that the marker on another enantiomer better detects, the situation may be so.

[0124] In one embodiment, the linker is a purified isomer based on the R configuration of the X element in Formula V (supra). In another embodiment, the linker is a purified isomer based on the S configuration of the X element. In another embodiment, the linker is synthesized as an isomer based on the R configuration of the X element. In another embodiment, the linker is synthesized as an isomer based on the S configuration of the X element.

[0125] In some applications based on a single isomer of the X element in Formula V (supra), combinations of isomers can have advantageous properties. For example, but not intended to be limiting, a skilled person can aim to create a probe or probe set that selectively or specifically senses adenine and guanine bases on a target nucleic acid strand. In this case, when the R isomer is present at 70%, the R isomer based on the X element can advantageously interact with the adenine base on the target nucleic acid strand, and when the S isomer is present at 30%, the S isomer based on the X element can advantageously interact with the guanine base on the target nucleic acid strand. In one embodiment, the linker is a mixture of the R and S isomers based on the X element. In another embodiment, the mixture is 90% R isomer and 10% S isomer. In another embodiment, the mixture is 80% R isomer and 20% S isomer. In another embodiment, the mixture is 70% R isomer and 30% S isomer. In another embodiment, the mixture is 60% R isomer and 40% S isomer. In another embodiment, the mixture is 40% R isomer and 60% S isomer. In another embodiment, the mixture is 30% R isomer and 70% S isomer. In another embodiment, the mixture is 20% R isomer and 80% S isomer. In another embodiment, the mixture is 10% R isomer and 90% S isomer. Relatedly, in another embodiment, the mixture is other non-racemic fractions of R and S isomers.

[0126] Example

[0127] Example 1: Synthesis of 2-(3-aminopropyl)-1,3-propanediol linker reagent

[0128] The synthetic scheme for this synthesis is given in Figure 1 and summarized below.

[0129] (a) Synthesis of diethyl 2-(3-nitrilopropyl)malonate (1): The procedure used was adapted from the method of R. Adams and RM Kamm in Organic Syntheses, Coll. Vol. 1, 1941, p. 250, ed. Gilman & Blatt.

[0130] MaterialDiethyl malonate, 3-bromopropionitrile, and sodium ethoxide (21% ethanol solution) were purchased from Aldrich Chemical Company (Milwaukee, WI, USA). Anhydrous ethanol (200 proof) was from US Industrial Chemicals.

[0131] step : sodium ethoxide (0.1 mole) is diluted to final volume with absolute ethanol and is 100mL.Under agitation, drip the solution of diethyl malonate (0.1 mole) in 50mL absolute ethanol, use calcium chloride drying tube to protect reaction unit from moisture.At room temperature, continue to stir 1 hour.Then, under agitation, drip the solution of 3-bromopropionitrile in 50mL absolute ethanol, and at room temperature stir mixture and spend the night.Gained solution is filtered to remove precipitated sodium bromide, concentrate and be extracted in ether (50mL).Then this solution water (50mL) is extracted, use anhydrous magnesium sulfate drying, and be condensed into oily matter. Thin layer chromatography on silica gel plates with chloroform as the mobile phase produced three spots after visualization with iodine vapor: Rf 0.58, 0.51, and 0.38, which were subsequently identified as diethyl malonate, diethyl 2-(3-nitrilopropyl)malonate (1), and 2,2-bis-(nitrilopropyl)malonate, respectively. After several days in the refrigerator, crystals separated from the crude oil, were filtered off, dissolved in toluene (10 mL), and reprecipitated by adding hexane to give 3.28 g of a white solid (12% yield); thin layer chromatography (as described above), Rf 0.38. The structure of this compound was determined by 1 H NMR (CDCl3) confirmed that it was 2,2-bis-(nitrilopropyl)malonate (6): δ 1.30 (t, 6H), 2.26 (t, 4H), 2.47 (t, 4H), 4.27 (q, 4H). The filtered oil was vacuum distilled to obtain the title compound (1) (boiling point (bp) 99-103°C, 0.3 mmHg) with a yield of 20%; 1 H NMR analysis: δ 1.24 (t, 6H), 2.19 (q, 2H), 2.47 (t, 2H), 3.46 (t, 1H), 4.18 (q, 4H).

[0132] (b) Synthesis of 2-(3-aminopropyl)-1,3-propanediol (2).

[0133] Material Lithium aluminum hydride (1.0 M in diethyl ether) was purchased from Aldrich. Other materials are described in the previous examples, see above.

[0134] step : 2-(3-nitrilopropyl)-diethyl malonate (1) (3.21 g, 15.1 mmol) was added dropwise to a stirred solution of lithium aluminum hydride (0.1 mol in 100 mL of ether) in anhydrous ether (50 mL) under nitrogen. The resulting mixture was refluxed for 2 hours and then stirred at room temperature overnight. Next, 2.5 mM aqueous sodium hydroxide solution (100 mL) was slowly added to quench the unreacted hydride. The mixture was stirred for 2 hours, the ether layer was decanted and discarded (the product remained in the water layer). The white gel-like solid was removed from the water layer by centrifugation, washed with water, the aqueous supernatant and washings were combined, and concentrated into a syrup by rotary evaporation under reduced pressure. Thin layer chromatography (Analtech reverse phase plates, aqueous mobile phase, visualized with ninhydrin reagent) gave a major spot identified as the title compound (2), Rf 0.48, and a small spot attributed to the condensation by-product, Rf 0.29. The title compound (2) was purified by cation exchange chromatography (Dowex 50X8, 0.5M HCl mobile phase) with an overall yield of 50%. 1 H NMR analysis (D2O): δ 1.36 (apparent quartet, 2H)), 1.68 (m, 3H), 2.97 (t, 2H), 3.57 (d, 4H).

[0135] (c) Synthesis of 2-(3-N-trifluoroacetamidopropyl)-1,3-propanediol (3): The procedure was adapted from the method of RF Goldfinger in Methods in Enzymology, vol. 12, 1967, p. 317, edited by HW Hirs.

[0136] Material : S-ethyl trifluorothioacetate was from Aldrich. Other materials are described in the previous examples, supra.

[0137] step: 2-(3-Aminopropyl)-1,3-propanediol (2) (3 mmol) was dissolved in water (25 mL). The pH of the solution was reduced to 9.5 by the dropwise addition of 6N HCl. The following reaction was carried out in a fume hood: S-trifluorothioacetic acid ethyl ester (2 mL) was added dropwise to the vigorously stirred solution; the pH was maintained between 9.5 and 10.0 by the dropwise addition of 6N KOH. After 30 minutes, another milliliter of S-trifluorothioacetic acid ethyl ester was added, and the pH was maintained as described above. The mixture was stirred for an additional 45 minutes. Next, the pH was adjusted to 7 using 6N KOH, and the mixture was concentrated to dryness by rotary evaporation under reduced pressure. The residue was stirred with acetone (20 mL) and filtered to remove the potassium acetate precipitate. The filtrate was concentrated to a syrup, redissolved in acetone (2 mL), and then applied to a flash chromatography column containing 40 g of silica gel (40 μm average particle size, from JT Baker Chemical Co., Phillipsburg, NJ, USA). The column was eluted with a 50:50 solution (v / v) of dichloromethane / acetone (500 mL) to yield 25 mL fractions. The fractions were analyzed for product content by spotting a 2 μL aliquot onto a silica gel plate, spraying it with a 10% aqueous piperidine solution, allowing it to stand for 15 minutes, drying it with a heat gun, and then treating it with a ninhydrin reagent. Omitting the piperidine spray treatment prevented the colorimetric reaction with ninhydrin, confirming trifluoroacetylation of the primary amine. Using this procedure, the product was found between fractions 13 and 18; these fractions were combined and concentrated by rotary evaporation to yield a colorless oil with an Rf of 0.4 (silica gel thin layer chromatography using the same solvent system and visualization method as above).

[0138] (d) Synthesis of 1-O-DMT-2-(3-N-trifluoroacetylaminopropyl)-1,3-propanediol (4).

[0139] Material : Dimethoxytrityl chloride was purchased from Aldrich. Other materials are described in the previous examples, see above. Dichloromethane was refluxed, distilled over calcium hydride and stored at 4 angstroms. Pyridine was distilled over potassium hydroxide pellets and p-toluenesulfonate and stored under dry nitrogen.

[0140] step: 2-(3-N-trifluoroacetamidopropyl)-1,3-propanediol (3) (362 mg, 1.58 mmol) was evaporated to dryness several times with dry pyridine under reduced pressure and then further dried under full vacuum for several hours. The residue was then dissolved in 10 mL of anhydrous pyridine under dry nitrogen. Dimethoxytrityl chloride (401 mg, 1.18 mmol) in anhydrous dichloromethane (1.5 mL) was added with stirring, and the resulting solution was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure and the residue was dissolved in chloroform (50 mL). The solution was extracted three times with 5% aqueous sodium bicarbonate solution and then dried over anhydrous magnesium sulfate. The resulting solution was concentrated to an oil, redissolved in 2 mL of chloroform, and fractionated by flash chromatography as described above, except that chloroform / ethyl acetate / pyridine (95:5:0.2 v / v / v) was used as the mobile phase. The fractions were analyzed by thin layer chromatography on silica gel plates using the same solvent system. Spots with Rf values ​​of 0.27, 0.87, and 0.93 observed with HCl fume were identified as the 1-(dimethoxytrityl) product (4), dimethoxytrityl, and 1,3-bis-(dimethoxytrityl) byproduct, respectively. The 1,3-bis-(dimethoxytrityl) byproduct can be hydrolyzed to the title compound (4) by shaking with 4% dichloroacetic acid in a saturated aqueous dichloromethane solution. The product (4) was isolated by evaporating the solvent from the appropriate fractions and drying under full vacuum to give a foam (370 mg, 44%).

[0141] (e) Synthesis of 1-O-DMT-2-(3-N-trifluoroacetamidopropyl)-3-O-(methyl-N,N-diisopropylphosphoramido)-1,3-propanediol (5).

[0142] Material N,N-diisopropylethylamine and N,N-diisopropylmethylphosphoramide chloride were purchased from Aldrich. Other materials are described in the previous examples, see above. Dimethylformamide was refluxed, distilled over calcium hydride, and stored at on molecular sieves.

[0143] step:1-O-DMT-2-(3-trifluoroacetamidopropyl)-1,3-propanediol (4,300 mg, 0.56 mmol) was evaporated to dryness several times with anhydrous pyridine and dissolved in 10 mL of anhydrous dimethylformamide. The following reaction was carried out under dry nitrogen: N,N-diisopropylethylamine (245 μL, 1.3 mmol) was added under stirring, followed by N,N-diisopropylmethylphosphoramide chloride (140 μL, 0.7 mmol). The reaction mixture was stirred for 2 hours. Then, the mixture was concentrated under reduced pressure and dissolved in dichloromethane (50 mL). The solution was extracted 3 times with 5% aqueous sodium bicarbonate solution, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure to an oil. The starting material (4) was converted into the corresponding phosphoramidite (5) by 31 P NMR (CDCl3, trimethyl phosphate, external standard): δ (ppm) 147.9 confirmed. Purity estimated to be greater than 70%.

[0144] Example 2: Synthesis of 2,2-bis-(3-aminopropyl)-1,3-propanediol linker reagent

[0145] This example consists of monomers for incorporation into the phosphodiester backbone of a synthetic oligonucleotide with two aminopropyl linkers for attachment of multiple tags. The synthesis principle is given in FIG2 .

[0146] Material : Unless otherwise specified, the materials are the same as those shown in Example 1.

[0147] (a) Synthesis of 2,2-bis-(3-aminopropyl)-1,3-propanediol (7): The following steps are a brief description of a modification of the method described in Example 1(b).

[0148] step Diethyl 2,2-bis-(nitrilopropyl)malonate (6) (2.00 g, 7.51 mmol), the synthesis of which is described in Example 1(a), was dissolved in anhydrous diethyl ether (80 mL). The resulting solution was added dropwise to a stirred solution of lithium aluminum hydride (0.1 mol) in diethyl ether (100 mL). After 15 minutes, the mixture was heated at reflux for 2 hours and then stirred at room temperature overnight. Workup and recovery of the crude product were carried out as described above in Example 1(b). Thin layer chromatography, also as described in Example 1(b), gave a major spot (Rf ~ 0.2).

[0149] (b) Synthesis of 2,2-bis-(3-trifluoroacetamidopropyl)-1,3-propanediol (8).

[0150] step: 2,2-Bis-(3-aminopropyl)-1,3-propanediol (7) (3.7 mmol) was dissolved in water (25 mL) and the pH was adjusted to about 10 with 6N HCl. 1 mL of ethyl S-trifluorothioacetate was added with vigorous stirring; the pH was maintained between 9.5 and 10.0 by dropwise addition of 6N KOH. Similarly, 1.0 mL of additional ethyl S-trifluorothioacetate was added twice at 30 minute intervals. The mixture was concentrated to an oil by rotary evaporation under reduced pressure and then dissolved in acetone (30 mL). The precipitated potassium acetate was removed by filtration. The product (8) was purified by silica gel flash chromatography using a dichloromethane / acetone (50:50 v / v) mobile phase as described in Example 1(c). Using the same solvent system, the material purified by silica gel thin layer chromatography gave a single spot (Rf 0.7); visualization was performed by treatment with piperidine followed by ninhydrin as in Example 1(c). The yield was 510 mg (1.48 mmol).

[0151] (c) Synthesis of 1-O-DMT-2,2-bis-(trifluoroacetamidopropyl)-1,3-propanediol (9).

[0152] step : 2,2-bis-(3-trifluoroacetamidopropyl)-1,3-propanediol (8) (510 mg, 1.48 mmol) was dried by evaporating anhydrous pyridine several times by rotary evaporation under reduced pressure, and then dissolved in 5 mL of anhydrous pyridine under nitrogen. A solution of dimethoxytrityl chloride (376 mg, 1.11 mmol) in anhydrous dichloromethane (1.5 mL) was added under stirring under nitrogen, and the mixture was stirred for 1 hour. The mixture was concentrated under reduced pressure and dissolved in chloroform (50 mL). The solution was extracted 3 times with saturated sodium bicarbonate aqueous solution and once with saturated sodium chloride aqueous solution. The solution was then dried over anhydrous magnesium sulfate and concentrated to an oil by rotary evaporation under reduced pressure. The oil was then dissolved in 2 mL of chloroform and separated by flash chromatography on silica gel using chloroform / ethyl acetate / pyridine (80:20:0.2 v / v / v) as described above. The product (9) was identified by thin layer chromatography on silica gel using the same solvent system and visualized with HCl fumes (Rf 0.3); concentrated under reduced pressure and dried under full vacuum to give a light yellow foam (517 mg, 52%).

[0153] (d) Synthesis of 1-O-DMT-2,2-bis-(3-trifluoroacetamidopropyl)-3-O-(methyl-N,N-diisopropylphosphoramido)-1,3-propanediol (10).

[0154] Steps: 1-O-DMT-2,2-bis-(trifluoroacetamidopropyl)-1,3-propanediol (9) (136 mg, 0.2 mmol) was dried by co-evaporation with dry pyridine (3 mL) three times. The residue was dissolved in anhydrous dichloromethane (1.5 mL) under argon and N,N-diisopropylethylamine (175 μL, 1.0 mmol) was added under stirring. Then, N,N-diisopropylmethylphosphoramide chloride (80 μL, 0.4 mmol) was added and the reaction was stirred for 1 hour. The resulting mixture was diluted with ethyl acetate / triethylamine (98:2, 50 mL) and extracted twice with saturated aqueous sodium bicarbonate solution (25 mL). The organic layer was dried over anhydrous magnesium sulfate and concentrated to an oil (240 mg). The conversion to phosphoramidite (10) was carried out by 31 P NMR (CDCl3, trimethoxyphosphate, external standard): δ (ppm) 145.5. Purity estimated to be greater than 60%.

[0155] Example 3: Synthesis of 3-amino-1,2-propanediol-based linker reagent

[0156] The synthesis is illustrated in Figure 3 and described below.

[0157] (a) Synthesis of 3-(trifluoroacetylamino)-1,2-propanediol (11).

[0158] Material : 3-amino-1,2-propanediol and ethyl S-trifluorothioacetate were purchased from Aldrich. Other materials are described in the previous examples, see above.

[0159] step : Ethyl S-trifluorothioacetate (5.13 mL, 45 mmol) was added to a rapidly stirred mixture of 3-amino-1,2-propanediol (2.32 mL, 30 mmol) and ethyl acetate (5.0 mL). After a few minutes, the mixture became homogeneous. After 1 hour, the reaction solution was shaken with petroleum ether (100 mL) to obtain an oil, which was separated and concentrated by rotary evaporation under reduced pressure. The substance was analyzed by thin layer chromatography on silica gel plates using ethyl acetate / dichloromethane (2:1) as the mobile phase. The plate was first visualized with ninhydrin reagent, which showed traces of unreacted amine starting material at the beginning. Next, the plate was visualized by the following steps: spraying with 10% piperidine aqueous solution, drying with a heat gun after 15 minutes, and then treating with ninhydrin reagent. In the case of treatment with ninhydrin reagent, a major spot (Rf 0.28) was clearly observed, which was estimated to account for more than 95% of the material. Purification of material associated with the major spot (11) was achieved by preparative thin layer chromatography.

[0160] (b) Synthesis of 3-(trifluoroacetylamino)-1-O-DMT-1,2-propanediol (12).

[0161] Material : Materials as described in Example 1(d), supra.

[0162] step :3-(Trifluoroacetylamino)-1,2-propanediol (11) (1.87 g, 10 mmol) was dried by three co-evaporations with anhydrous pyridine (10 mL) under reduced pressure. Then, the substance was dissolved in anhydrous pyridine (10 mL). Then, a solution of dimethoxytrityl chloride (3.73 g, 11 mmol) in anhydrous pyridine (10 mL) was added dropwise under nitrogen with stirring. After about 1 hour, methanol (0.2 mL) was added. The resulting solution was diluted with ethyl acetate (80 mL), extracted twice with saturated sodium bicarbonate aqueous solution (30 mL), and extracted twice with water (20 mL). The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain 6 g of crude oil. As described above, 1.1 g (one and one-tenth g) of crude material was separated by silica gel flash chromatography using dichloromethane / ethyl acetate / pyridine (10:1:0.01 v / v / v). The fractions were analyzed by thin layer chromatography on silica gel plates using the same solvent system. Visualization of the spots with HCl smoke revealed two minor components with Rf values ​​of 0.94 and 0.87 and a major component with Rf 0.53, which were identified as 1,2-bis-(dimethoxytrityl) by-product, dimethoxytriacontol, and the predicted product (12), respectively. The fractions with Rf 0.53 obtained by thin layer chromatography as described above were combined and concentrated to dryness by rotary evaporation under reduced pressure to give 0.72 g of (12), the structure of which was determined by 1 H NMR confirmed that the overall yield of (12) was 80%, based on the yield from the flash column.

[0163] (c) Synthesis of 3-(trifluoroacetylamino)-1-O-DMT-2-O-(methyl-N,N-diisopropylphosphoramido)-1,2-propanediol (13).

[0164] Material : Materials are as described in Example 1, supra.

[0165] step: 3-(Trifluoroacetylamino)-1-O-DMT-1,2-propanediol (12) (196 mg, 0.4 mmol) was dissolved in anhydrous dichloromethane (1.5 mL) containing diisopropylethylamine (348 μL, 2 mmol). N,N-diisopropylmethylphosphoramide chloride (200 μL, 1 mmol) was added dropwise with stirring under argon. After 1 hour, ethyl acetate (50 mL) containing 1% triethylamine was added, and the resulting solution was extracted 3 times with saturated sodium bicarbonate aqueous solution. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure to an oil. 31 P NMR (CD3CN, trimethoxyphosphate, external standard): δ (ppm) 147.5 The purity of this material was estimated to be greater than 95%. The crude sample was used directly to add the linker to the oligonucleotide. Example 3 (A): Synthesis of a 6-amino-1,2-hexanediol-based linker reagent

[0166] The synthesis is illustrated in Figure 4 and described below.

[0167] (a) Synthesis of 1,2-(isopropylidinium)-1,2,6-hexanetriol (14).

[0168] Material : 1,2,6-Hexanetriol and 2,2-dimethoxypropane were purchased from Aldrich.

[0169] step : 1,2,6-Hexanetriol (1.00 g, 7.45 mmol), anhydrous acetone (10 mL) and concentrated sulfuric acid (30 μL) were added to a 50 mL round-bottom flask along with a magnetic stirring bar. The flask was purged with nitrogen and a rubber septum was attached to exclude moisture. Next, 2,2-dimethoxypropane (3.00 mL, 24.4 mmol) was slowly added to the stirred solution via syringe over a period of 30 minutes. Stirring was continued for 2 hours. Anhydrous sodium carbonate (150 mg) was added to quench the reaction, and the contents were then stirred overnight. Finally, the solution was filtered and concentrated by rotary evaporation under reduced pressure to give a light yellow syrup (14) (1.6 g). The material was used in the next step without purification.

[0170] (b) Synthesis of 1,2-(isopropylidinyl)-6-(p-toluenesulfonyl)-1,2,6-hexanetriol (15)

[0171] Material : p-Toluenesulfonyl chloride was purchased from Aldrich.

[0172] stepThe crude isopropylated material (14) (about 7.45 mmol) from the previous step was dissolved in anhydrous acetone (15 mL). Next, p-toluenesulfonyl chloride (2.8 g, 14.9 mmol) and anhydrous pyridine (5 mL) were added and the contents were stirred at room temperature for 3 hours while removing moisture. The solvent was then removed by rotary evaporation under reduced pressure and the residue was distributed between dichloromethane (25 mL), dried over anhydrous magnesium sulfate, filtered, and then concentrated to dryness by rotary evaporation under reduced pressure to give 2.8 g of crystalline solid (15). The crude product was purified by flash chromatography on silica gel as described above using chloroform as the mobile phase. The fractions were analyzed by thin layer chromatography on fluorescent silica gel plates using the same solvent. Spots were observed under UV light. The fractions containing the product (Rf 0.50) were combined and concentrated by rotary evaporation under reduced pressure to give an oil (15) (2.37 g) with an overall yield of 97%.

[0173] (c) Synthesis of 1,2-(isopropylidinyl)-6-azido-1,2-hexanediol (16).

[0174] step : The toluenesulfonate (15) (2.37 g, 7.22 mmol) from the previous step was dissolved in anhydrous dimethylformamide (30 mL). Sodium azide (1.64 g, 25.2 mmol) and a magnetic stirring bar were added, and a reflux condenser and a calcium chloride drying tube were connected. The mixture was then stirred in a water bath at 60-65° C. for 3 hours. Stirring was continued overnight at room temperature. The precipitate was then removed by centrifugation, and the resulting solution was concentrated to a final volume of approximately 5 mL by rotary evaporation under reduced pressure. The concentrated solution was distributed between chloroform (50 mL) and water (15 mL). The organic layer was further washed with water (15 mL), dried over anhydrous magnesium sulfate, filtered, and concentrated to an amber oil (16) by rotary evaporation under reduced pressure. The crude product was then used in the next step without further purification.

[0175] (d) Synthesis of 6-amino-1,2-hexanediol (17).

[0176] Material Lithium aluminum hydride (1.0 M) in diethyl ether was purchased from Aldrich.

[0177] step: Under argon atmosphere, anhydrous ether (10mL) and an ether solution (15mL) of lithium aluminum hydride (1.0M) are added in a 250mL round-bottom flask. Then under argon, a solution of the crude azide (16) (about 7mmol) in anhydrous ether (25mL) from the previous step is added by addition funnel under stirring. After completion of the addition, the mixture is stirred under argon. After completion of the addition, the mixture is stirred under reflux for 90 minutes. The gained slurry is diluted with ether (25mL), and the following solutions are added in the order shown under stirring: water (1mL), 5N NaOH (1mL) and water (1mL). The mixture is then filtered through a medium glass filter. The filtrate is distilled and concentrated at room temperature, and then high vacuum is used to obtain a light yellow oil. Next, water (10.8mL) and 88% formic acid (14.2mL) are added. The gained mixture is placed at room temperature overnight, then heated at 70-75°C for 2 hours. The solution was concentrated to a syrup by rotary evaporation under reduced pressure, dissolved in water (50 mL), and applied to a cation exchange column (H type, 50 mL bed volume, Bio-Rad Labs, CA, USA) containing AG50W-X8 resin. The column was eluted with 1N HCl. The fractions containing the amine product were visualized by spotting, spraying ninhydrin reagent on a silica gel TLC plate and heating as described above. The fractions containing the product were merged and concentrated to give a syrup by rotary evaporation under reduced pressure. The syrup was further co-evaporated with methanol by rotary evaporation under reduced pressure to give a light yellow needle-like substance (17) in the form of a hydrochloride salt.

[0178] (e) Synthesis of 6-N-(9-fluorenylmethoxycarbonyl)amino-1,2-propanediol (18).

[0179] Material 9-Fluorenylmethylsuccinimidyl carbonate (Fmoc) and N-hydroxysuccinimide (NHS) were purchased from Bachem, Inc. (Torrance, CA, USA).

[0180] step: The 6-amino-1,2,-hexanediol hydrochloride (17) obtained in the previous step was dissolved in water (10 mL) and adjusted to a final pH of 8.7 with 5N NaOH. Sodium bicarbonate (588 mg, 7 mmol), Fmoc-NHS (2.76 g, 7 mmol) and acetone (10 mL) were added. The suspension was stirred at room temperature overnight, after which all the Fmoc-NHS entered the solution. The reaction mixture was concentrated by rotary evaporation under reduced pressure to remove acetone. 1N HCl (50 mL) and ethyl acetate (150 mL) were added, and the mixture was transferred to a separatory funnel. The organic layer was separated and washed with 0.1N HCl (50 mL) and then with water (2×50 mL). The organic layer was then dried over anhydrous magnesium sulfate, filtered, and concentrated to an oil. Chloroform / acetone (50:50) was used as the mobile phase and the product was purified by flash chromatography on silica gel as described above. The fractions were analyzed by thin layer chromatography on fluorescent silica gel plates using the same solvent system. The spots were observed under UV light. The fractions containing the product (Rf 0.25) were combined and the solvent was removed by rotary evaporation under reduced pressure to give 1.20 g of a white crystalline solid (18). The overall yield was 45% based on the amount of 1,2,6-hexanetriol starting material.

[0181] (f) Synthesis of 1-O-DMT-6-N-(fluorenylmethoxycarbonyl)-6-amino-1,2-hexanediol.

[0182] step : The product (18) (0.5 g, 1.41 mmol) of the previous step was co-evaporated with anhydrous pyridine (3×3 mL) and then dissolved in anhydrous pyridine (8 mL) under argon. A solution of dimethoxytrityl chloride (0.5736 g, 1.69 mmol) in anhydrous dichloromethane (2 mL) was added by syringe with stirring over a few minutes. Stirring was continued at room temperature for 2 hours, and then methanol (100 μL) was added to quench the reaction. The solvent was removed by rotary evaporation under reduced pressure, and the residue was dissolved in chloroform (100 mL). The resulting solution was transferred to a separatory funnel and washed with saturated aqueous sodium bicarbonate solution (3×20 mL) and then with 5 M sodium chloride (20 mL). The organic layer was then dried over anhydrous magnesium sulfate, filtered and concentrated to an oil by rotary evaporation under reduced pressure. The product was purified by flash chromatography on silica gel as described above using a dichloromethane / ethyl acetate / triethylamine (95:5:0.5) solvent system. Fractions were analyzed by thin layer chromatography on silica gel plates using the same solvent; spots were visualized by placing the plates in HCl fumes. Fractions containing product (19) (Rf 0.35) were combined and the solvent removed by rotary evaporation under reduced pressure to give a foam (910 mg, 100% of theoretical yield).

[0183] (g) Synthesis of 1-O-DMT-6-N-(fluorenylmethoxycarbonyl)-2-O-(methyl-N,N-diisopropylphosphoramido)-6-amino-1,2-hexanediol (20):

[0184] Material : Materials are described in previous Examples, supra.

[0185] step : N,N-diisopropyl-methoxyphosphinyl chloride (102 μL, 0.513 mmol) was added dropwise to a stirred solution of (19) (225 mg, 0.34 mmol) and N,N-diisopropylethylamine (236 μL, 1.36 mmol) in anhydrous dichloromethane (3 mL) under an argon atmosphere. After 90 minutes, the reaction mixture was diluted into ethyl acetate containing 2% triethylamine (50 mL) and washed with saturated aqueous sodium bicarbonate solution (2×25 mL). The organic layer was dried over anhydrous magnesium sulfate, filtered, and concentrated to dryness by rotary evaporation under reduced pressure. The residue was dissolved in toluene (2 mL) and added dropwise to petroleum ether at -20°C with rapid stirring. The resulting mixture was then stored at -20°C for 16 hours. It was then warmed to room temperature and the supernatant was decanted. The precipitated product (20) was then dried in vacuo to give 160 mg (58% yield). The purity of this material was confirmed by thin layer chromatography on silica gel plates using a solvent system of dichloromethane / ethyl acetate / triethylamine (10:1:0.1) and visualized under UV light (Rf 0.9, compared to Rf 0.25 of the starting material).

[0186] Extended analogs of the linker reagents described in Example 3(A) above were also generated. The structures of these analogs are shown in Figure 5 (21-24). The preparation of these analogs is described in the following examples.

[0187] Example 3(B): Synthesis of 3-N-(glycidyl)-amino-1,2-propanediol-based linker reagent (21)

[0188] The scheme of this synthesis is outlined in FIG6 .

[0189] (a) Synthesis of 3-N-[N-(fluorenylmethoxycarbonyl)glycidyl]-amino-1,2-propanediol (25).

[0190] Material N-(Fluorenylmethoxycarbonyl)-glycine-N-hydroxysuccinimide (Fmoc-glycine-NHS) was purchased from Bachem. Other reagents have been described above.

[0191] step: 3-Amino-1,2-propanediol (91 mg, 1 mmol) was added to an acetone solution (7 mL) of Fmoc-glycine-NHS (394 mg, 1 mmol). To this solution was added an aqueous solution (5 mL) of sodium bicarbonate (84 mg, 1 mmol). The reaction mixture was stirred at room temperature for 16 hours. Thin layer chromatography using silica gel plates and a dichloromethane / methanol / acetic acid (20:2:0.1) solvent system showed that the reaction was complete. The product (25) was produced in the flask as a precipitate, which was filtered off and dried in vacuo over phosphorus pentoxide for 2 days. The yield was 310 mg (84%).

[0192] (b) Synthesis of 1-O-DMT-3-N-[N-(fluorenylmethoxycarbonyl)-glycidyl]-amino-1,2-propanediol (26).

[0193] Material : Materials are described in previous Examples, supra.

[0194] step Compound (25) (185 mg, 0.5 mmol) was dried by co-evaporation with anhydrous pyridine (3 × 3 mL). It was then dissolved in anhydrous pyridine (3 mL) and a solution (4 mL) of dimethoxytrityl chloride (222 mg, 0.57 mmol) in a 1:1 mixture of dichloromethane / pyridine was added dropwise with stirring. Stirring was continued for 1.5 hours, and the reaction was monitored by silica gel thin layer chromatography using a dichloromethane / methanol (8:1) solvent system. The reaction was quenched by adding methanol (0.2 mL); stirring was continued for 10 minutes. Pyridine was removed by rotary evaporation under reduced pressure. The residue was dissolved in dichloromethane (150 mL) and washed with saturated aqueous sodium bicarbonate solution (2 × 50 mL) and then with water (50 mL). After drying over anhydrous magnesium sulfate, the dichloromethane solution was removed by rotary evaporation under reduced pressure. The residue was purified by flash chromatography on silica gel using a dichloromethane / ethyl acetate (11:5) solvent system containing 0.1% pyridine according to the method described above. The product-containing fractions were identified by silica gel thin layer chromatography as described above. These fractions were combined and the solvent removed by rotary evaporation under reduced pressure to give 250 mg (26) (75% yield).

[0195] (c) Synthesis of 1-O-DMT-2-O-(N,N-diisopropylamino-methoxyphosphonamido)-3-N-[N-(fluorenylmethoxycarbonyl)-glycidyl]-amino-1,2-propanediol (21).

[0196] Material : Materials are described in previous Examples, supra.

[0197] stepCompound (26) (235 mg, 0.35 mmol) was dried by co-evaporation with anhydrous pyridine (2 × 3 mL). Then, it was dissolved in anhydrous dichloromethane (2 mL) and N, N-diisopropylethylamine (244 μL, 1.4 mmol) was added. Then, N, N-diisopropylamino-chloromethoxyphosphine (105 μL, 0.53 mmol) was added dropwise under stirring under an argon atmosphere. Using dichloromethane / ethyl acetate / triethylamine (10:5:0.5) solvent system, it was found by silica gel thin layer chromatography that the reaction was complete after 20 minutes. Then, the reaction mixture was diluted with saturated sodium bicarbonate aqueous solution (2 × 25 mL) into ethyl acetate (ethyl). After drying over anhydrous magnesium sulfate, the ethyl acetate layer was removed by rotary evaporation under reduced pressure. The residue was redissolved in ethyl acetate (3 mL) and poured into hexane (150 mL) at -25 ° C. The precipitate was filtered and dried in vacuo to afford 210 mg (21) (72%). 31 P-NMR (CDCl3, trimethyl phosphate, external standard): (ppm) 147.5 (d). The structure is 1 The results were also confirmed by H-NMR analysis.

[0198] Example 3(C): Synthesis of linker reagents (22, 23) based on 3-N-(4-aminobutyryl)-amino-1,2-propanediol and 3-N-(6-aminohexanoyl)-amino-1,2-propanediol

[0199] The synthetic steps specific to this example are schematically illustrated in Figure 7 and described below.

[0200] (a) Synthesis of N-fluorenylmethoxycarbonyl protected forms of 4-aminobutyric acid and 6-aminohexanoic acid (N-Fmoc-4-aminobutyric acid (27) and N-Fmoc-6-aminohexanoic acid (28)).

[0201] Material : 4-aminobutyric acid and 6-aminohexanoic acid were purchased from Aldrich. Fmoc-NHS was described in Example 3(A).

[0202] step These syntheses were carried out according to the method described in A. Paquet (Can. J. Chem., 1982, 60, 976).

[0203] (b) Coupling of either N-Fmoc-4-aminobutyric acid or N-Fmoc-6-aminohexanoic acid with 3-amino-1,2-propanediol.

[0204] MaterialTrimethylacetyl chloride was purchased from Aldrich. Other materials are described in the previous examples, see above.

[0205] step : Either compound (27) or (28) (1 mmol) was first dried by co-evaporation with pyridine (2×3 mL). The residue was then dissolved in a mixture of anhydrous dimethylformamide (3 mL) and anhydrous tetrahydrofuran (3 mL). The resulting solution was cooled in an ice bath, and N,N-diisopropylethylamine (1 mmol) was added, followed by the slow addition of trimethylacetyl chloride (1 mmol) with stirring. Stirring was continued in an ice bath for 45 minutes. Subsequently, a solution of 3-amino-1,2-propanediol (1.2 mmol) in anhydrous dimethylformamide (3 mL) was added, and the resulting mixture was warmed to room temperature and stirred for 1 hour. The reaction was monitored by silica gel thin layer chromatography using a dichloromethane / methanol / acetic acid (10:1:0.1) solvent system. Based on this analysis, it was determined that the reaction had proceeded to approximately 90% completion. The reaction mixture was then concentrated by rotary evaporation under reduced pressure, diluted with ethyl acetate (100 mL) and transferred to a separatory funnel. The organic solution was washed with saturated aqueous sodium bicarbonate solution (2×50 mL) and water (50 mL). After drying over anhydrous magnesium sulfate, the organic layer was concentrated to dryness by rotary evaporation under reduced pressure. Typically, the resulting product was determined to be greater than 95% pure and was used in subsequent steps without further purification. However, in cases where purification was required, purification was performed by silica gel flash chromatography using a dichloromethane / methanol (40:1) solvent system as described in the previous examples. The purity of (29) and (30) was determined by 1 The results were confirmed by H-NMR analysis.

[0206] (c) 1-O-Dimethoxytritylation of (29) and (30):

[0207] The materials and procedures used for this synthesis are as described in Example 3(B), part (b). The purity of these materials was determined by 1 H-NMR confirmed.

[0208] (d) Converting the compounds mentioned in section (c) above into the corresponding 2-O-(N,N-diisopropylmethyl)phosphoramidites (22 and 23):

[0209] The materials and procedures used for this synthesis are as described in Example 3(B), part (c). The purity of products (22) and (23) was determined by 31 P-NMR confirmed.

[0210] Example 3(D): Synthesis of further extended analog linker reagents based on 3-N-(6-aminohexanoyl)-amino-1,2-propanediol

[0211] The unique steps of this synthesis are schematically illustrated in Figure 8 and described below.

[0212] (a) Synthesis of 1-O-DMT-3-N-(6-aminohexanoyl)-amino-1,2-propanediol (32).

[0213] Material : Compound (31) was prepared as described in Example 3(C), part (c).

[0214] step Compound (31) (0.89 g, 1.1 mmol) was ammonolyzed with concentrated ammonium hydroxide (10 mL) and pyridine (10 mL) at room temperature overnight. An aliquot from the reaction was spotted onto a silica TLC plate and treated with ninhydrin to monitor the deprotection of the primary amine. The reaction mixture was then concentrated to dryness by rotary evaporation under reduced pressure, and the resulting residue (32) was used in the subsequent step without purification.

[0215] (b) Coupling of compound (32) and compound (28).

[0216] Material : Compound (28) was synthesized according to the procedure described in Example 3(C), part (a).

[0217] step N-Fmoc-aminocaproic acid (28) (1.1 mmol) was reacted with trimethylacetyl chloride (1.1 mmol) according to the procedure described in Example 3(C), part (b) above. Next, a solution of compound (32) (1.1 mmol) in anhydrous dimethylformamide was added, again according to the procedure in Example 3(C), part (b). The resulting adduct (33) was purified by flash chromatography on silica gel using a chloroform / methanol (30:1) solvent system as described in the previous example. The yield of the product was 250 mg (23%).

[0218] (c) Compound (33) is converted into the corresponding 2-O-(N,N-diisopropylamino)-methoxyphosphoramidite (34).

[0219] step: This method is essentially the same as that described in Example 3(B), part (c) above. Thus, compound (33) (240 mg, 0.285 mmol) was reacted with N,N-diisopropylaminochloromethoxyphosphine (73 μL, 0.371 mmol) in anhydrous dichloromethane (3 mL) containing N,N-diisopropylethylamine (198 μL, 1.14 mmol). The reaction was worked up by diluting the reaction with 2% triethylamine in ethyl acetate (50 mL) and extracting with saturated aqueous sodium bicarbonate solution (25 mL) and water (25 mL). The resulting organic layer was dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure. The residue was dissolved in a few milliliters of ethyl acetate and precipitated into hexane (150 mL) as described in the previous examples. The yield of (34) was 200 mg and its purity was determined by 1 H-NMR and 32 The results were confirmed by P-NMR spectroscopy. Example 4: Automated ligation of 2-(3-aminopropyl)-1,3-propanediol-based linkers to synthetic oligonucleotides

[0220] The ligation of a linker reagent to various synthetic oligonucleotides will now be described. The linker reagent is described in Example 1, reagent (5), and will be referred to as "L1" hereinafter.

[0221] (a) Conjugation of the "L1" reagent to the 5'-end of the deoxyoligonucleotide. A deoxyoligonucleotide having the sequence "5'-GCTCGTTGCGGGACTTAACCCAACAT-3'" (SEQ ID NO 1) was synthesized on a controlled-pore glass support using an Applied Biosystems, Inc. (Foster City, CA, USA) 380A DNA synthesizer using standard 3'-β-cyanoethylphosphoramidite chemistry. The 5'-dimethoxytrityl group was removed, and a solution of "L1" (0.1 M) in anhydrous acetonitrile was coupled twice using a standard coupling cycle. The percent coupling of the first and second additions of "L1" relative to the amount of full-length deoxyoligonucleotide was quantified by measuring the absorbance at 498 nm of the released dimethoxytrityl group at the end of each coupling cycle; these values ​​were determined to be 29% and 42%, respectively. 5'-(L1)- and 5'-(L1)-(L1)-oligonucleotides were purified by gel electrophoresis on a 20% polyacrylamide gel containing 7 M urea. The corresponding bands were visualized by UV shadowing and were estimated to migrate slower on the gel, with a spacing approximately 1.5 times the spacing between the corresponding additional nucleotides. These bands were excised, and the linker-modified deoxynucleotides were recovered and purified by standard methods.

[0222] (b) Coupling of the "L1" reagent to the 3'-end of the deoxyoligonucleotide. For this synthesis, a Teflon oxidizable solid support (Molecular Biosystems, Inc., San Diego, CA, USA, catalog #OSS-01) was used. When the deoxyribonucleotide is cleaved from the support, the compound used in the first coupling cycle remains at the 3'-end along with the 3'-terminal phosphate group. Using standard phosphoramidite chemistry on an Applied Biosystems, Inc. 380A DNA synthesizer, a solution of "L1" (0.2 M) in anhydrous acetonitrile was coupled to the support using three coupling cycles. Next, the same deoxyoligonucleotide sequence (SEQ ID NO 1) as presented in part (a) above was added using the same coupling chemistry. For the first, second, and third couplings, the affinity of the "L1" reagent to the deoxyoligonucleotide was determined as described above. 1 The initial coupling percentages of the reagents were 40%, 63% and 63%, respectively. After removing the terminal dimethoxytrityl group from the resulting trimer, a deoxyoligonucleotide having the same sequence as in 4(a) was ligated using standard phosphoramidite chemistry. The support material was then removed from the synthesizer and treated with concentrated ammonium hydroxide at 55°C for 16 hours. Next, the support was washed three times with water and treated with 50 mM sodium periodate in 20 mM sodium phosphate buffer (pH 7.4) at room temperature for 2.5 hours. Finally, the support was washed several times with water and treated with 10% aqueous n-propylamine at 55°C for 3 hours. The resulting solution was applied to a 20% polyacrylamide gel containing 7 M urea and separated by electrophoresis. The corresponding 3'-(L1)-(L1)-(L1) deoxyoligonucleotide was recovered as described above.

[0223] (c) "L1" was coupled to the 3'-terminus of a deoxyoligonucleotide having the sequence "5'-AAATAACGAACCCTTGCAGGTCCTTTCAACTTTGAT-3'" (SEQ ID NO 2). The synthesis method was the same as that described in part (b), except that a Biosearch 8750 DNA synthesizer was used.

[0224] (d) "L1" was coupled to the 3'-end of a deoxyoligonucleotide having the sequence "5'-CAGTCAAACTCTAGCCATTACCTGCTAAAGTCATTT-3'" (SEQ ID NO 3). Similarly, the method described in part (b) was used, except that the automated portion of the synthesis was performed on a Biosearch 8750 DNA synthesizer.

[0225] (e) containing a 2-(3-aminopropyl)-1,3-propanediol linker ("L 1 Hybridization and melting temperatures (Tm) of synthetic deoxyoligonucleotide probes.

[0226] Material Two L1 derivatives of a 33-nucleotide-long deoxyoligonucleotide probe were synthesized by methods similar to those described above: "L1-insertion" had L1 inserted between nucleotide residues 21 and 22 (numbering from the 5'-end); and "L1-replacement" had L1 between nucleotide residues 20 and 22 as a replacement for residue 21. Both probes had sequences complementary to Chlamydia trachomatis ribosomal RNA ("target rRNA"). Probes were used 125 I was labeled using a standard protocol developed by Gen-Probe Incorporated; hydroxyapatite (HAP) was from Behring Diagnostic (Calbiochem Division, La Jolla, CA, USA); sodium dodecyl sulfate (SDS), sodium phosphate (monobasic and dibasic salts), and hydrochloric acid were reagent grade and purchased from Fisher Scientific Corp.; Betagel (liquid scintillant) was from West Chem (San Diego, CA, USA). All other materials were reagent grade. Unless otherwise noted, operations were performed in 1.5 mL screw-capped polypropylene Eppendorf tubes.

[0227] Hybridization was performed as follows : 48 μL 1M sodium phosphate (pH 6.8), 10 μL 1% SDS (v / v), 10 μL 125I-labeled probe (approximately 200,000 CPM), 29.5 μL of water, and 2.5 μL of rRNA solution (0.5 μg, "target") or 2.5 μL of water ("control") were mixed and incubated at 60°C for 1 hour. Next, a 10 μL aliquot was diluted into 1 mL of 0.12 M sodium phosphate (pH 6.8) / 0.02% SDS / 0.02% sodium azide and vortexed for 5 seconds. The diluted aliquot was incubated in a water bath heated from room temperature to 80°C; aliquots were removed at the specified temperature and stored on ice. The sample was then passed through a hydroxyapatite column equilibrated with 0.12 M sodium phosphate (pH 6.8) / 0.02% SDS / 0.02% sodium azide, and the eluate was counted by scintillation using standard procedures. (The percentage of counts that remained bound to the column corresponds to the hybridization probe). The Tm value is calculated as the temperature at which 50% of the initially formed hybrid is thermally denatured into single-stranded species. 1 -The Tm of the inserted probe is 69°C with L 1 - The Tm of the replaced probe is 66° C. The data show that both probes hybridize to the intended target rRNA, with the Tm of the "insertion" probe being approximately 3 degrees higher than the Tm of the "replacement" probe.

[0228] Example 5: Demonstration of the ability of "L1" modified deoxyoligonucleotides to be labeled with biotin and fluorescein

[0229] (a) Use 32 P-labeled 5'-(L1)- and 5'-(L1)-(L1)-modified deoxyoligonucleotides "5'-L1-GCTCGTTGCGGGACTTAACCCAACAT-3'" (SEQ ID NO 1) and "5'-L1-L1-GCTCGTTGCCCCACTTAACCCAACAT-3'" (SEQ ID NO 3).

[0230] Material :α- 32 ATP was purchased from New England Nuclear (DuPont, Boston, MA, USA). Terminal deoxynucleotidyl transferase (TdT) and 5X tailing buffer were products of Bethesda Research Laboratories (Gaithersburg, MD, USA).

[0231] 20 pmol of 5'-(L 1 )-(L 1) modified oligonucleotides and 16.5 pmol of α- 32 ATP (specific activity 3000 Ci / mmol) and 40 units of TdT were reacted in 20 μL 1X tailing buffer for 1 hour. 32 P-labeled oligonucleotides were placed in Nensorb-20 (TM) Purification was performed on a Sigma-Aldrich column (New England Nuclear, DuPont Corp., Boston, MA, USA) according to the manufacturer's protocol, which is incorporated herein by reference.

[0232] (b) make 32 P-labeled 5'-(L 1 )-(L 1 )-oligonucleotides were reacted with biotin-ε-aminohexanoic acid N-hydroxysuccinimide ("Bio-X-NHS", Calbiochem-Behring Corp., San Diego, CA, USA). Streptavidin-agarose was purchased from Bethesda Research Laboratories, and D(+)-biotin was purchased from Calbiochem-Behring Corp.

[0233] 1 pmol of each modified oligonucleotide was reacted with 2.5 mM Bio-X-NHS in 125 mM borate buffer (pH 9) containing 12.5% ​​dimethyl sulfoxide for 1.5 hours. A small aliquot of the resulting reaction mixture was then tested for binding to streptavidin-agarose in 50 mM sodium phosphate (pH 7.4) / 2 mM ethylenediaminetetraacetic acid / 0.5 M sodium chloride in the presence ("non-specific binding") or absence ("specific binding") of 0.2 mg / mL D(+) biotin. Bound material was quantified by scintillation counting: for oligonucleotides 5'-(L 1 ), the nonspecific binding percentage was 0.3%, and the specific binding percentage was 71.8%; for oligonucleotide 5'-(L 1 )-(L 1 ), the nonspecific binding percentage was 0.5%, and the specific binding percentage was 90.3%.

[0234] The binding of biotin to these L-terminal oligomers was also confirmed by electrophoretic separation of oligomers from aliquots of the reaction mixture on 20% polyacrylamide / 7M urea gels. 1 Ligation of modified oligomers. Representative bands were visualized by autoradiography, showing nearly quantitative conversion to the biotinylated form, which migrated slower than the non-biotinylated control.

[0235] (c) 3'-L1-modified deoxyoligonucleotide {"5'-AAATAACGAACCCTTGCAGGTCCTTTCAACTTTGAT-L 1 -3'" (SEQ ID NO 2) and "5'-CAGTCAAACTCTAGCCATTACCTGCTAAAGTCATTT-L 1 -3'" (SEQ ID NO 4)} were labeled with fluorescein isothiocyanate and biotin-X-NHS, respectively. The oligonucleotides were first labeled with [.γ.- 32 [P] Adenosine triphosphate was labeled using T4-polynucleotide kinase according to the method of Maxam and Gilbert (Proc. Natl. Acad. Sci., USA, vol. 74, p. 560, 1977).

[0236] The first modified oligonucleotide was reacted with fluorescein isothiocyanate (FITC, Sigma Chemical Co., St. Louis, MO, USA). 40 pmol of this oligomer was treated with 90 mM FITC in 0.1 M borate buffer (pH 9) containing 90% dimethyl sulfoxide for 12 hours. The reaction mixture was then separated by electrophoresis on a 20% polyacrylamide / 7 M urea gel. Bands were visualized by autoradiography. The top band of each lane was excised, and the FITC-labeled oligomer was recovered from the gel and purified.

[0237] Binding assays using anti-FITC antibody-derived solid supports were used to confirm the attachment of fluorescein to the oligonucleotides. Anti-FITC magnetic microspheres were purchased from Advanced Magnetics, Inc. (Cambridge, MA, USA), catalog #4310. Purified fluorescein was tested in the presence ("nonspecific binding") or absence ("specific binding") of 20 mM hydrolyzed FITC. 32 An aliquot of P-labeled FITC-modified oligonucleotide was mixed with 20 μL of anti-FITC microspheres in 0.5 mL of buffer solution (50 mM sodium phosphate, pH 7.4 / 2 mM EDTA / 0.5 M sodium chloride). After 1 hour, the microspheres were removed by magnetic separation, and the supernatant was counted by Cherenkov radiation to determine the amount of bound material: nonspecific binding was 0.1% and specific binding was 80.2%.

[0238] The second modified oligonucleotide was reacted with biotin-X-NHS. 40 pmol of this oligomer was treated with 10 mM Bio-X-NHS in 0.1 M borate buffer (pH 9) containing 20% ​​dimethyl sulfoxide for 1 hour. The resulting biotinylated oligomer was purified by polyacrylamide gel electrophoresis as described above. The presence of biotin attached to this oligonucleotide was confirmed by analyzing binding on streptavidin-agarose as described in this example: the nonspecific binding percentage was 0.3% and the specific binding percentage was 87.4%.

[0239] Example 6: 3'-L 1 Resistance of modified deoxyoligonucleotides to phosphodiesterase-catalyzed hydrolysis

[0240] Material Phosphodiesterase from Crotalus durissus was purchased from Boehringer-Mannheim Biochemicals (Indianapolis, IN, USA). This enzyme catalyzes exonucleolytic cleavage from the 3'-end of oligonucleotides. Synthetic deoxyoligonucleotides with the sequence "5'-AAATAACGAACCCTTGCAGGTCCTTTCAACTTTGAT-3'" (SEQ ID NO 2) were synthesized using standard phosphoramidite chemistry on an Applied Biosystems 380A DNA synthesizer. Deoxyoligonucleotides with the same sequence but with a 3'-L attached were synthesized according to the procedure given in Example 4. 1 Connector probe.

[0241] Both oligonucleotides were used 32 P was labeled according to the method described in Example 5. Approximately 350,000 CPM of each labeled oligonucleotide was added to a 3×10 -5 or 3×10 -6 The reaction was carried out in 10 μL of buffer (0.1 M tris (hydroxymethyl) aminomethane) hydrochloride, pH 8.0 / 20 mM magnesium chloride) containing 1 unit of phosphodiesterase. 1.5 μL (one and 1 / 2 μL) aliquots were removed at time intervals of 5 minutes, 10 minutes, 15 minutes, and 30 minutes; the reaction was quenched by adding 3 μL of 0.1N sodium hydroxide. Next, 5 μL of 90% formamide containing bromophenyl blue and xylene cyanol FF dye was added to each aliquot, and the resulting samples were separated by electrophoresis on a 20% polyacrylamide / 7M urea gel. The gel was then analyzed by autoradiography.

[0242] The 3'-L1 modified oligonucleotides were found to be more than 95% resistant to phosphodiesterase-catalyzed hydrolysis after 30 minutes for both enzyme concentrations tested. However, essentially no full-length unmodified oligonucleotides were visible on the gel, indicating that the enzyme typically cleaves oligomers that do not contain the 3'-L1 group.

[0243] Example 7: Automated Introduction of 2,2-Bis-(3-aminopropyl)-1,3-propanediol-Based Linker Reagents into Synthetic Oligonucleotides

[0244] Example 2: The linker reagent described in Reagent 10 (hereinafter referred to as "L2") is inserted between the bases of the synthetic oligonucleotide to produce the sequence "5'-CGTTACTCGGATGCCCAAAT(L 2 )ATCGCCACATTCG-3'" (SEQ ID NO 5). The method used was similar to that described in Example 4(a), except that the "L2" solution (0.1 M in acetonitrile) was reacted in the thirteenth coupling cycle instead of the last coupling cycle described in Example 4(a). The coupling efficiency with "L2" was approximately 30%, as estimated from the amount of dimethoxytrityl group released (see Example 4(a)).

[0245] Example 8: Automated Conjugation of 3-Amino-1,2-Propanediol-Based Linker Reagents to Synthetic Oligonucleotides

[0246] According to the procedure given in Example 4(a), this linker (reagent (13), hereinafter referred to as "L3") was introduced into a synthetic oligonucleotide having the sequence "5'-CCCGCACGTCCCTATT(L3)AATCATTACGATGG-3'" (SEQ ID NO 6). In this example, a solution of "L3" (0.3 M in anhydrous acetonitrile) was reacted in the fifteenth coupling cycle; the coupling efficiency of this step was approximately 60%, as estimated based on the release of the dimethoxytrityl group (see Example 4(a)).

[0247] Example 8(A): Automatic introduction of linker reagents (20), (21), (22), (23) and (24) into synthetic oligonucleotides

[0248] The linker reagents (20-24) (synthesized as described above in Examples 3(A)-3(D)) were introduced as described above in Example 4, part (a). The corresponding linkers associated with these reagents are referred to as "L4", "L5", "L6", "L7" and "L8" respectively below. Therefore, in the specific case corresponding to the use of one of the above reagents, a 0.12-0.2M solution of the reagent in anhydrous acetonitrile was loaded at position #6 of an Applied Biosystems 380A DNA synthesizer. The reagents were introduced into the oligonucleotide polymer using a standard phosphoramidite coupling protocol. A series of oligonucleotides were prepared with lengths ranging from 17 to 35 bases, with each linker L4-L8 inserted at a different position within the sequence. The coupling efficiency associated with these reagents was between 75% and 98%, as measured by the release of dimethoxytrityl groups at the end of the coupling cycle.

[0249] Example 9: Labeling of amine linker arm probes with acridinium esters and subsequent purification.

[0250] A 25 mM stock solution of acridinium ester N-hydroxysuccinimide labeling reagent (for composition, see I. Weeks et al., Clin. Chem., Vol. 29, p. 1474, 1983) was prepared in distilled dimethyl sulfoxide. The desired amount of polymer produced in Example 4, 7, or 8(A) was evaporated to dryness in a 1.5 mL conical polypropylene tube. The following composition was constructed by adding the following ingredients in the order listed: 3 μL of water, 1 μL of 1 M 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (pH 8.0), 4 μL of dimethyl sulfoxide (distilled), and 2 μL of 25 mM acridinium ester N-hydroxysuccinimide labeling reagent in dimethyl sulfoxide (distilled). The mixture was vortexed, spun in a microcentrifuge for 2 seconds (to bring the contents to the bottom of the tube), and incubated at 37°C for 20 minutes. At this point, the following components were added to the reaction mixture in the order listed: 3.0 μL of 25 mM acridinium ester N-hydroxysuccinimide labeling reagent in dimethyl sulfoxide (distilled), 1.5 μL of water, and 0.5 μL of 1 M 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (pH 8.0). The mixture was vortexed again, spun, and incubated at 37°C for an additional 20 minutes. Unreacted label was quenched using a 5-fold excess of lysine by adding 5 μL of 0.125 M lysine in 0.1 M 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (pH 8.0), 50% dimethyl sulfoxide and incubated at room temperature for 5 minutes.

[0251] At this point, the acridinium ester-labeled oligomers were purified using the following method. 30 μL of 3 M sodium acetate (pH 5.0), 245 μL of water, and 5 μL of glycogen as a carrier (the glycogen was pretreated to remove any nuclease activity) were added to the 20 μL quenched reaction mixture. The sample was vortexed briefly and 640 μL of anhydrous ethanol was added. The sample was vortexed briefly and incubated on ice for 5-10 minutes, then centrifuged at 15,000 rpm in a microcentrifuge for 5 minutes. The supernatant was carefully removed and the precipitate was redissolved in 20 μL of 0.1 M sodium acetate (pH 5.0), 0.1% SDS. The sample was further purified by ion exchange high performance liquid chromatography (HPLC) as follows: 20 μL of the redissolved precipitate was injected into a Nucleogen-DEAE60-7 ion exchange HPLC column installed in an IBM 9533 HPLC system. All buffers used in this process were made of HPLC grade water, acetonitrile and sodium acetate, as well as reagent grade glacial acetic acid and lithium chloride. In addition, all buffers were filtered through a 0.45 μm pore size Nylon-66 filter before use. In the specific case of a nucleotide / non-nucleotide polymer having a total of 26 monomer units, only one of which is a non-nucleotide monomer unit, the following elution scheme was adopted. Buffer A was 20 mM sodium acetate (pH 5.5), 20% acetonitrile; buffer B was 20 mM sodium acetate (pH 5.5), 20% acetonitrile and 1 M lithium chloride. Elution was performed using a linear gradient from 55% buffer A, 45% buffer B to 30% buffer A, 70% buffer B over 25 minutes at a flow rate of 1 mL / min. During the run, the absorbance at 260 nm was monitored; 0.5 mL fractions were collected in 1.5 mL conical polypropylene tubes. Immediately after the run, 5 μL of 10% SDS was added to each tube, and each tube was then vortexed (this was to ensure that the acridinium ester-labeled probe did not adsorb to the tube wall). A 0.5 μL aliquot was taken from fractions 21-42 and added to 200 μL of water in a 12×75 mm tube (a separate pipette tip was used for each aliquot to avoid carryover problems). The chemiluminescence of each aliquot was then determined in a Berthold Clinilumat by automated injection of 200 μL of 0.25 N nitric acid, 0.1% hydrogen peroxide, followed by the addition of 200 μL of 1 N sodium hydroxide after a 1 second delay and reading for 10 seconds.

[0252] Fractions 29-33 were ethanol precipitated by adding 5 μL of glycogen to each fraction, vortexing, adding 1 mL of ethanol to each fraction, vortexing, incubating on ice for 5-10 minutes, and centrifuging at 15,000 rpm in a microcentrifuge for 5 minutes. Each supernatant was carefully removed, and each pellet was redissolved in 20 μL of 0.1 M sodium acetate (pH 5), 0.1% SDS, and the individual fractions were then combined.

[0253] Example 10: N-(Fluorenylmethoxycarbonylamino)hexanoylamino-O 1 -DMT-O 2 Synthesis of 3-Cyanoethoxydiisopropylphosphinyl-1,2-propanediol

[0254] The synthesis of single propanediol enantiomers of cyanoethyl derivatives of 3-amino-1,2-propanediol isomers based on the linker reagent (23) of 3-N-(6-aminohexanoyl)-amino-1,2-propanediol is described below.

[0255] Example 10(A): N-(Fluorenylmethoxycarbonylamino)hexanoylamino-O 1 -DMT-O 2 Synthesis of cyanoethoxydiisopropylphosphinyl-(S)-3-amino-1,2-propanediol (38)

[0256] (a) Synthesis of N-fluorenylmethoxycarbonyl (Fmoc)-protected 6-aminohexanoic acid (N-Fmoc-6-aminohexanoic acid, compound (28)).

[0257] Material : 9-Fluorenylmethyl N-succinimidyl carbonate was from Sigma-Aldrich (St. Louis, MO, USA). Other materials are described in the previous examples, supra.

[0258] step : The synthesis was essentially as described in Example 3(C)(a) and Figure 7a The product was prepared as shown. Briefly, 1.68 g (5 mmol) of 9-fluorenylmethyl N-succinimidyl carbonate was added to a stirred solution of 656 mg (5 mmol) of 6-aminocaproic acid and 420 mg (5 mmol) of sodium bicarbonate in a mixture of water (7 mL) and acetone (7 mL). After stirring overnight at ambient temperature, the mixture was acidified to pH 2 with concentrated hydrochloric acid and the acetone was removed in vacuo. The product was dissolved in chloroform and washed with 0.1 N HCl and water. The combined organic phases were concentrated to dryness by rotary evaporation under reduced pressure, and the residue was crystallized from dichloromethane-hexane to give compound (28) as a white solid (1.66 g, 4.70 mmol, 94%). Liquid chromatography mass spectrometry (LCMS) analysis showed that the purity of the product was 99.9% and m / z was 354.2 (M+1).

[0259] (b) Coupling of N-Fmoc-6-aminohexanoic acid (28) with (S)-3-amino-1,2-propanediol to form compound (36).

[0260] Material : (S)-3-Amino-1,2-propanediol was from Sigma-Aldrich. Other materials are described in the previous examples, supra.

[0261] step : The synthesis was carried out as follows, similar to the description in Example 3(C)(b) and as Figure 9a As shown. Compound (28) (1.3 g, 3.68 mmol) was first dried by co-evaporation with pyridine (2×5 mL). The residue was then dissolved in a mixture of anhydrous dimethylformamide (2.5 mL) and anhydrous tetrahydrofuran (2.5 mL). The resulting solution was cooled to an ice bath at 0–5°C, N,N-diisopropylethylamine (0.7 mL, 4.02 mmol) was added at this temperature, and then trimethylacetyl chloride (1 mmol) was slowly added over 5 minutes with stirring at the same temperature. Stirring was continued in the ice bath for 45 minutes to form compound (35), which was used without isolation. Next, a solution of (S)-3-amino-1,2-propanediol (4.02 g, 4.41 mmol) in anhydrous dimethylformamide (2.5 mL) was added at 0-5°C over 5 minutes, and the resulting mixture was allowed to warm to room temperature and stirred for 3 hours. LCMS analysis indicated that the reaction was 91% complete. The reaction mixture was then concentrated by rotary evaporation under reduced pressure, diluted with ethyl acetate (100 mL) and transferred to a separatory funnel. The organic solution was washed with saturated aqueous sodium bicarbonate solution (2×50 mL) and water (50 mL). After drying over anhydrous magnesium sulfate, the organic layer was concentrated to dryness by rotary evaporation under reduced pressure. The crude product was dissolved in methanol (2 mL) and applied to a flash chromatography column containing 40 grams of silica gel. The column was eluted with a 40:1 solution (v / v) of dichloromethane / methanol (500 mL) and 25 mL fractions were extracted simultaneously. The fractions were analyzed for product content by thin layer chromatography, and the fractions containing the product were combined and concentrated by rotary evaporation under reduced pressure. The reaction produced 1.289 g (3.02 mmol, 82%) of compound (36) as a white solid with an LCMS analytical purity of 98.0% and an m / z of 427.3 (M+1).

[0262] (c) 1-O-Dimethoxytritylation of compound 36 to form 1-O-DMT-3-N-[N-(fluorenylmethoxycarbonyl)-hexyl]-(S)-amino-1,2-propanediol (37).

[0263] Material : Materials are described in the previous examples, supra.

[0264] step : The synthesis was carried out as follows, similar to the description in Example 3(C)(c), and as Figure 9b As shown. Compound (36) (0.644 g, 1.51 mmol) was dried by co-evaporation with anhydrous pyridine (2 x 5 mL). It was then dissolved in anhydrous pyridine (6.5 mL) and a solution of 4,4'-dimethoxytriphenylmethyl chloride (0.588 g, 1.74 mmol) in a 9:1 mixture of dichloromethane / pyridine (6.5 mL) was added dropwise with stirring at ambient temperature. When the reaction was complete as monitored by silica gel thin layer chromatography using a dichloromethane / methanol (8:1) solvent system, stirring was continued for 2 hours. The reaction was quenched by the addition of methanol (0.2 mL); stirring was continued for 10 minutes. Pyridine was removed by rotary evaporation under reduced pressure. The residue was dissolved in dichloromethane (150 mL) and washed with saturated aqueous sodium bicarbonate solution (2 x 50 mL) and then with water (50 mL). After drying over anhydrous magnesium sulfate, the dichloromethane solution was removed by rotary evaporation under reduced pressure. The residue was purified by flash chromatography on silica gel using a dichloromethane / ethyl acetate (11:5) solvent system containing 0.1% pyridine according to the above method. The fractions containing the product were identified by thin layer chromatography on silica gel as described above. These fractions were combined and concentrated by rotary evaporation under reduced pressure to give 0.96 g (1.32 mmol, 87% yield) of the intermediate 1-O-DMT-3-N-[N-(fluorenylmethoxycarbonyl)-hexyl]-(S)-amino-1,2-propanediol (37) as a white solid. A single spot on TLC analysis confirmed the purity of the material.

[0265] (d) 1-O-DMT-3-N-[N-(fluorenylmethoxycarbonyl)-hexyl]-(S)-3-amino-1,2-propanediol (37) was converted to N-(fluorenylmethoxycarbonylamino)hexanoylamino-O 1 -DMT-O 2 -Cyanoethoxydiisopropylphosphinyl-(S)-3-amino-1,2-propanediol (38).

[0266] Material : Materials are described in the preceding Examples, supra.

[0267] step : The synthesis was carried out as follows, similar to the description in Example 3(c)(d), and as Figure 9cAs shown. 1-O-DMT-3-N-[N-(fluorenylmethoxycarbonyl)-hexyl]-(S)-3-amino-1,2-propanediol (37) (0.84 g, 1.15 mmol) was dried by co-evaporation with anhydrous pyridine (2×6 mL). It was then dissolved in anhydrous dichloromethane (6 mL) and N,N-diisopropylethylamine (0.8 mL, 4.59 mmol) was added with stirring at ambient temperature. Subsequently, 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (0.385 mL, 1.72 mmol) was added dropwise under stirring over 5 minutes under an argon atmosphere. The reaction was found to be complete after 20 minutes by silica gel thin layer chromatography using a dichloromethane / ethyl acetate / triethylamine (10:5:0.5) solvent system. The reaction mixture was then diluted into ethyl acetate and washed with saturated aqueous sodium bicarbonate solution (2×25 mL). After drying over anhydrous magnesium sulfate, the ethyl acetate layer was concentrated to dryness by rotary evaporation under reduced pressure. The residue was redissolved in ethyl acetate (3 mL) and poured into hexane (150 mL) at -25°C. The precipitate was filtered and dried under vacuum for 5 hours to give 0.882 g (0.950 mmol, 82% yield) of the desired product, N-(fluorenylmethoxycarbonylamino)hexanoylamino-O-(4-fluorophenyl)-1-methyl-1-oxo-1-yl)-1-oxo ... 1 -DMT-O 2 -cyanoethoxydiisopropylphosphinyl-(S)-3-amino-1,2-propanediol (38) is a white foam. 1 H-NMR (benzene-d6) analysis was confirmed: (ppm) 1.08-1.17 (m, 20H), 1.47-1.54 (m, 2H), 1.71-1.74 (m, 1H), 1.84-1.87 (m, 2H), 2.91-2.95 (m, 1H), 3.32, 3.34 (2s, 6H), 3.40-3.50 (m, 4H), 3.66 -3.75(m,2H),4.04(q,1H),4.45-4.48(m,2H),5.48(t,0.5H),5.75(t,0.5H),6.79( t,4H),7.08(t,1H),7.19-7.24(m,6H),7.48-7.52(m,6H),7.60(d,2H),7.67(t,2H).

[0268] Example 10(B): Synthesis of N-(fluorenylmethoxycarbonylamino)hexanoylamino-O-DMT-O 2 -Cyanoethoxydiisopropylphosphinyl-(R)-3-amino-1,2-propanediol (39)

[0269] N-(Fluorenylmethoxycarbonylamino)hexanoylamino-O 1 -DMT-O 2-cyanoethoxydiisopropylphosphinyl-(R)-3-amino-1,2-propanediol (39), such as Figure 10 The synthesis was performed essentially the same as reagent (38) except that (R)-3-amino-1,2-propanediol (Sigma-Aldrich) was used instead of (S)-3-amino-1,2-propanediol. Only the results are shown below.

[0270] (b) N-Fmoc-6-aminohexanoic acid (28) was coupled with (R)-3-amino-1,2-propanediol to form compound (40).

[0271] Material : (R)-3-Amino-1,2-propanediol was from Sigma-Aldrich. Other materials are described in the previous examples, supra.

[0272] result The reaction produced 1.3 g (3.05 mmol, 83%) of compound (40) as a white solid with a purity of 98.3% by LCMS analysis and an m / z of 427.3 (M+1).

[0273] (c) 1-O-Dimethoxytritylation of compound (40) to form 1-O-DMT-3-N-[N-(fluorenylmethoxycarbonyl)-hexyl]-(R)-amino-1,2-propanediol (41).

[0274] Material : Materials are described in the preceding Examples, supra.

[0275] result The reaction produced 1.01 g (1.38 mmol, 90%) of the intermediate 1-O-DMT-3-N-[N-(fluorenylmethoxycarbonyl)-hexyl]-(R)-amino-1,2-propanediol (41) as a white solid. TLC analysis confirmed the purity of the material.

[0276] (d) 1-O-DMT-3-N-[N-(fluorenylmethoxycarbonyl)-hexyl]-(R)-3-amino-1,2-propanediol (41) was converted to N-(fluorenylmethoxycarbonylamino)hexanoylamino-O 1 -DMT-O 2 -Cyanoethoxydiisopropylphosphinyl-(R)-3-amino-1,2-propanediol (39).

[0277] Material : Materials are described in the preceding Examples, supra.

[0278] resultThe reaction produced 1.00 g (1.08 mmol, 96%) of the desired product N-(fluorenylmethoxycarbonylamino)hexanoylamino-O 1 -DMT-O 2 -cyanoethoxydiisopropylphosphinyl-(R)-3-amino-1,2-propanediol (39) is a white foam. 1 H-NMR analysis (benzene-d6) was confirmed: (ppm) 1.03 (d, 4H), 1.10-1.15 (m, 10H), 1.15 (t, 6H), 1.50-1.54 (m, 2H), 1.69-1.74 (m, 1H), 1.85-1.88 (m, 2H), 2.92-2.96 (m, 1H), 3.33, 3.34 (2s, 6H), 3.43-3.49 (m,4H),3.67-3.73(m,2H),4.05(q,1H),4.45-4.48(m,2H),5.52(t,0.5H),5.78(t,0.5H), 6.79(t,4H),7.07(t,1H),7.19-7.25(m,6H),7.48-7.52(m,6H),7.60(d,2H),7.67(t,2H).

[0279] Example 11: Shorter and Longer N-(Fluorenylmethoxycarbonylamino)alkylamido-O 1 -DMT-O 2 Synthesis of 3-Cyanoethoxydiisopropylphosphinyl-1,2-propanediol

[0280] Example 11(A): Shorter and Longer N-(Fluorenylmethoxycarbonylamino)alkylamido-O 1 -DMT-O 2 Synthesis of cyanoethoxydiisopropylphosphinyl-(S)-3-amino-1,2-propanediol (40, 41, 42, 43)

[0281] N-(fluorenylmethoxycarbonylamino)alkylamido-O with shorter and longer alkyl groups 1 -DMT-O 2 -Cyanoethoxydiisopropylphosphinyl-(S)-3-amino-1,2-propanediol (40, 41, 42, 43) Figure 11 They were synthesized essentially as reagent (38) in Example 10(A), except that 4-aminobutyric acid, 5-aminopentanoic acid, 7-aminoheptanoic acid, or 8-aminooctanoic acid (Sigma-Aldrich) was used instead of 6-aminohexanoic acid. As can be seen from this example, it will be appreciated by those skilled in the art that products with shorter or longer alkyl groups can be readily synthesized.

[0282] Example 11(B): Shorter and Longer N-(Fluorenylmethoxycarbonylamino)alkylamido-O 1 -DMT-O 2 Synthesis of cyanoethoxydiisopropylphosphinyl-(R)-3-amino-1,2-propanediol (44, 45, 46, 47)

[0283] N-(fluorenylmethoxycarbonylamino)alkylamido-O with shorter and longer alkyl groups 1 -DMT-O 2 -Cyanoethoxydiisopropylphosphinyl-(R)-3-amino-1,2-propanediol (44, 45, 46, 47) Figure 11 They were synthesized essentially as reagent (39) in Example 10(B), except that 4-aminobutyric acid, 5-aminopentanoic acid, 7-aminoheptanoic acid, or 8-aminooctanoic acid (Sigma-Aldrich) was used instead of 6-aminohexanoic acid. As can be seen from this example, it will be appreciated by those skilled in the art that products with shorter or longer alkyl groups can be readily synthesized.

[0284] Example 12: N-Fmoc-O 1 -DMT-O 2 Synthesis of 3-amino-1,2-propanediol by diisopropylamino-cyanoethoxyphosphinyl

[0285] Example 12(A): N-Fmoc-O 1 -DMT-O 2 Synthesis of -cyanoethoxydiisopropylaminophosphinyl-(S)-3-amino-1,2-propanediol (48)

[0286] N-Fmoc-O 1 -DMT-O 2 -Cyanoethoxydiisopropylaminophosphinyl-(S)-3-amino-1,2-propanediol (48) Figure 12 It was synthesized essentially as described by PS Nelson et al. (Nucleic Acids Res., vol. 17, p. 7179, 1989), except that (S)-3-amino-1,2-propanediol (Sigma-Aldrich) was used instead of (±)-3-amino-1,2-propanediol.

[0287] Example 12(B): N-Fmoc-O 1 -DMT-O 2 Synthesis of cyanoethoxydiisopropylaminophosphinyl-(R)-3-amino-1,2-propanediol (49)

[0288] N-Fmoc-O 1 -DMT-O 2 -cyanoethoxydiisopropylaminophosphinyl-(R)-3-amino-1,2-propanediol (49) is shown in Figure 12 It was synthesized essentially as described by PS Nelson et al. (Nucleic Acids Res., vol. 17, p. 7179, 1989), except that (R)-3-amino-1,2-propanediol (Sigma-Aldrich) was used instead of (±)-3-amino-1,2-propanediol.

[0289] Example 13: N-Fmoc-O 1 -DMT-O 3 Synthesis of 2-(aminomethyl)-1,3-propanediol by diisopropylamino-2-cyanoethoxyphosphinyl

[0290] Example 13(A): N-Fmoc-O 1 -DMT-O 3 Synthesis of 2-(aminomethyl)-1,3-propanediol (50)

[0291] N-Fmoc-O 1 -DMT-O 3 -cyanoethoxydiisopropylaminophosphinyl-(S)-2-(aminomethyl)-1,3-propanediol (50) is shown in Figure 13 , which was synthesized essentially as described in Example 12, except that 2-(aminomethyl)-1,3-propanediol (Sigma-Aldrich) was used instead of (±)-3-amino-1,2-propanediol. However, there was an additional step during the synthesis where the hydroxyl group was protected with a dimethoxytrityl group resulting in chirality of carbon 2 and the intermediate becoming N-Fmoc-O 1 -DMT-(±)-2-(aminomethyl)-1,3-propanediol racemic mixture. The enantiomers of this mixture are chromatographed on an alkyl, aryl or chiral phase column or layer medium. 1 -DMT-(R)-2-(aminomethyl)-1,3-propanediol isomer and used in the final synthetic step to obtain N-Fmoc-O 1 -DMT-O 2 -cyanoethoxydiisopropylaminophosphinyl-(S)-2-(aminomethyl)-1,3-propanediol (50). Alternatively, N-Fmoc-O 1The racemic mixture of -DMT-(±)-2-(aminomethyl)-1,3-propanediol was not separated into its enantiomers. Instead, the mixture was used in the final step to synthesize the diastereoisomer N-Fmoc-O 1 -DMT-O 3 From this mixture, N-Fmoc-O is chromatographed on an alkyl, aryl or chiral column or layer medium. 1 -DMT-O 3 -cyanoethoxydiisopropylaminophosphinyl-(S)-2-(aminomethyl)-1,3-propanediol (50) was reacted and collected as the product.

[0292] Example 13(B): N-Fmoc-O 1 -DMT-O 3 Synthesis of cyanoethoxydiisopropylaminophosphinyl-(R)-2-(aminomethyl)-1,3-propanediol (51)

[0293] N-Fmoc-O 1 -DMT-O 3 -cyanoethoxydiisopropylaminophosphinyl-(R)-2-(aminomethyl)-1,3-propanediol (51) is shown in Figure 13 and was synthesized essentially as described in Example 12, except that 2-(aminomethyl)-1,3-propanediol (Sigma-Aldrich) was used instead of (±)-3-amino-1,2-propanediol. The same additional steps as in Example 13(A) or alternative steps were used in this example, except that in this example, N-Fmoc-O 1 -DMT-O 3 -cyanoethoxydiisopropylaminophosphinyl-(R)-2-(aminomethyl)-1,3-propanediol (51) as the product.

[0294] Example 14: N-Fmoc-O 1 -DMT-O 3 Synthesis of 2-aminobutyl-1,3-propanediol by diisopropylaminophosphinyl-cyanoethoxy

[0295] Example 14(A): N-Fmoc-O 1 -DMT-O 3 Synthesis of -cyanoethoxydiisopropylaminophosphinyl-(S)-2-(4-aminobutyl)-1,3-propanediol (52)

[0296] N-Fmoc-O 1 -DMT-O 3-cyanoethoxydiisopropylaminophosphinyl-(S)-2-(4-aminobutyl)-1,3-propanediol (52) is shown in Figure 14 , and it was synthesized essentially as described by PS Nelson et al. (Nucleic Acids Res., Vol. 20, p. 6253, 1992). However, there is an additional step in the synthesis whereby the hydroxyl group is protected with a dimethoxytrityl group resulting in chirality of carbon 2 and the intermediate becoming N-Fmoc-O 1 -DMT-(±)-2-(4-aminobutyl)-1,3-propanediol racemic mixture. The enantiomers of this mixture are chromatographed on an alkyl, aryl or chiral phase column or layer medium. 1 -DMT-(R)-2-(4-aminobutyl)-1,3-propanediol isomer and used in the final synthetic step to obtain N-Fmoc-O 1 -DMT-O 3 -cyanoethoxydiisopropylaminophosphinyl-(S)-2-(4-aminobutyl)-1,3-propanediol (52). Alternatively, N-Fmoc-O 1 The racemic mixture of -DMT-(±)-2-(4-aminobutyl)-1,3-propanediol was not separated into its enantiomers. Instead, the mixture was used in the final step to synthesize the diastereoisomer N-Fmoc-O 1 -DMT-O 3 -cyanoethoxydiisopropylaminophosphinyl-(±)-2-(4-aminobutyl)-1,3-propanediol mixture. From this mixture, N-Fmoc-O is chromatographed on an alkyl, aryl or chiral phase column or layer medium. 1 -DMT-O 2 -cyanoethoxydiisopropylaminophosphinyl-(S)-2-(4-aminobutyl)-1,3-propanediol (52) was reacted and collected as the product.

[0297] Example 14(B): N-Fmoc-O 1 -DMT-O 3 Synthesis of -cyanoethoxydiisopropylaminophosphinyl-(R)-2-(4-aminobutyl)-1,3-propanediol (53)

[0298] N-Fmoc-O 1 -DMT-O 3 -cyanoethoxydiisopropylaminophosphinyl-(R)-2-(4-aminobutyl)-1,3-propanediol (53) is shown in Figure 14In this example, the synthesis was performed essentially as described by PS Nelson et al. (Nucleic Acids Res., Vol. 20, p. 6253, 1992). The same additional steps as those in Example 14(A) or alternative steps were used, but in this example, N-Fmoc-O was collected. 1 -DMT-O 3 -cyanoethoxydiisopropylaminophosphinyl-(R)-2-(4-aminobutyl)-1,3-propanediol (53) as the product.

[0299] Example 15: N-(Fluorenylmethoxycarbonylamino)propionylamino-O 1 -DMT-O 3 Synthesis of 2-Cyanoethoxydiisopropylphosphinyl-2-amino-1,3-propanediol

[0300] Example 15(A): N-(Fluorenylmethoxycarbonylamino)propionylamino-O 1 -DMT-O 3 Synthesis of cyanoethoxydiisopropylphosphinyl-(S)-2-amino-1,3-propanediol (54)

[0301] N-(Fluorenylmethoxycarbonylamino)propionylamino-O 1 -DMT-O 3 -cyanoethoxydiisopropylphosphinyl-(S)-2-amino-1,3-propanediol (54) is shown in Figure 15 and was synthesized essentially as described in P.S. Nelson et al. (U.S. Patent No. 8,394,948, issued March 12, 2013). However, there is an additional step in which the hydroxyl group is protected with a dimethoxytrityl group, resulting in chirality of carbon 2 and the intermediate becoming N-(fluorenylmethoxycarbonylamino)propionylamino-O 1 -DMT-(±)-2-amino-1,3-propanediol racemic mixture. The enantiomers of the mixture were chromatographed on an alkyl, aryl or chiral phase column or layer medium. The N-(fluorenylmethoxycarbonylamino)propionylamino-O 1 -DMT-(R)-2-amino-1,3-propanediol isomer and used in the final synthetic step to give N-(fluorenylmethoxycarbonylamino)propionylamino-O 1 -DMT-O 3 -cyanoethoxydiisopropylphosphinyl-(S)-2-amino-1,3-propanediol (54). Alternatively, N-(fluorenylmethoxycarbonylamino)propionylamino-O 1The racemic mixture of -DMT-(±)-2-amino-1,3-propanediol was not separated into its enantiomers. Instead, the mixture was used in the final step to synthesize the diastereomeric N-(fluorenylmethoxycarbonylamino)propionylamino-O 1 -DMT-O 3 From this mixture, N-(fluorenylmethoxycarbonylamino)propionylamino-O-(fluorenylmethoxycarbonylamino)propionylamino) ... 1 -DMT-O 3 -cyanoethoxydiisopropylphosphinyl-(S)-2-amino-1,3-propanediol (54) and collected as the product.

[0302] Example 15(B): N-(Fluorenylmethoxycarbonylamino)propionylamino-O 1 -DMT-O 3 Synthesis of cyanoethoxydiisopropylphosphinyl-(R)-2-amino-1,3-propanediol (55)

[0303] N-(Fluorenylmethoxycarbonylamino)propionylamino-O 1 -DMT-O 3 -cyanoethoxydiisopropylphosphinyl-(R)-2-amino-1,3-propanediol (55) is shown in Figure 15 and synthesized essentially as described in P.S. Nelson et al. (U.S. Patent No. 8,394,948, issued March 12, 2013). In this example, the same additional steps or alternative steps as in Example 15(A) were used, but in this example, N-(fluorenylmethoxycarbonylamino)propionylamino-O 1 -DMT-O 3 -cyanoethoxydiisopropylphosphinyl-(R)-2-amino-1,3-propanediol (55) as the product.

[0304] Example 16: N-(Fluorenylmethoxycarbonylamino)hexanoylamino-O 1 -DMT-O 2 Automated ligation of cyanoethoxydiisopropylphosphinyl-3-amino-1,2-propanediol linkers (38, 39) to oligonucleotides

[0305] The ligation of the linker reagents described in Example 10 to various synthetic oligonucleotides to yield 5'-3' 3-amino-1,2-propanediol linkers will now be described.

[0306] (a) The linker reagent described in Example 10(A), reagent (38), hereinafter referred to as "L9," was inserted between the bases of the synthetic oligonucleotide using standard 3'-β-cyanoethylphosphoramidite chemistry to produce the sequence "5'-GATCTGAGCGTCG(L9)ACGTCGTGACATG-3'" (SEQ ID NO 7). The method used was similar to that described in Example 4(a), except that a solution of "L9" (0.1 M in acetonitrile) was reacted in the thirteenth coupling cycle, rather than the last coupling cycle as described in Example 4(a).

[0307] (b) "L9" was introduced between the bases of the synthetic oligonucleotide using standard 3'-β-cyanoethylphosphoramidite chemistry, generating the sequence "5'-GATCTGAGCGTCA(L9)GCGTCGTGACATG-3'" (SEQ ID NO 8). The method used was similar to that described in Example 16(a).

[0308] (c) Using standard 3'-β-cyanoethylphosphoramidite chemistry, the linker reagent described in Example 10(B), reagent (39), hereinafter referred to as "L10", was inserted between the bases of the synthetic oligonucleotide to produce the sequence "5'-GATCTGAGCGTCG(L10)ACGTCGTGACATG-3'" (SEQ ID NO 7), a diastereomer of the sequence in Example 16(a). The method used was similar to that described in Example 16(a), except that "L10" was used instead of "L9". The structure near the linker is as shown in FIG. Figure 16 As shown in (56).

[0309] (d) "L10" was introduced between the bases of the synthetic oligonucleotide using standard 3'-β-cyanoethylphosphoramidite chemistry, generating the sequence "5'-GATCTGAGCGTCA(L10)GCGTCGTGACATG-3'" (SEQ ID NO 8), an epimer of the sequence in Example 16(b). The method used was similar to that described in Example 16(c).

[0310] Example 17: N-(Fluorenylmethoxycarbonylamino)hexanoylamino-O 1 -DMT-O 2 Automated ligation of cyanoethoxydiisopropylphosphinyl-3-amino-1,2-propanediol linkers (38, 39) to oligonucleotides

[0311] The ligation of the linker reagents described in Example 10 to various synthetic oligonucleotides to produce 5' to 3' 2'-amino-1,2-propanediol linkers will now be described.

[0312] (a) Introduction of the linker reagent "L9" was inserted between the bases of the synthetic oligonucleotide using 5'-β-cyanoethyl phosphoramidite chemistry, resulting in the sequence "5'-GATCTGAGCGTCG(L9)ACGTCGTGACATG-3'" (SEQ ID NO 7). The method used was similar to that described in Example 16(a), except that 5'-β-cyanoethyl phosphoramidite was used instead of 3'-β-cyanoethyl phosphoramidite.

[0313] (b) "L9" was introduced between the bases of the synthetic oligonucleotide using 5'-β-cyanoethylphosphoramidite chemistry, generating the sequence "5'-GATCTGAGCGTCA(L9)GCGTCGTGACATG-3'" (SEQ ID NO 8). The method used was similar to that described in Example 17(a).

[0314] (c) Introduction of the linker reagent "L10" was inserted between the bases of the synthetic oligonucleotide using 5'-β-cyanoethyl phosphoramidite chemistry to produce the sequence "5'-GATCTGAGCGTCG(L10)ACGTCGTGACATG-3'" (SEQ ID NO 7), an epimer of the sequence in Example 17(a). The method used was similar to that described in Example 17(a), except that the protocol for "L10" was used instead of "L9". The structure near the linker is shown in FIG. Figure 16 As shown in (57).

[0315] (d) "L10" was introduced between the bases of the synthetic oligonucleotide using 5'-β-cyanoethylphosphoramidite chemistry, generating the sequence "5'-GATCTGAGCGTCA(L10)GCGTCGTGACATG-3'" (SEQ ID NO 8), an epimer of the sequence in Example 17(b). The method used was similar to that described in Example 17(c).

[0316] Example 18: Labeling of amine linker arm probes with acridinium esters and purification

[0317] A 25 mM stock solution of acridinium ester N-hydroxysuccinimide labeling reagent (for composition, see I. Weeks et al., Clin. Chem., Vol. 29, p. 1474, 1983) was prepared in anhydrous dimethyl sulfoxide (AMRESCO, Solon, OH, USA). The polymer produced in Example 16 was purified and desalted by anion exchange HPLC. The polymer was concentrated by precipitation in a 1.5 mL conical polypropylene tube as follows: 1-10 nmol of polymer was combined with water to 85 μL, 5 μL of 40 mg / mL glycogen (Affymetrix, Santa Clara, CA, USA), and 10 μL of 2 M sodium chloride (Alfa Aesar, Ward Hill, MA, USA), vortexed, and 250 μL of cold ethanol (Electron Microscopy Sciences, Hatfield, PA, USA) was added, vortexed, and incubated overnight at 4°C; centrifuged at 12,000 × g in a microcentrifuge for 30 minutes at room temperature, the supernatant was removed, 500 μL of cold 70% ethanol was added, the supernatant was removed, and the tube was air-dried at room temperature for 1-2 hours.

[0318] The following labeling compositions were constructed by adding the components to the polymer pellet and processing in the order listed: 8 μL of 50% dimethyl sulfoxide in 0.125 M 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (pH 8.0; AMRESCO), vortexed, 2 μL of 25 mM acridinium ester N-hydroxysuccinimide labeling reagent (Toronto Research Chemicals, Toronto, Ontario, Canada) in dimethyl sulfoxide, vortexed, spun in a microcentrifuge for 2 seconds (to bring the contents to the bottom of the tube), incubated at 37° C. for 20 minutes, 3 μL of 25 mM acridinium ester N-hydroxysuccinimide labeling reagent in dimethyl sulfoxide, vortexed, 2 μL of 0.25 M 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (pH 8.0; AMRESCO), vortexed, 8.0), vortex to mix, and incubate at 37°C for 20 minutes.

[0319] The labeled polymer was separated from unreacted acridinium ester label in 15 μL of reaction as follows: 15 μL of reaction, 75 μL of water, and 10 μL of 2 M sodium chloride were mixed, vortexed, 250 μL of cold ethanol was added, vortexed, and incubated at 20°C for 60 minutes; centrifuged at 12,000 × g for 10 minutes at room temperature in a microcentrifuge, the supernatant was removed, 500 μL of cold 70% ethanol was added, the supernatant was removed, and the pellet was air-dried at room temperature for 1-2 hours; 50 μL of probe reagent (10 mM succinic acid (TCI America, Portland, OR, USA), 0.1% lithium dodecyl sulfate (Chem-Impex, Wood Dale, IL, USA), pH 5.0) was added, vortexed, incubated at room temperature for 30 minutes in the dark, mixed, and stored at -20°C.

[0320] Regardless of the purification method used, the acridinium ester-labeled polymers were quantified for nucleic acid content and specific luminescent activity according to N.C. Nelson et al. (Detection of Acridinium Esters by Chemiluminescence in "Nonisotopic Probing, Blotting, and Sequencing," Kricka, L.J., (Ed.), pp. 391-428, 1995, Academic Press).

[0321] (b) Alternatively, unlabeled polymer was concentrated by precipitation in a 1.5 mL conical polypropylene tube as follows: 1-10 nmol of polymer was combined with water to 40 μL-100 μL, glycogen to approximately 2 mg / mL, and sodium chloride to approximately 0.2 M, vortexed, 2.5 times more cold ethanol than the aqueous phase was added, vortexed, and incubated at 4°C overnight or at -20°C for 60-120 minutes; centrifuged at 12,000 × g in a microcentrifuge at room temperature for 30 minutes, removed the supernatant, added approximately 500 μL of cold 70% ethanol, removed the supernatant, and air-dried at room temperature for 1-2 hours.

[0322] (c) Alternatively, the labeled polymer is separated from the unreacted acridinium ester label by precipitation in a 1.5 mL conical polypropylene tube as follows: combine the labeling reactions, add water to 40 μL-100 μL, sodium chloride to approximately 0.2 M, vortex to mix, add 2.5 times more cold ethanol than the water / dimethyl sulfoxide fraction, vortex to mix, and incubate at -20°C for 60-120 minutes; centrifuge at 12,000×g in a microcentrifuge at room temperature for 10 minutes, remove the supernatant, add 200-500 μL of cold 70% ethanol, remove the supernatant, and air-dry the pellet at room temperature for 1-2 hours; add 20-50 μL of probe reagent (approximately 10 mM succinic acid, approximately 0.1% lithium dodecyl sulfate, approximately pH 5.0), vortex to mix, incubate at room temperature in the dark for 30 minutes, mix, and store at -20°C.

[0323] In the case of further purification of the acridinium ester-labeled polymer, the method was the same as described in Example 9.

[0324] Example 19: Hybridization of Acridinium Ester-labeled Polymers with Nucleic Acids

[0325] Acridinium ester-labeled polymers were combined with nucleic acids in a solution containing sequences complementary to the polymer (complete matching), sequences complementary to the polymer except one position (single nucleotide mismatches), and sequences substantially non-complementary to the polymer. Furthermore, acridinium ester-labeled polymers were treated in the same manner, except that other nucleic acid sequences were not present. The materials identical to Example 18 were from the same source.

[0326] (a) The hybridization reagents for these binding reactions consisted of buffers, salts, metal ion chelators, and surfactants: 0.1 M succinic acid, 0.4 M lithium chloride (AMRESCO), 0.001 M ethylenediaminetetraacetic acid (Alfa Aesar), 0.001 M ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (JTBaker), and 5% lithium dodecyl sulfate, with the pH adjusted to 4.9 with lithium hydroxide (Acros Organics, NJ, USA).

[0327] (b) Hybridization reaction was performed as follows. 1 pmol of acridinium ester-labeled polymer and 5 pmol of a matching or mismatched nucleic acid sequence, or no additional nucleic acid sequence, were mixed in a total volume of 200 μL using the final concentration of the hybridization reagent described in Example 19(a). The mixture was incubated at 60°C for 15 minutes and then at room temperature for 15 minutes to form a hybridization product.

[0328] (c) Alternative formulations of hybridization reagents include buffers, salts, metal ion chelators, and surfactants in the following concentration ranges: 0.02-0.40 M succinic acid, 0.05-0.8 M lithium chloride, 0-0.02 M ethylenediaminetetraacetic acid, 0-0.02 M ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid, and 1-10% lithium dodecyl sulfate, with the pH adjusted to 4.5-5.5 with lithium hydroxide.

[0329] (d) Other alternative formulations of hybridization reagents include buffers other than succinic acid (e.g., citric acid or acetic acid), anions other than chloride (e.g., bromide or sulfate), and cations other than lithium (e.g., sodium or potassium). The concentration ranges of these components are as shown in Example 19(c).

[0330] (e) Alternative hybridization steps are used. These steps use approximately 0.001 to 10 pmol of acridinium ester-labeled polymer and approximately 1 amol to 100 pmol of matched or mismatched nucleic acid sequence. Volumes range from approximately 10 μL to approximately 1,000 μL. Formulations are as described in Examples 19(a), 19(c), and 19(d). These mixtures are incubated at approximately 30-70°C for approximately 5-60 minutes, or even up to approximately 16-24 hours, and then at room temperature for approximately 1-60 minutes to form hybridization products.

[0331] (f) Other alternative steps for hybridization: The mixture is incubated at about 30-70°C for about 5-60 minutes and then cooled at a controlled rate of about 0.1-5°C / min until it reaches a predetermined temperature to form hybridization products.

[0332] Example 20: Controlled Hydrolysis of Acridinium Ester Probes

[0333] Modification of the acridinium ester attached to the polymer by hydrolysis produces acridinium carboxylic acid that is non-chemiluminescent. Controlled hydrolysis of the acridinium ester probe is based on the observation that acridinium ester-labeled polymers that are not bound to other nucleic acids or that are bound to partially mismatched nucleic acids have a greater degree of modification than those bound to nucleic acids that are perfectly matched near the linker. After a short time interval, the degree of modification is measured to determine the relative effectiveness of the reaction. A greater degree of modification is evidenced by a lower chemiluminescent signal, while a lesser degree of modification is evidenced by a higher chemiluminescent signal.

[0334] (a) As described in Examples 19(a) and 19(b), acridinium ester-labeled polymers were bound to perfectly matched or mismatched nucleic acids (or similarly reacted but without other nucleic acids). These solutions were diluted 10-fold, mixed, and then 25 μL aliquots were dispensed into 12×75 mm polystyrene tubes. Controlled hydrolysis of the acridinium ester probe was performed by adding 250 μL of an alkaline hydrolysis reagent {0.15 M sodium tetraborate (Alfa Aesar), pH 8.5, 5% (v / v) Triton X-100 (IBI Scientific, Peosta, IA, USA)} to each tube, vortexing the tubes and incubating at 60°C for various times, up to 15 minutes. The tubes were then cooled in an ice-water bath for 1 minute and in a room temperature water bath for 1 minute. The outside of each tube was wiped with a damp cloth and then inserted into a photometer (e.g., Leader 50). The chemiluminescence of each aliquot was then measured by automatically injecting 200 μL of the first initiation reagent {0.0044% (v / v) aqueous nitric acid (EMD, Billerica, MA, USA) and 0.1% (v / v) hydrogen peroxide (EMD)}, followed by injecting 200 μL of the second initiation reagent {4% (w / v) aqueous sodium hydroxide (AMRESCO)} after a 2-second delay and reading the chemiluminescence for 2 seconds. Under these conditions, after approximately 3 minutes, the decline in signal transitioned to a nonlinear or second linear phase that declined at a slower rate.

[0335] (b) Controlled hydrolysis of acridinium ester probes was performed as in Example 20(a), except that 150 to 500 μL of the hydrolysis reagent was added to each tube.

[0336] (c) Controlled hydrolysis of the acridinium ester probe was performed as in Example 20(a), except that the hydrolysis reagent consisted of 0.05-0.5 M sodium tetraborate, pH 7.5-9.1, 0.5-5% Triton X-100.

[0337] (d) Controlled hydrolysis of the acridinium ester probe was performed as in Example 20(a), except that the tube containing the reactants was incubated at a temperature of about 40°C to about 70°C for a time of about 0.5 minutes to about 100 minutes.

[0338] (e) Controlled hydrolysis of the acridinium ester probe was performed as in Example 20(a), except that after incubation at elevated temperature, the tubes containing the reaction were cooled by incubation at room temperature for about 1-15 minutes, in a room temperature water bath for about 1-15 minutes, or in a cold water bath (below room temperature but above 0° C.) for about 1-15 minutes. Alternatively, controlled hydrolysis of the acridinium ester probe was performed as in Example 20(a), except that after incubation at elevated temperature, the tubes were read without cooling.

[0339] (f) Controlled hydrolysis of the acridinium ester probe is performed as in Example 20(a), except that the chemiluminescence of each aliquot is then measured by automatically injecting about 50-400 μL of about 0.0001-0.01% (v / v) aqueous nitric acid and about 0.02-2% (v / v) hydrogen peroxide, followed by about 50-400 μL of 1-8% (w / v) aqueous sodium hydroxide.

[0340] (g) Controlled hydrolysis of the acridinium ester probe is performed as in Example 20(a), except that the chemiluminescence is collected after a delay of about 0-100 seconds between the injection of the first and second initiating reagents, and the chemiluminescence is collected for about 1-10 seconds.

[0341] (h) Controlled hydrolysis of the acridinium ester probe was performed as in Example 20(a), except that the chemiluminescence was collected in a luminometer other than a Leader50, such as a LeaderI, a Leader450, a LeaderHC, a LeaderHC+, an Optocomp I (MGM Instruments), an Optocomp II, a Lumat 3 LB 9508 single tube photometer (Berthold Technologies, Oak Ridge, TN, USA) or others.

[0342] (i) Controlled hydrolysis of the acridinium ester probe is performed as in Example 20(a), except that the reactions are in the wells of a microtiter plate (e.g., 96-well) and the chemiluminescence is collected in a plate luminometer, such as a PHERAstar reader (BMG Labtech, Cary, NC, USA), a MicroLumatPlus reader (Berthold Technologies), a GloMax Microplate reader (Promega, Madison, WI, USA), or others.

[0343] (j) As described in Examples 19(a) and 19(b), acridinium ester-labeled polymers were bound to perfectly matched or mismatched nucleic acids (or reacted similarly but without other nucleic acids). These solutions were diluted 10-fold, mixed, and 100 μL aliquots were dispensed into 12×75 mm polystyrene tubes. Controlled hydrolysis of the acridinium ester probe was performed by adding 250 μL of an alkaline hydrolysis reagent (0.15 M sodium tetraborate, pH 8.5, 5% (v / v) Triton X-100) to each tube, vortexing the tubes and incubating at 60°C for various times, up to 15 minutes. The tubes were then cooled in an ice-water bath for 1 minute and in a room temperature water bath for 1 minute. The outside of each tube was wiped with a damp cloth and then inserted into a photometer (e.g., Leader 50). The chemiluminescence of each aliquot was then measured by automatically injecting 200 μL of a first initiation reagent (0.0044% (v / v) aqueous nitric acid and 0.1% (v / v) hydrogen peroxide) and then, after a 2-second delay, injecting 200 μL of a second initiation reagent (4% (w / v) aqueous sodium hydroxide) and reading the chemiluminescence for 2 seconds. Under these conditions, after approximately 3 minutes, the decline in the signal transitioned to a nonlinear or second, linear phase that declined at a slower rate.

[0344] Example 21: N-(Fluorenylmethoxycarbonylamino)hexanoylamino-O 1 -Cyanoethoxydiisopropylphosphinyl-O 2 Synthesis of -DMT-(±)-3-amino-1,2-propanediol

[0345] Example 21(A): N-(Fluorenylmethoxycarbonylamino)hexanoylamino-O 1 -Cyanoethoxydiisopropylphosphinyl-O 2 Synthesis of DMT-(S)-3-amino-1,2-propanediol (58)

[0346] N-(Fluorenylmethoxycarbonylamino)hexanoylamino-O 1 -Cyanoethoxydiisopropylphosphinyl-O 2 -DMT-(S)-3-amino-1,2-propanediol (58) Figure 17As shown, it is synthesized essentially as reagent (38), except that after amine protection and coupling of N-Fmoc-6-aminocaproic acid with (S)-3-amino-1,2-propanediol, the primary oxygen is protected with tert-butyldimethylsilyl chloride, the secondary oxygen is protected with 4,4'-dimethoxytriphenylmethyl chloride, the tert-butyldimethylsilyl group is removed with fluoride, and the primary oxygen is activated with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite. Silylation and desilylation are performed by the general procedures of TW Green and PGM Wuts (New York, Wiley-Interscience Press, Protective Groups in Organic Synthesis, Wiley-Interscience, New York, pp. 127-141 and 708-711, 1999). One of ordinary skill in the art can devise alternatives to selectively protect primary and secondary alcohols with the desired groups to produce reagent 58. Only the different steps are described.

[0347] (c1) 1-O-tert-butyldimethylsilylation of compound (36) to form 1-O-(tert-butyldimethylsilyl)-3-N-[N-(fluorenylmethoxycarbonyl)-hexyl]-(S)-amino-1,2-propanediol.

[0348] Material : tert-Butyldimethylsilyl chloride, imidazole and dimethylformamide were from Sigma-Aldrich. Other materials are described in previous examples, supra.

[0349] step To compound (36) dissolved in dimethylformamide were added 2.5 equivalents of imidazole and 1.2 equivalents of tert-butyldimethylsilyl chloride. The solution was mixed at room temperature for 30 minutes to produce 1-O-(tert-butyldimethylsilyl)-3-N-[N-(fluorenylmethoxycarbonyl)-hexyl]-(S)-amino-1,2-propanediol.

[0350] (c2) 2-O-Dimethoxytritylation of 1-O-(tert-butyldimethylsilyl)-3-N-[N-(fluorenylmethoxycarbonyl)-hexyl]-(S)-amino-1,2-propanediol to form 1-O-(tert-butyldimethylsilyl)-2-O-DMT-3-N-[N-(fluorenylmethoxycarbonyl)-hexyl]-(S)-amino-1,2-propanediol.

[0351] Material : Materials are described in the preceding Examples, supra.

[0352] step: This reaction is essentially the same as Example 10(A)(c), except that the 2-hydroxyl group of 1,2-propylene glycol is dimethoxytritylated.

[0353] (c3) Selective Removal of the tert-butyldimethylsilyl Protecting Group from 1-O-(tert-butyldimethylsilyl)-2-O-DMT-3-N-[N-(fluorenylmethoxycarbonyl)-hexyl]-(S)-amino-1,2-propanediol.

[0354] Material Tetrabutylammonium fluoride, tetrahydrofuran, and dipotassium phosphate were from Sigma-Aldrich. Other materials are described in previous examples, supra.

[0355] step 1-O-(tert-Butyldimethylsilyl)-2-O-DMT-3-N-[N-(fluorenylmethoxycarbonyl)-hexyl]-(S)-amino-1,2-propanediol was dissolved in tetrahydrofuran and 0.1 M aqueous potassium phosphate solution, pH 7.1. Tetrabutylammonium fluoride (0.5 equivalents) was added and mixed with the solution at room temperature for 30 minutes to provide 2-O-DMT-3-N-[N-(fluorenylmethoxycarbonyl)-hexyl]-(S)-3-amino-1,2-propanediol.

[0356] (d) Conversion of 2-O-DMT-3-N-[N-(fluorenylmethoxycarbonyl)-hexyl]-(S)-3-amino-1,2-propanediol to N-(fluorenylmethoxycarbonylamino)hexanoylamino-O 1 -Cyanoethoxydiisopropylphosphinyl-O 2 -DMT-(S)-3-amino-1,2-propanediol (58).

[0357] Material : Materials are described in the preceding Examples, supra.

[0358] step The reaction was essentially the same as in Example 10(A)(d), except that the 1-hydroxyl group of 1,2-propylene glycol was phosphinylated.

[0359] Example 21(B): N-(Fluorenylmethoxycarbonylamino)hexanoylamino-O 1 -Cyanoethoxydiisopropylphosphinyl-O 2 Synthesis of DMT-(R)-3-amino-1,2-propanediol (59)

[0360] N-(Fluorenylmethoxycarbonylamino)hexanoylamino-O 1 -Cyanoethoxydiisopropylphosphinyl-O 2-DMT-(R)-3-amino-1,2-propanediol (59) Figure 17 As shown, it was synthesized essentially as reagent (58), except that a (R)-3-amino-1,2-propanediol precursor was used.

[0361] Example 22: N-(Fluorenylmethoxycarbonylamino)hexanoylamino-O 1 -DMT-O 2 Automated ligation of cyanoethoxydiisopropylphosphinyl-3-amino-1,2-propanediol linkers (58, 59) to oligonucleotides

[0362] Ligation of the linker reagent described in Example 21 to various synthetic oligonucleotides is described to produce a 5' to 3' 2'-amino-1,2-propanediol linker.

[0363] (a) The linker reagent described in Example 21(A), reagent (58), hereinafter referred to as "L11," was inserted between the bases of a synthetic oligonucleotide using 3'-β-cyanoethylphosphoramidite chemistry to produce the sequence "5'-GATCTGAGCGTCG(L11)ACGTCGTGACATG-3'" (SEQ ID NO 7). The method used was similar to that described in Example 16(a).

[0364] (b) "L11" was introduced between the bases of the synthetic oligonucleotide using 3'-β-cyanoethylphosphoramidite chemistry, generating the sequence "5'-GATCTGAGCGTCA(L11)GCGTCGTGACATG-3'" (SEQ ID NO 8). The method used was similar to that described in Example 22(a).

[0365] (c) The linker reagent described in Example 21(B)—reagent (59), hereinafter referred to as "L12"—was inserted between the bases of the synthetic oligonucleotide using 3'-β-cyanoethylphosphoramidite chemistry to produce the sequence "5'-GATCTGAGCGTCG(L12)ACGTCGTGACATG-3'" (SEQ ID NO 7), a diastereomer of the sequence in Example 22(a). The method used was similar to that described in Example 22(a), except that the protocol for "L12" was used instead of "L11." The structure near the linker is shown in FIG. Figure 16 As shown in (57).

[0366] (d) "L12" was introduced between the bases of the synthetic oligonucleotide using 3'-β-cyanoethylphosphoramidite chemistry to produce the sequence "5'-GATCTGAGCGTCA(L12)GCGTCGTGACATG-3'" (SEQ ID NO 8), an epimer of the sequence in Example 22(b). The method used was similar to that described in Example 22(c).

[0367] Example 23: Application of controlled hydrolysis of acridinium ester probes in specific sequences

[0368] Controlled hydrolysis of acridinium ester probes was performed as in Example 20(a) using oligonucleotides from Examples 16(a) and 16(c), all labeled with acridinium ester and purified as described in Example 18. 6 The relative light unit (RLU) signal starts and the decrease in chemiluminescence after 3 minutes of hydrolysis is as follows (only the nucleotides around the linker site are shown here):

[0369] Table 1

[0370]

[0371] Unexpectedly, the performance of the different enantiomeric linkers under hybridization conditions when hybridizing to a perfectly matched sequence between the 5'-GA-3' positions indicated that L10 was significantly more stable to the modification, as evidenced by an excess of >140,000 RLU for oligonucleotides composed of L10 compared to L9. The performance of the same enantiomeric linker under hybridization conditions when hybridizing to a mismatched sequence between the 5'-GA-3' positions indicated that L10 was less stable to the modification, as evidenced by an excess of >4,000 RLU for oligonucleotides composed of L9 compared to L10. Overall, the discrimination ratio (DR = RLU perfect match / RLU mismatch) at 3 minutes was much greater for the sequence with L10 (DR = 174) than for the same sequence with L9 (DR = 64).

[0372] (b) Controlled hydrolysis of acridinium ester probes was performed as in Example 20(a) using oligonucleotides from Examples 16(b) and 16(d), all labeled with acridinium ester and purified as described in Example 18. 6 The RLU signal starts and the chemiluminescence decreases after 3 minutes of hydrolysis as follows (only the nucleotides around the linker site are shown here):

[0373] Table 2

[0374]

[0375] Also unexpectedly, the performance of the different enantiomeric linkers under hybridization conditions when hybridizing to a perfectly matched sequence between the 5'-AG-3' positions indicated that L10 was significantly more stable to modification, as evidenced by an excess of >78,000 RLUs for oligonucleotides composed of L10 compared to L9. The performance of the same enantiomeric linkers under hybridization conditions when hybridizing to a mismatched sequence between the 5'-AG-3' positions indicated that L10 was more susceptible to modification, as evidenced by an excess of >9,000 RLUs for oligonucleotides composed of L9 compared to L10. Overall, the DR at 3 minutes was much greater for the sequence with L10 (DR = 82) than for the same sequence with L9 (DR = 36).

[0376] (c) Controlled hydrolysis of the acridinium ester probe was performed as in Example 20(j) using the oligonucleotide from Example 16(d), labeled with an acridinium ester and purified as described in Example 18. 6 The RLU signal starts and the chemiluminescence decreases after 3 minutes of hydrolysis as follows (only the nucleotides around the linker site are shown here):

[0377] Table 3

[0378]

[0379] The performance of the L10 enantiomeric linker in hybridizing to perfectly matched and mismatched sequences between the 5'-AG-3' positions was demonstrated by comparable chemiluminescent signals and DR (=80) when the hybridizing sequences were in 100 μL of hybridization reagent (Example 23(c)) or 25 μL of hybridization reagent (Example 23(b)).

[0380] These examples demonstrate that one linker enantiomer (R) has a higher reporter signal (RLU) than the other linker enantiomer (S). Furthermore, the higher DR of the R linker enantiomer compared to the S linker enantiomer indicates better discrimination between matched and mismatched sequences. Furthermore, controlled hydrolysis of the acridinium ester probe yields consistent results across a range of hybridization reagent conditions.

[0381] Example 24: Synthesis of a Linking Reagent Composed of 4-Aminobutane-1,3-diol

[0382] Example 24(A): N-Fmoc-O 1 -DMT-O 3 Synthesis of cyanoethoxydiisopropylaminophosphinyl-(S)-4-aminobutane-1,3-diol (60)

[0383] N-Fmoc-O 1 -DMT-O 3-Cyanoethoxydiisopropylaminophosphinyl-(S)-4-aminobutane-1,3-diol (60) is shown in FIG18 and was synthesized essentially as in the above example except that (S)-4-aminobutane-1,3-diol was used as the starting diol.

[0384] Example 24(B): N-Fmoc-O 1 -DMT-O 3 Synthesis of -cyanoethoxydiisopropylaminophosphinyl-(R)-4-aminobutane-1,3-diol (61)

[0385] N-Fmoc-O 1 -DMT-O 3 -Cyanoethoxydiisopropylaminophosphinyl-(R)-4-aminobutane-1,3-diol (61) is shown in FIG18 and was synthesized essentially as in the above example except that (R)-4-aminobutane-1,3-diol was used as the starting diol.

[0386] Example 24(C): N-Fmoc-O 1 -Cyanoethoxydiisopropylaminophosphinyl-O 3 Synthesis of DMT-(S)-4-aminobutane-1,3-diol (62)

[0387] N-Fmoc-O 1 -Cyanoethoxydiisopropylaminophosphinyl-O 3 -DMT-(S)-4-aminobutane-1,3-diol (62) is shown in FIG18 and was synthesized essentially as in the above example except that (S)-4-aminobutane-1,3-diol was used as the starting diol.

[0388] Example 24(D): N-Fmoc-O 1 -Cyanoethoxydiisopropylaminophosphinyl-O 3 Synthesis of DMT-(R)-4-aminobutane-1,3-diol (63)

[0389] N-Fmoc-O 1 -Cyanoethoxydiisopropylaminophosphinyl-O 3 -DMT-(R)-4-aminobutane-1,3-diol (63) is shown in FIG18 and was synthesized essentially as in the above example except that (R)-4-aminobutane-1,3-diol was used as the starting diol.

[0390] Example 25: Controlled Adduct Formation of Acridinium Ester Probes.

[0391] Adduct formation on the acridinium ester connected to the polymer can result in a product that is not chemiluminescent (PW Hammond et al., Journal of Bioluminescence and Chemiluminescence (J.Biolum.Chemilum.), Vol. 6, p. 35, 1991; LJ Arnold Jr. et al., U.S. Patent No. 4,950,613; M. Becker et al., U.S. Patent No. 5,731,148). Acridinium ester-labeled polymers that are not bound to other nucleic acids or bound to partially mismatched nucleic acids produce a greater degree of adduct formation, while acridinium ester-labeled polymers that are bound to nucleic acids that are fully matched near the joint produce a lower degree of adduct formation. After a very short time interval, the degree of modification is measured to determine the relative effectiveness of the adduct formation reaction. A greater degree of adduct formation modification is demonstrated by a lower chemiluminescent signal, while a lower degree of modification is demonstrated by a higher chemiluminescent signal. These observations are used as the basis for controlled adduct formation determination.

[0392] (a) Acridinium ester-labeled polymers can be bound to fully matched or mismatched nucleic acids (or similarly reacted but in the absence of other partially matched nucleic acids) in 100 μL volumes in 12×75 mm polystyrene tubes as described in Example 19. Controlled adduct formation is performed by adding 100 μL of adduct-forming composition (0.01 M sodium sulfite, 0.03 M sodium tetraborate, pH 8.7) to each tube, vortexing the tubes and incubating at room temperature for 60 seconds. During this time, wipe the outside of each tube with a damp cloth and then insert it into a photometer (e.g., Leader 50). At 30 seconds, the chemiluminescence of each aliquot was measured by automatically injecting 200 μL of the acidic oxidant Trigger 1 formulation (0.0063% (v / v) nitric acid in water and 0.1% (v / v) hydrogen peroxide), and then after a 2-second delay, by automatically injecting 200 μL of the basic Trigger 2 formulation (4% (w / v) sodium hydroxide in water) and reading the chemiluminescence for 2 seconds. The chemiluminescence from hybridized perfectly matched nucleic acids is very high and stable, while the chemiluminescence from the probe in the absence of complementary nucleic acids is very low and stable.

[0393] (b) Controlled Adduct Formation was performed as in Example 25(a), except that 10 to 500 μL of the adduct-forming composition was added to each tube.

[0394] (c) Controlled Adduct Formation was performed as in Examples 25(a) and 25(b), except that the adduct-forming composition consisted of 0.005-0.20 M sodium sulfite, 0.05-0.5 M sodium tetraborate, pH 7.5-9.1, and 0-5% Triton X-100.

[0395] (d) Controlled adduct formation is carried out as in Examples 25(a), 25(b) and 25(c), except that the adduct formation is followed by automated injection of about 50-400 μL of about 0.0001-0.01% (v / v) aqueous nitric acid and about 0.02-2% (v / v) hydrogen peroxide and about 50-400 μL of 1-8% (w / v) aqueous sodium hydroxide.

[0396] (e) Controlled adduct formation was performed as in Examples 25(a), 25(b), 25(c) and 25(d), except that the chemiluminescence was collected after a delay of about 0-10 seconds between the injection of Trigger 1 and Trigger 2, and the chemiluminescence was collected for about 1-10 seconds.

[0397] (f) Controlled Adduct Formation

[0146] The process was carried out as in Examples 25(a), 25(b), 25(c), 25(d) and 25(e), except that the interval between the addition of the adduct-forming composition and the hydrogen peroxide / alkaline solution was about 1-200 seconds.

[0398] (g) Controlled adduct formation is performed as in Examples 25(a), 25(b), 25(c), 25(d), 25(e) and 25(f), except that about 50-400 μL of the adduct-forming composition is automatically injected into the tube, followed by a delay of about 1-200 seconds and injection of about 50-400 μL of an injection solution comprising about 1-8% (w / v) aqueous sodium hydroxide solution and about 0.02-2% (v / v) hydrogen peroxide.

[0399] (h) Controlled adduct formation is carried out as in Examples 25(a), 25(b), 25(c), 25(d), 25(e), 25(f) and 25(g), except that the adduct-forming composition consists of about 0.01-0.20 M of tetrahydrothiophene, propyl mercaptan, benzyl mercaptan, sulfite, glycol sulfite, hyposulfite, metabisulfite, thiosulfate, thiophosphate, metabisulfite salt or mercaptoethanesulfonic acid.

[0400] (i) Controlled adduct formation was performed as in Examples 25(a), 25(b), 25(c), 25(d), 25(e), 25(f), 25(g), and 25(h), except that the chemiluminescence was collected in a luminometer other than Leader50, such as a LeaderI, a Leader450, a LeaderHC, a LeaderHC+, an Optocomp I (MGM Instruments), an Optocomp II, a Lumat 3LB 9508 single tube photometer (Berthold Technologies, Oak Ridge, TN, USA) or others.

[0401] (j) Controlled adduct formation is carried out as in Examples 25(a), 25(b), 25(c), 25(d), 25(e), 25(f), 25(g) and 25(h), except that the reactants are in the wells of a multiwell plate (e.g., 96-well) and the chemiluminescence is collected in a plate luminometer, such as a PHERAstar reader (BMG Labtech, Cary, NC, USA), a MicroLumatPlus reader (Berthold Technologies), a GloMax Microplate reader (Promega, Madison, WI, USA), or others.

[0402] Example 26: Connection of 1-O-DMT-3-N-[N-(Fluorenylmethoxycarbonyl)-hexyl]-(S)-amino-1,2-propanediol and 1-O-DMT-3-N-[N-(Fluorenylmethoxycarbonyl)-hexyl]-(R)-amino-1,2-propanediol linkers to controlled pore glass (CPG).

[0403] Linkers can be introduced into one, the other, or both nucleic acid termini by using a linker phosphoramidite in the final introduction step, using a cleavable linker attached to a solid support, or both. Particularly useful solid supports include macroporous polystyrene and controlled pore glass (CPG) particles.

[0404] (a) 2 mmol of the precursor {1-O-DMT-3-N-[N-(fluorenylmethoxycarbonyl)-hexyl]-(S)-amino-1,2-propanediol (37) (Example 10(A)) or 1-O-DMT-3-N-[N-(fluorenylmethoxycarbonyl)-hexyl]-(R)-amino-1,2-propanediol (Example 10(B)} was dissolved in 5 mL of 1,2-dichloroethane. To this mixture were added 240 mg of succinic anhydride (2.4 mmol), 122 mg of dimethylaminopyridine (DMAP, 1.0 mmol) and 0.56 mL of triethylamine (4.0 mmol). The combined mixture was heated to 50°C for 45 minutes. 75 mL of ethyl acetate was added and the entire amount was transferred to a separatory funnel. The organic layer was washed with 3×35 mL of ethanol. 5% ice-cold citric acid, 3×35mL water, 1×35mL brine washed and dried over anhydrous sodium sulfate. Add 2mL pyridine and evaporate in vacuo. The residue is dissolved in 100mL dichloromethane. To this solution, 0.56mL triethylamine (4.0mmol), 270mg 1-hydroxybenzotriazole (HBT, 2.0mmol) and 25g 1,000 angstrom CPG are added. Finally, 885mg benzotriazole-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP, 2.0mmol) are added and immediately vortex mixed. Subsequently, it is stirred on an orbital shaker for 2 hours. The modified CPG is collected by filtration and thoroughly washed with dimethylformamide (DMF) 3 times, methanol 5 times and ether 2 times. The solid material is dried under high vacuum for 1 hour.

[0405] The resulting CPG was capped with a pyridine-acetic anhydride-DMAP solution and reacted for 0.25-1.0 hours. The CPG was collected by filtration and washed thoroughly with pyridine twice, DMF three times, dichloromethane four times, methanol five times, and ether twice. The washed material was dried under high vacuum overnight to obtain modified CPG, which was connected to 1-O-DMT-3-N-[N-(fluorenylmethoxycarbonyl)-hexyl]-(S)-amino-1,2-propanediol and 1-O-DMT-3-N-[N-(fluorenylmethoxycarbonyl)-hexyl]-(R)-amino-1,2-propanediol, respectively.

[0406] Table 8

[0407]

[0408] The above examples have demonstrated that reagents of the present invention can be prepared, each of which has a non-nucleotide backbone, a first and a second coupling group, and a ligand. Specifically, reagent (5) has a non-nucleotide propyl backbone, bonded thereto with a first coupling group methyl-N,N-diisopropylphosphoramido, a second coupling group 1-hydroxyl protected by dimethoxytrityl (DMT), and a ligand in the form of an aminopropyl linker arm protected by trifluoroacetyl. Reagent (10) is identical to reagent (5), except that reagent (10) has two identical ligands (in the form of two protected linker arms trifluoroacetylaminopropyl). Reagent (13) is also similar to reagent (5), except that the non-nucleotide backbone is an ethyl group rather than a propyl group, and the length of the linker arm is shorter, the linker arm being only a methylamine protected by trifluoroacetyl rather than a similarly protected aminopropyl group.

[0409] As also demonstrated above, a single reagent of the present invention can be used to specifically provide only a joint arm at any preselected position on a nucleotide polymer, without introducing unwanted nucleotides. As also described, by coupling a backbone with nucleotides and another backbone (which is then coupled with another backbone, and so on, to produce a chain), the reagent of the present invention can provide a series of adjacent ligands (e.g., a label or a joint arm that can be connected to a label) in a nucleotide polymer. Therefore, a plurality of adjacent labels can be connected to a probe, thereby improving the sensitivity of a hybridization assay using the probe.

[0410] In addition, the use of the non-nucleotide backbone of one or a series of sequential connections of the present invention can also be used for bridging between two nucleotide sequences complementary to the corresponding sequence on the target nucleotide polymer on the probe, and in test sample, the non-nucleotide backbone can be bridged by single different nucleotide or different sequences.Therefore, the target nucleotide polymer can actually be the group consisting of two or more nucleotide polymers, and the nucleotide polymer is made up of a common target nucleotide sequence, and the common target nucleotide sequence is made up of a single different nucleotide bridge, or is made up of different non-target nucleotide sequences. Even if it is a target sequence to a single target sequence, a probe with a complementary sequence can also be prepared, and the complementary sequence has a non-nucleotide monomer unit with a labeling group, which is coupled between any two nucleotides. In this case, the probe hybridizes the target nucleotide sequence in a normal manner, and the difference is that the monomer unit with the labeling group tends to make itself exist in the mode that does not interfere with the above-mentioned hybridization (that is, it tends to " loop out (loop out) " of mixed structure). This arrangement is particularly advantageous when wishing to utilize the intercalation effect, for example, to possibly improve the specificity of the probe. Of course, compared with the existing probe using the nucleotide monomer unit, the present invention uses this fact of the non-nucleotide monomer unit to significantly reduce the interference of the aforementioned type.

[0411] As described above, compounds (4), (9), and (12) can be attached to a solid phase synthesis support via their primary hydroxyl groups. When used in the synthesis of another polymer, the resulting derivatized support results in the non-nucleotide monomer unit being attached to the 3'-terminus of the resulting nucleotide / non-nucleotide polymer. Alternatively, if the oligonucleotide is synthesized 5'-to-3', the non-nucleotide monomer unit will be located at the 5'-terminus of the resulting nucleotide / non-nucleotide polymer.

[0412] It should be understood that various variations of the above invention are possible. For example, the ligand may actually be a label or intercalator provided on the reagent of the present invention prior to coupling with the nucleotides of the nucleotide polymer. In addition, as described above, in addition to the protecting groups of the specific embodiments described above, various other protecting groups may also be used. However, it is particularly preferred to use one of the trifluoroacetyl and 9-fluorenylmethoxycarbonylamino protecting groups because they are cleaved under the same alkaline conditions used in known standard oligonucleotide synthesis to deprotect the amine to deprotect the exocyclic nucleotide amine (usually in concentrated ammonium hydroxide at 50°C for 1 to 12 hours). Similarly, the use of dimethoxytrityl 5'-hydroxy protection, methyl or β-cyanoethyl phosphite O protection, and N,N-diisopropyl as the phosphite leaving group during coupling can make the reagent fully compatible with current standard oligonucleotide solid phase synthesis techniques to minimize the need for any additional special steps.

[0413] Other variations and modifications of the above embodiments of the present invention may occur to those skilled in the art. Therefore, the present invention is not limited to those embodiments described in detail above.

[0414] All references cited in the above disclosure are incorporated by reference in their entirety. Sequence Listing <110> K.A. Brown <120> Diastereomeric ligation reagents for nucleotide probes <130> BBA-4 <160> 8 <170> SIPOSequenceListing 1.0 <210> 1 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 1 gctcgttgcg ggacttaacc caacat 26 <210> 2 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 2 aaataacgaa cccttgcagg tcctttcaac tttgat 36 <210> 3 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 3 cagtcaaact ctagccatta cctgctaaag tcattt 36 <210> 4 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 4 gctcgttgcc ccacttaacc caacat 26 <210> 5 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 5 cgttactcgg atgcccaaat atcgccacat tcg 33 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 6 cccgcacgtc ccctattaatc attacgatgg 30 <210> 7 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 7 gatctgagcg tcgacgtcgtgacatg 26 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Synthetic Oligonucleotide <400> 8 gatctgagcg tcagcgtcgtgacatg 26

Claims

1. A diastereomeric linking reagent comprising a phosphoramidite-based compound having the formula: wherein each diastereomeric linking reagent is present in the form of its pure enantiomer.

2. The linking reagent according to claim 1, wherein The compound is the R enantiomer or the S enantiomer.

3. An oligonucleotide incorporating a ligation reagent as claimed in any one of the preceding claims.

4. A method for preparing a linking reagent according to any one of claims 1 to 2, comprising the following steps: (a) Synthesis of linkers and (b) purification of isomers.

5. A method for producing an oligonucleotide, wherein the oligonucleotide is introduced with a linking reagent according to any one of claims 1 to 2, the method comprising the following steps: (a) coupling the reactive phosphorus group of the linking reagent to a first nucleotide or strand or nucleotides under DNA synthesis conditions, and (b) removing the protecting group to allow coupling of a second nucleotide or the activated phosphorus group of a second linking reagent.

6. Use of the diastereomeric linking reagent according to any one of claims 1 to 2 in the preparation of nucleotide probes.

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