JPH06206889A - Organophosphorus binder for synthesizing amino-induced polymer - Google Patents

Abstract

PURPOSE: To provide the linking agents, comprising specific organophosphor compounds which can covalently bond organic portion of fluorescent dyes or the like to DNA fragments, oligonucleotides, and are useful for automatic DNA synthesis, sequence analysis and for constituting gene probe or the like.

CONSTITUTION: The object linking agents comprising 2-methoxy-3-tri- fluoroacetyl-1,3-2-oxazaphoshur cyclopentane of the formula or the like. In the formula, (j) is 2 or 3; R₁ is trihaloacetyl; R₄ is 1-6C lower alkyl.

COPYRIGHT: (C)1994,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION This invention relates generally to organophosphorus compounds, and more particularly to organophosphorus binders for synthesizing amino-derivatized polymers, especially oligonucleotides.

[0002]

Genes and gene control regions can now be routinely characterized and studied at the molecular level. This is possible due to some recent advances in technology associated with engineering and modifying deoxyribonucleic acid (DNA). Of particular importance are advances in DNA sequencing (Methods in Enzymology, Gr.
Maxam and Gilbert in ossman and Moldave, Vol. 65 (Academic Press, New York, 1980).
(Gilbert) 's “Sequencing End-Labeled DNAwith Base-
Specific Chemical Cleavages ", and Smith" D
NA Sequence Analysis by Primed Synthesis ", p. 499 to 560 and 560 to 580, respectively; Isolation of a number of host restriction enzymes, Methods in Enzymology, Wu, Vol. 68 (Aca).
demicPress, New York, 1979) in Roberts (Robe
rts) “Dictionary of Restriction Endonucleases”; and construction of vectors for cloning and amplification of defined DNA sequences, eg Methods in Enzymology, Wu
Vol.68 (AcademicPress New York, 1979) Bolivar and Backman "Plasmid"
s of Escherichia coli as Cloning Vectors ").

[0003]

Many of these new techniques require that DNA fragments or oligonucleotides be labeled or attached to a polymeric support. DNA sequencing techniques and gene probes that can be used to aid in the placement of naturally occurring genes of commercial or chemical importance require the use of labeled oligonucleotides. Until recently, all DNA sequencing techniques relied on radioactive labels to distinguish oligonucleotide fragments separated by electrophoresis. Radioactive labels are highly sensitive and can be introduced without steric hindrance and other chemical side effects. However, their use poses a laboratory health risk and requires them to undergo special handling and disposal. Furthermore, their use is not available for the practical identification of nucleotide bases with different nucleotide-specific radiolabels, and radiodetection techniques such as autoradiography and scintillation counting are too time consuming, thus making oligonucleotides Not suitable for rapid automated sequencing of. As a result, other non-radioactive labeling techniques have been sought, which rely on the ability to covalently attach fluorescent or chromogenic molecules to oligonucleotides such as fluorescent and colorimetric labeling.

Chu et al., “Derivatization
of Unprotected Polynucleotides "Nucleic Acids Rese
Arch, Vol. 11, p. 6513-6529 (1983), discloses a method of attaching an amine to the terminal 5'-phosphate of an oligonucleotide. One purpose of this method is to provide a means of attaching organic labeled molecules to oligonucleotides by amine linkages. This method involves treating the oligonucleotide with carbodiimide.

Chollet and Kawawash
ima) is "Biotin-Labeled Synthetic Oligodeoxyribonucle
otides: Chemical Synthesis and Uses as Hybridizat
ionProbes ", Nucleic Acids Research, Vol 13, p. 152
9-1541 (1985) discloses the use of a method of attaching biotin such as Ju to the 5'-phosphate of an oligonucleotide. The reported yields of 50-70% are lower than required for use in automated synthesizers,
Carbodiimides are capable of causing undesired modification of the oligonucleotide base during the reaction.

[0006] Smith et al.
of Oligonucleotides Containingan Aliphatic Amino G
roup at the 5 'Terminis: Synthesis of Fluorescent
DNA Primers for Use in DNA Sequence Analysis ", Nuc
Leic Acids Research, Vol. 13, p. 2399-2412 (1985) discloses a protected amino-derivatized nucleotide phosphoramidite for attaching fluorescent or colorimetric labels to oligonucleotide fragments. Although this coupling agent is very useful for coupling base-specific labels to the 5'-end of oligonucleotides, protected-amine phosphoramidites are not easily purified.

Connolly and Ride
r) is "Chemical Synthesis of Oligonucleotides Conta
ining a Free Sulphydryl Group and Subsequent Attac
hment of Thiol Specific Probes ", Nucleic Acids Re
search, Vol. 13, p. 4485-4502 (1985), discloses the synthesis of an oligonucleotide having a trityl-protected sulfur attached to the 5'phosphate of the oligonucleotide via a 2, 3 or 6 carbon chain. To do.

Apart from binding the labeling agent to the oligonucleotides, various molecules are supported on the polymer such as catalysts, enzymes, microorganisms, affinity reagents, immunoadsorbents for both preparative and analytical purposes. Immobilization on the body is of great interest. For example, shot (Sc
hott), Affinity Chromatography (Mercel Dekker, Inc.
New York (1984) and Mosback, Meth
ods in Enzymology, Vol. 44, "Immobilized Enzymes",
(Academic Press New York 1976). Of particular interest in this field are means for covalently immobilizing molecules and cells.

[0009]

SUMMARY OF THE INVENTION The compounds of the present invention include novel binders consisting of 2-substituted-3-protected-1,3,2-oxazaphosphacycloalkanes.
The present invention relates to organic moieties such as fluorescent and chromogenic dyes to DNA fragments and oligonucleotides, especially single-stranded DNA and RN.
It relates to compounds useful for binding A and for binding DNA fragments, oligonucleotides, proteins and the like to polymeric supports. These compounds and their complexes are useful in automated and manual DNA and RNA synthesis and sequence analysis, gene probe construction, affinity techniques and the like. In particular, the cyclic embodiment of the binding agent of the present invention advantageously overcomes the drawbacks associated with currently available binding methods by providing a more easily purified binding agent.

The binding compounds of the present invention include 2-substituted-3-protected-1,3,2-oxazaphosphacycloalkanes represented by the general formula:

[0011]

[Chemical 2]

Wherein j is 2 or 3 and R ₁ is trihaloacetyl. R ₄ is lower alkyl containing up to 6 carbon atoms. The term "lower alkyl" as used in the present invention refers to straight and branched chain alkyl groups containing 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, tert-butyl, isobutyl, sec-butyl, neopentyl. , Tert-pentyl and the like.

The following compounds can be purified using the binding compounds of the invention. The complex includes a phosphite triester and a phosphate triester and diester compound of the formula:

[0014]

[Chemical 3]

Wherein i is 0 or 1 (i = 0 indicates a phosphite, and i = 1 indicates a phosphite) j is 2 or 3 and R ₁ is trihaloacetyl. Where R ₄ is lower alkyl containing up to 6 carbon atoms, and W represents an oligonucleotide, or an oligonucleotide attached to a polymeric support.

Oligonucleotides include single-stranded or double-stranded RNA fragments and single-stranded and double-stranded DNA.
Including fragments. Preferably the binder is attached to the terminal 5'carbon of the oligonucleotide, the terminal 3'carbon of the oligonucleotide or the terminal 2'carbon of RNA. More preferably, the binding agent is attached to the terminal 5'carbon atom of the oligonucleotide, most preferably the binding agent is single-stranded DNA.
Is joined to the terminal 5'carbon of the fragment.

The polymeric support may have various forms and compositions. The polymeric support can be derived from natural materials, synthetically modified natural materials and synthetic materials. Of particular interest are polysaccharides, especially cross-linked polysaccharides such as agarose available as Sepharose, dextran available as Sephadex and Sephacyl, cellulose, starch and the like (Sepharose, Sep
hadex and Sephacyl are branded products of Pharmacia Fine Chemicals). Other materials include polyacrylamide, polystyrene, polyvinyl alcohol,
Examples thereof include a copolymer of hydroxyethyl methacrylate and methyl methacrylate, silicone, Teflon glass, and cells. In addition to solid supports in the form of particles,
The polymer support also contains an internal liquid and may be in the form of liquid particles consisting of a lipophilic or amphipathic membrane which serves to define the space. Such particles include vesicles, cells,
Examples thereof include liposomes. Preferably A'represents an insoluble polymer support having hydroxyl functionality. The binder of the present invention is attached to a polymeric support having hydroxyl functionality by the following method of attaching a binder to oligonucleotides, which is generally described below.

On the left-hand side of general formula II (surrounding H and R ₁ ).
Indicates that this embodiment includes both protected and deprotected forms of the compound. Oligonucleotides can be prepared by standard techniques of affinity chromatography or by, for example, Calthers et al., US Pat. No. 4,458,0
It is bonded to the polymer support by the bonding means disclosed in, for example, 66 and 4,415,732.

Generally, the phosphoric acid triester compound of the present invention is prepared from the above phosphorous acid complex by oxidation, for example, water,
I in 2,6-lutidine and tetrahydrofuran
Obtained by oxidation with ₂ . Oxidation is extremely fast (1-2 minutes). The phosphodiester complexes of the present invention can be prepared from the above phosphoric acid triesters by standard techniques such as R
_{When 4} is methyl, the phosphoric acid diester is obtained from the phosphoric acid triester by treatment with thiophenol / triethylamine for about 30 minutes. The general method for synthesizing the phosphoramidide precursor of general formula I comprises the following steps. Halo-substituted-N, N- represented by the formula:
Di-substituted-O-substituted phosphine:

[0020]

[Chemical 4]

Wherein X is halogen, usually chloro, and R ′, R ″ and R ₄ are as defined above, and the amino-protected alcohol amine is defined by the formula:

[0022]

[Chemical 5]

(Wherein R ₁ and j have the same meanings as described above), which absorbs the halogen acid released during the reaction.
The reaction is carried out in an aprotic solvent such as dichloromethane containing a nucleophilic base such as a trialkylamine such as N, N-diisopropylethylamine. Preferably the reaction is carried out under an inert atmosphere such as argon.
Acidic conditions in the reaction mixture should be avoided because the acid protonates the amine of the phosphoramidite product to make it reactive. Non-nucleophilic bases reduce the potential for side reactions between bases and activated phosphoramidites.

After reacting the above materials, the reaction mixture, hereinafter referred to as the first reaction mixture, is washed with a mildly basic solution to remove non-nucleophilic base salts. Finally, the first reaction mixture is dried using, for example, MgSO ₄ , Na ₂ SO _4, etc. to obtain the phosphoramidite precursor. Heterocycles of general formula I are obtained by heating a suitable precursor to form a second reaction mixture and then removing the heterocycle from the mixture. The amount of heating required, i.e. temperature and duration, will vary depending on the different embodiments of the invention, but preferably the heating will be about 25-250 ° C precursor.
More preferably about 25-150 ° C, most preferably about 25-150 ° C.
Increasing to a temperature in the range of ~ 100 ° C. The choice of separation method depends on the nature of the substituents R ₁ and R ₄ . For example, if the total molecular weight of the substituents is low enough, as a rough approximation, the heating and separating steps can be accomplished by distillation. Other separation methods include crystallization and chromatography. Preferably, the oligonucleotide-binder complex of the present invention is formed by conjugating the binder to oligonucleotides synthesized by the solid phase synthesis method developed by Caruthers and his coworkers. (Eg, Caruthers et al., P. 1-17, Genetic Enginee
ring, Vol.4, Setlow and Hollaender (Plenum Pres
s, New York 1982), and Carousser et al. (Caruthe
rs et al.), U.S. Pat. No. 4,458,066].

Ligation of the binding agent is carried out as the last step of the synthetic process, ie the binding agent is attached to the oligonucleotide as if it were a subunit of a nucleotide in Carousel et al.

[0026]

The following examples serve to illustrate the invention. The values of reagent concentrations, temperatures, and other variable parameters are merely illustrative of the application of the present invention and are not to be considered limiting thereof. Example I. 2-methoxy-3-trifluoroacetyl-
Phosphor of 1,3,2-oxazaphosphacyclopentane
Synthesis of ruamidide precursor Chloro-N, N-diisopropylaminomethoxyphosphine (5.0 ml, Aldrich Chemical Co., Milwaukee,
(Commercially available from Wisconsin) at 0 ° C. in N- (2-hydroxyethyl)-in dichloromethane (about 40 ml).
To a stirred solution of 2,2,2-trifluoroacetamide (3.9g) and diisopropylethylamine (5.0ml) was added dropwise under argon. N- (2-hydroxyethyl)-
Add 2,2,2-trifluoroacetamide to Lazarus
rus) and Benkovic J. Am. Chem. S.
oc., Vol. 101, p. 4300-4312 (1979). That is, ethyl trifluoroacetate (56.8 g, 0.4 mol) in 50 ml chloroform was added.
To a stirred solution of 24.4 g (0.4 mol) ethanolamine in 50 ml chloroform was added dropwise. Add this solution at room temperature to 5
Stirred for hours, removed solvent by rotary evaporation and distilled at 115 ° C. (4.3 Torr) to give 58.5 g of oil which crystallized by scratching. After stirring for 0.5 h at room temperature, the reaction mixture is washed once with 0.2 M potassium carbonate solution and once with brine, dried (MgSO ₄ ),
Concentrated under reduced pressure to give N- (2- (N ', N'-diisopropylaminomethoxyphosphinyloxy) ethyl) -2,
2,2-trifluoroacetamide was obtained as a colorless liquid (7.77g). ¹ H-NMR (CD ₂ Cl ₂ ): δ3.6 (6H, m), 3.4 (3H, d,
J = 12), 1.2 (12H, d, J = 6.5) ³¹ P-NMR (CD ₂ Cl ₂ , ¹ H-decoupled): δ149 (s)

Example II. 2-methoxy-3-trifluor
Roacetyl-1,3,2-oxazaphosphacyclohexyl
Synthesis of Sun Phosphoramidite Precursor Chloro-N, N-diisopropylaminomethoxyphosphine (3.7 ml) at 0 ° C. in N (20 ml) in dichloromethane (about 20 ml).
-(3-Hydroxypropyl) -2,2,2-trifluoroacetamide (2.9 g, 3-amino-1-propanol and ethyl trifluoroacetate from Lazarus and Bencovic, J. Amer. Chem. Soc., Vol. 101, p. 4300-
4312 (1979) and a stirred solution of diisopropylethylamine (3.7 ml) were added dropwise under argon to the stirred solution. After stirring at room temperature for 3 hours, the reaction mixture was poured into hexane (100 ml) and stirred. The mixture is allowed to settle, the supernatant is separated and concentrated under reduced pressure to give N- (3- (N
′, N′-Diisopropylaminomethoxyphosphonyloxy) propyl) -2,2,2-trifluoroacetamide was obtained as a colorless liquid (5.2 g). ³¹ P-NMR (CH ₂ Cl ₂ , ¹ H-decoupled): δ149 (s)

Example III. 2-Methoxy-3-triflu
Oroacetyl-1,3,2, -oxazaphosphacyclo
Synthesis of pentane N- (2- (N ' , N'-diisopropylaminomethoxyphosphinyloxy) ethyl) -2,2,2-trifluoroacetamide (7.7 g) was distilled (5 at 0.8 Torr
(8 to 59 ° C.), 2-methoxy-3-trifluoroacetyl-1,3,2-oxazaphosphacyclopentane was quantitatively obtained as a colorless liquid. IR (membrane): 1705, 1420, 1230, 1200, 1160, 1020, 96
^{5 cm -1 1 H-NMR (} CD 2 Cl 2): δ4.45 (2H, m), 3.65 (2H,
m) 3.60 (3H, d, J = 12) ³¹ P-NMR (CD ₂ Cl ₂ , ¹ H-decoupled): δ 132 (s),
125 (q, J = 61), MS: m / e 217 (M ⁺ ), 197, 148, 136, 123, 120, 109,
92, 79, 70 (100), 69, 62

Example IV. 2-methoxy-3-trifluor
Roacetyl-1,3,2-oxazaphosphacyclopen
To the 5'end of the tan oligonucleotide 2-methoxy-3-trifluoroacetyl-1,3,2
-Coupling of oxazaphosphacyclopentane to the 5'hydroxyl of the oligonucleotide was performed using the Applied Biosystem 380A DNA Synthesizer (Applied Biosys
tems, Foster City, CA) or similar equipment. Caruthers et a
l.), U.S. Pat. No. 4,458,066; Carousser et al., U.S. Pat. No. 4,415,732; and Carousser et al., "New Methods for Sy
nthesizing Deoxyoligonucleotides) '', Genetic Engi
neering, Vol. 4, pp. 1-17 (Plenum Press, New York, 1982), Applied Biosystems 380A DNA.
A detailed description of the chemistry used by the synthesizer is given. Therefore, these documents apply mutatis mutandis to their explanations. 2-Methoxy-3-trifluoroacetyl-1,3,2-oxazaphosphacyclopentane is 0.
Combined with 5 M tetrazole / acetonitrile solution.
Used as a 2M solution in acetonitrile to form the active reagent in the synthesis cycle. The normal synthesizer cycle was modified as a fourth order upon addition of the active reagent.
The active reagent was added twice with a waiting time of 1 hour after each addition. The coupling yield was about 95%. Conventional deprotection with thiophenol / triethylamine and then ammonium hydroxide gave the 5'-aminoethylphosphate oligonucleotide. Active reagent is 0.2M 2-methoxy-
Similar yields were obtained with an acetonitrile solution containing 3-trifluoroacetyl-1,3,2-oxazaphosphacyclopentane and 0.1M 4-dimethylaminopyridine. In this case, the modified activator reagent was added once and allowed to react for about 15 minutes.

Example V. 2-methoxy-3-trifluor
Roacetyl-1,3,2-oxazaphosphacyclopen
The ligation of the tan to the 3'end of the oligonucleotide was carried out in the Example except that the oligonucleotide was generally synthesized in the 3'direction according to the method described by Caruthers et al. US Pat. No. 4,458,066. It was achieved in substantially the same way as IV. [Roughly speaking, the difference is that the oligonucleotide is 3'-protected nucleotide 5'N, N-diisopropylaminophosphoramidide instead of 5'-protected nucleotide 3'N, N-diisopropylaminophosphoramidide. Is to be synthesized from. Alternatively, the oligonucleotide is corana (K
horana) and Itakura's phosphotriester method in the 3'direction (ie, Khorana, Sci.
ence, Vol. 203, p, 614-625 (1979); Itakura et al.
J. Biol. Chem., Vol. 250, p. 4592-4600, both of which are applied mutatis mutandis) or modifications by others, such as Letsinger and Mahadera.
n), J. Am. Chem. Soc. Vol. 187, p. 3526- (1965)
]. In each case, the binder is attached as a final addition instead of nucleotides.

Example VI. Fluorescein isothiocyanate
(FITC) aminoethyl phosphate oligo
Conjugation to nucleotides Fluorescein-6-isothiocyanate in dimethylformamide (25 μl at a concentration of 10 mg / ml, eg Mole
cular Probes, Inc., available from Junction City, Oreg.) 5′-aminoethyl phosphate TCCCAGTCACGACGTT (0.200 μmol, in water (200 μl) and 1M NaHCO ₃ / Na ₂ CO ₃ buffer, pH 9.0 (25 μl). Applied Biosyste
Added to a solution of crude material made on a ms 380A DNA synthesizer, where T = thymidine, C = cytidine, G = guanosine, and A = adenosine. The resulting solution was stored at room temperature in the dark for at least 6 hours.
The reaction mixture was equilibrated to remove unbound dye
10ml Sephadex (Trademark of Pharmacia Fine Chemicals) G
Water was passed through a -25 (medium) column. Bands of colored material within the excluded volume were collected. The crude 5'-fluorescein aminoethyl phosphate oligonucleotide was loaded onto a Vydac C18 column (No. 218T54) by HPLC (eg, Perkin-Elmer Series 4 or similar instrument) with 10-20% acetonitrile / 0.1M triethyl. Purified in a linear gradient of ammonium acetate, pH 7.0.

[0032]

According to the present invention, organic moieties such as fluorescent and chromogenic dyes are attached to DNA fragments and oligonucleotides, especially single-stranded DNA and RNA, and D
It is useful for attaching NA fragments, oligonucleotides, proteins, etc. to polymer supports and has applications in automated and manual DNA and RNA synthesis and sequence analysis, gene probe construction, affinity technology and the like.

 ─────────────────────────────────────────────────── ─── Continued Front Page (72) Inventor Wu, Sam Lee 94061 Redwood City Carlos Road 450 (72) Inventor Smith, Lloyd M. United States California 91030 South Pasadena Meridian Avenue 612

Claims (3)

[Claims] 1. An organophosphorus binder for synthesizing amino-derivatized polymers of the formula: [Chemical 1] Wherein: j is 2 or 3 and R ₁ is trihaloacetyl. R ₄ is lower alkyl containing up to 6 carbon atoms. 2. The organophosphorus binder according to claim 1, which is 2-methoxy-3-trifluoroacetyl-1,3,2-oxazaphosphacyclopentane. 3. The organophosphorus binder according to claim 1, which is 2-methoxy-3-trifluoroacetyl-1,3,2-oxazaphosphacyclohexane.