WO1992001673A1 - Synthesis and uses of spin labelled ribonucleosides and ribonucleotides - Google Patents
Synthesis and uses of spin labelled ribonucleosides and ribonucleotides Download PDFInfo
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- WO1992001673A1 WO1992001673A1 PCT/GB1991/001146 GB9101146W WO9201673A1 WO 1992001673 A1 WO1992001673 A1 WO 1992001673A1 GB 9101146 W GB9101146 W GB 9101146W WO 9201673 A1 WO9201673 A1 WO 9201673A1
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- HUYBKFNGEQERND-WKIYYKSKSA-O CC(C)(CC1(CC2(C)C)OC3[C@H](N(C=CC(N4)=O)C4=O)O[C@H](CO)C3O1)N2[OH2+] Chemical compound CC(C)(CC1(CC2(C)C)OC3[C@H](N(C=CC(N4)=O)C4=O)O[C@H](CO)C3O1)N2[OH2+] HUYBKFNGEQERND-WKIYYKSKSA-O 0.000 description 1
- 0 CCC(C)(CC(*C12)(CC3(CI)C=I)OC1[C@@](COC(c1ccccc1)=O)O[C@]2N(C=CC(N1)=O)C1=O)N3OC(C)=O Chemical compound CCC(C)(CC(*C12)(CC3(CI)C=I)OC1[C@@](COC(c1ccccc1)=O)O[C@]2N(C=CC(N1)=O)C1=O)N3OC(C)=O 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B59/00—Introduction of isotopes of elements into organic compounds ; Labelled organic compounds per se
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
- C07D211/92—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with a hetero atom directly attached to the ring nitrogen atom
- C07D211/94—Oxygen atom, e.g. piperidine N-oxide
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
- C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
- C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
Definitions
- This invention relates to the preparation of spin labelled compounds from novel precursors and their use as probes. More specifically, the invention provides conformationally restricted spin labelled
- ribonucleosides and ribonucleotides which are useful, for example, in protein orientation studies.
- the present invention aims to provide such compounds.
- R represents a CH 3 or CD 3 group
- R 1 represents an alkyl group
- R 2 represents an alkyl or aryl group; and N may be either an 14 N or 15 N atom,
- R 1 may be any alkyl group and R 2 may be any alkyl or aryl group. It will be appreciated, however, that the presence of relatively small alkyl and aryl groups is usually to be preferred.
- X represents hydrogen, a mono-, di- or
- Y represents a purine or pyrimidine base, with the further proviso that when R represents CD3, the methylene hydrogen atoms of the six-membered
- piperidine ring are hydrogen or deuterium.
- either of X or Y may be radiolabel led.
- group X as a phosphate ester derivative examples include the 3-thiotriphosphate, the
- the spin labelled compound (2) incorporates the skeleton of the
- the spin labelled nucleotide and nucleoside compounds of this invention may comprise either a purine or a pyrimidine base.
- group Y can represent adenine, guanine, cytosine, uracil, thymine or 5-methylcytosine.
- a particularly preferred spin labelled compound of the invention has the
- Steps i), ii) and iii) involve relatively conventional chemistry, so that suitable reactants and process conditions will be readily appreciated by those skilled in the art.
- the initial reduction of step i) may be performed, for example, in the presence of ascorbic acid.
- the product is acylated in step ii), for example using acetic anhydride, and then converted in step iii) to a ketal.
- step iv) the ketal is converted to an enol ether.
- This latter product is the desired precursor compound (1) suitable for use in the synthesis of the spin labelled
- Step (iv) proceeds with unexpected
- Steps i) to iv) are illustrated in Example 1 below.
- Z is a protecting group for the 5'-hydroxyl position and which may either equate to group X (as hereinbefore defined, except when X represents hydrogen) or be capable of conversion or remo v a l to leave a group 1 in that position;
- R, R 1 , R 2 and Y are as previously defined, with the further proviso that when R represents CD 3 , the 2'- and 3'- hydroxyl hydrogen atoms in compound (8) may be deuterium in order that all four methylene hydrogen atoms of the six-membered piperidine ring of compound (9) are deuterium; vi) either a) where group 2 equates to group X, optionally further phosphorylating the compound (9) at that position, or b) treating the compound (9) such that Z is removed, and optionally phosphorylating, to leave a group X at that position; and
- step vi) will depend upon the nature of group Z.
- groups of the group Z when Z does not equate with X, include acyl (R 3 CO-, where R 3 may be alkyl, alkenyl, cycloalkyl or aryl), alkoxycarbonyl (R 3 OCO-, where R 3 is as defined above), allyl or substituted benzyl, such as 2-methoxybenzyl, o-nitrobenzyl or 1-(2-nitrophenyl)ethyl.
- Z may be a group that is retained in the final product, such as a phosphate (as in
- step vi) has the effect of converting group Z to group X.
- protecting group Z when protecting group Z is removed by alkaline hydrolysis (i.e. is acyl or alkoxycarbonyl), isolation of compound (9a), where X is hydrogen, is still possible because the protecting group is more susceptible to alkaline hydrolysis than the N-acyloxy group that is removed in step vii).
- step v) comprises reacting a precursor compound (1), where R, R 1 and R 2 are each CH 3 , with adenosine 5'-monophosphate (AMP) to produce a compound of the formula:-
- step vi) comprises pyrophosphorylating the product of step v) to produce a compound of the formula:-
- step vii) comprises deacetylation and air
- steps vi) and vii) the benzoyl and acetyl groups are sequentially removed and the resulting N-hydroxy compound oxidized in air to produce a compound of the formula (14):-
- a range of spin labelled compounds for use as probes, or as a part of such probes, can be derived from the precursor compound (1) of the present invention.
- the spin label is rigidly attached to nucleotide or nucleoside sugar residues and thereby, for example, rigidly oriented on proteins.
- This tight coupling of the spin label avoids the previously mentioned disadvantage of mobile nucleotide labels and thus opens up the possibility of new and improved orientation or structure studies of certain proteins.
- the use of such probes should enable betterinvestigation of how muscle cross-bridges move during contraction and relaxation, or how DNA interacts with DNA-binding proteins that control gene expression.
- the spin label precursor compounds (1) could be used to produce other types of labelled probes based on, for example, the common ribonucleoside 5' di- and 5'-triphosphates, caged ATP compounds (i.e. photo-labile derivatives of ATP from which ATP can be readily regenerated), pyridine nucleotides (e.g. NAD + , NADH), ribonucleotide analogues and ol igonucleotides.
- the spin label precursor compounds (1) could be used to produce other types of labelled probes based on, for example, the common ribonucleoside 5' di- and 5'-triphosphates, caged ATP compounds (i.e. photo-labile derivatives of ATP from which ATP can be readily regenerated), pyridine nucleotides (e.g. NAD + , NADH), ribonucleotide analogues and ol igonucleotides.
- Example 1 relates to the preparation of a spin label precursor compound
- Example 2 relates to the introduction of that molecule into AMP and further elaboration to produce a labelled compound suitable for use as a probe.
- Example 3 relates to the
- N-Acetoxy-2,2,6,6-tetramethyl-4-piperidone (6), 4-Oxo-2,2,6,6-tetramethyl piperidin-1-oxyl (20g, 188 mmol) was melted by gentle warming and treated with a solution of L-ascorbic acid (37.6g, 190 mmol) in water (320 ml). The solution was stirred vigorously at ambient temperature for 5 min, during which its colour changed rapidly from dark red to pale yellow. It was then diluted with saturated aqueous NaHCO 3 (800 ml) in a 5 litre conical flask and cooled in ice.
- AMP monohydrate (free acid form) (2.2g, 6 mmol) was suspended in dry dimethylformamide (50 ml) and the solvent was removed under vacuum ( ⁇ 1 mm Hg) at a bath temperature of 35°C. The procedure was repeated a further three times in order to remove traces of water from the nucleotide, and at the end of the final cycle the residue was thoroughly pumped under vacuum to ensure complete removal of the dimethylformamide.
- toluenesulphonic acid monohydrate (5.7g; 30 mmol) was similarly dried by repeated vacuum evaporation from anhydrous acetonitrile (4 ⁇ 50 ml), then dissolved in anhydrous acetonitrile (440 ml) together with the enol ether (1) (20g, 88 mmol).
- This solution was adde rapidly to the dried nucleotide and the resulting suspension was stirre for 7 days at room temperature.
- the undissolved fraction of the AMP was removed by filtration, the filtrate was diluted with 10 mM triethylammonium bicarbonate (TEAB) buffer, pH 7.4 (2 1) and the mixture was extracted with petroleum ether (3 ⁇ 400 ml).
- TEAB triethylammonium bicarbonate
- the AMP spiroketal (10) (120 umol) was pyrophosphorylated by known procedures 2 ' 3 and purified by ion-exchange chromatography on DEAE-cellulose as described above, using a column of void volume 150 ml, a linear gradient formed from 10 and 350 mM TEAB (each 600 ml) and a flow rate of 50 ml/h. Fractions were analysed by reverse-phase h.p.l.c. (Conditions as above). The retention time for the ATP spiroketal (11) was 1.83 min). Fractions containing pure product were combined and freed from buffer salts as above to afford the ATP spiroketal acetate (11) as its
Abstract
Compounds of formulae (1), wherein R represents a CH3 or CD3 group; R1 represents an alkyl group; R2 represents an alkyl or aryl group; and N may be either an ?14N or 15¿N atom, with the proviso that when R represents CD¿3?, the methylene and vinyl hydrogen atoms of the six-membered ring are deuterium; and (2), wherein R and N are as defined above, X represents hydrogen or a mono-, di- or triphosphate group, or a phosphate ester derivative; and Y represents a purine or pyrimidine base, with the proviso that when R represents CD3, the methylene hydrogen atoms of the six-membered piperidine ring are hydrogen or deuterium. Processes for the preparation of these compounds are also described. The spin labelled nucleotide and nucleoside compounds are suitable for use as probes, for example, in protein structure and orientation studies.
Description
SYNTHESIS AND USES OF SPIN LABELLED
RIBONUCLEOSIDES AND RIBONUCLEOTI DES
This invention relates to the preparation of spin labelled compounds from novel precursors and their use as probes. More specifically, the invention provides conformationally restricted spin labelled
ribonucleosides and ribonucleotides which are useful, for example, in protein orientation studies.
Spin labelled probes have proved useful in dynamic and orientation studies of proteins using electron spin resonance (ESR), such as monitoring the movement of protein cross-bridges during muscle contraction. A significant problem with existing spin labelled
nucleotide probes, however, is that the spin label moiety is mobile and thus does not give accurate
information about the position of the nucleotide probe on the protein which is under investigation.
The use of spin label molecules in nuclear magnetic resonance (NMR) work is also well known. Their
presence in labelled probes produces characteristic line broadening effects in the NMR spectra which can give important information during, for example, structural studies of a variety of biological macromolecules.
A number of reactions to produce various derivatives of nucleosides or nucleotides have previously been described. Hampton, J. Am. Chem. Soc, 83, 3640
(1961); Hampton et al., J. Am. Chem. Soc, 87, 5481 (1965); Reese et al., Tetrahedron, 26, 1023 (1970) and Chladek & Smrt, Coll. Czech. Chem. Commun., 28, 1301 (1963) illustrate a range of reactions that
essentially involve the acid-catalysed additions of ketones, ketals or enol ethers to the vicinal diol of the ribose ring. Grindley et al., Carbohydrate Res., 140, 215 (1985) describe the use of
1-ethoxycyclohexene and 2-methoxypropene to form cyclic ketals of a range of non-ribose sugars. Hai et al., J. Med. Chem., 25, 806 (1982) disclose the direct ketalisation of a nucleotide compound.
There is a need for spin labelled nucleoside and nucleotide analogues which overcome the aforementioned disadvantage of existing labelled probes. The present invention aims to provide such compounds.
According to the present invention there are provided compounds of the formula:-
R1 represents an alkyl group;
R2 represents an alkyl or aryl group; and N may be either an 14N or 15N atom,
with the proviso that when R represents CD3, the methylene and vinyl hydrogen atoms of the six-membered ring are deuterium.
In principle, R1 may be any alkyl group and R2 may be any alkyl or aryl group. It will be appreciated, however, that the presence of relatively small alkyl and aryl groups is usually to be preferred.
These compounds are useful as precursors of spin labelled nucleoside and nucleotide analogues. They have been found to be capable of reacting with
ribonucleosides and ribonucleotides to produce
spiroketal compounds that, on further modification, lead readily to spin labelled compounds suitable for use as probes.
According to a further embodiment of the present invention there is provided a spin labelled compound of the formula:-
wherein R is as defined above
X represents hydrogen, a mono-, di- or
triphosphate group, or a phosphate ester derivative; and
Y represents a purine or pyrimidine base, with the further proviso that when R represents CD3, the methylene hydrogen atoms of the six-membered
piperidine ring are hydrogen or deuterium.
If desired, either of X or Y may be radiolabel led.
Examples of group X as a phosphate ester derivative include the 3-thiotriphosphate, the
p3-1-(2-nitrophenyl)ethyl group or an ol igonucleotide that would produce spin labelled derivatives of ATP(ɣS), caged ATP or an ol igonucleotide.
As will be readily appreciated, the spin labelled compound (2) incorporates the skeleton of the
aforementioned type of compound (1).
The spin labelled nucleotide and nucleoside compounds of this invention may comprise either a purine or a pyrimidine base. Thus, in the above formula, group Y can represent adenine, guanine, cytosine, uracil, thymine or 5-methylcytosine. A particularly preferred spin labelled compound of the invention has the
following formula (wherein X is triphosphate, Y is adenine and R is as defined above):-
According to the present invention there is further provided a method for preparing a precursor compound (1) of the type defined above and which comprises the following sequence of reactions:-
Steps i), ii) and iii) involve relatively conventional chemistry, so that suitable reactants and process conditions will be readily appreciated by those skilled in the art. The initial reduction of step i) may be performed, for example, in the presence of ascorbic acid. The product is acylated in step ii), for example using acetic anhydride, and then converted in step iii) to a ketal. Finally, in step iv) the ketal is converted to an enol ether. This latter product is the desired precursor compound (1) suitable for use in the synthesis of the spin labelled
compounds. Step (iv) proceeds with unexpected
facility, because the unfavourable 1,3-diaxial
interactions present in the ketal are partially relieved in the enol ether. Steps i) to iv) are illustrated in Example 1 below.
According to the present invention there is still further provided a method for preparing a spin
labelled compound, involving the precursor compound (1) of the aforementioned type, which comprises the following sequences of reactions:-
wherein Z is a protecting group for the 5'-hydroxyl position and which may either equate to group X (as hereinbefore defined, except when X represents hydrogen) or be capable of conversion or remo v a l to leave a group 1 in that position; and
R, R1 , R2 and Y are as previously defined, with the further proviso that when R represents CD3, the 2'- and 3'- hydroxyl hydrogen atoms in compound (8) may be deuterium in order that all four methylene hydrogen atoms of the six-membered piperidine ring of compound (9) are deuterium; vi) either a) where group 2 equates to group X, optionally further phosphorylating the compound (9) at that position, or b) treating the compound (9) such that Z is removed, and optionally phosphorylating, to leave a group X at that position; and
It will be appreciated that the route followed in step vi) will depend upon the nature of group Z. Examples of the group Z, when Z does not equate with X, include acyl (R3CO-, where R3 may be alkyl, alkenyl, cycloalkyl or aryl), alkoxycarbonyl (R3OCO-, where R3 is as defined above), allyl or substituted benzyl, such as 2-methoxybenzyl, o-nitrobenzyl or 1-(2-nitrophenyl)ethyl. Z may be a group that is retained in the final product, such as a phosphate (as in
Example 2 below), or a group that is subsequently removed, such as an acyl group (as in Example 3 below). In any event, step vi) has the effect of converting group Z to group X. In compound (9) when protecting group Z is removed by alkaline hydrolysis (i.e. is acyl or alkoxycarbonyl), isolation of compound (9a), where X is hydrogen, is still possible because the protecting group is more susceptible to alkaline hydrolysis than the N-acyloxy group that is removed in step vii).
The purpose of the above sequences of reactions is to condense compound (1) with a ribonucleotide or
ribonucleoside and then to produce a spin labelled compound (2) which can be used as a probe in ESR or NMR work. The initial conversion to a spiroketal (9) is followed by an optional phosphorylation
or further phosphorylation, or a treatment such as hydrolysis, using conventional techniques.
Subsequently, the compound is subjected to alkaline hydrolysis of the N-acyloxy group and oxidation to produce the spin labelled compound (2) of the invention.
In a reaction to produce a particularly preferred spin labelled compound of this invention, step v) comprises reacting a precursor compound (1), where R, R1 and R2 are each CH3, with adenosine 5'-monophosphate (AMP) to produce a compound of the formula:-
step vi) comprises pyrophosphorylating the product of step v) to produce a compound of the formula:-
)
the step vii) comprises deacetylation and air
As an example to illustrate the applicability of the procedure to ribonucleosides, 5'-0-benzoyluridine is reacted with a precursor compound (1) as in step v) to produce compound (13):-
In steps vi) and vii) the benzoyl and acetyl groups are sequentially removed and the resulting N-hydroxy compound oxidized in air to produce a compound of the formula (14):-
Substantial difficulties were encountered in the construction of precursors to the desired spin
labelled compounds. Conventional acid catalysed condensations (as referenced above) of the ketone (6) or ketal (7) with ribonucleosides and ribonucleotides failed and even the enol ether (1) was substantially less reactive than the less sterically compressed
1-ethoxycyclohexene (the compound used by Grindley et al., see above). This lack of reactivity is accounted for by unfavourable 1,3-diaxial interactions which are created when an alcohol group adds to the enol ether (1) and the further element of ring strain when the spiroketal (9) is formed. A further surprising feature of the condensation is the fact that it only occurs in essentially non-polar solvents. Even the presence of 10% dimethylformamide in the reaction mixture
inhibits the reaction almost completely. This makes for considerable difficulty because of the almost total insolubility of ribonucleotides in non-polar solvents. Finally the purine series was difficult to synthesize because of the lability of the purine- ribose bond under the acid-catalyzed conditions of the synthesis.
Both Broensted-Lowry and Lewis acids were effective in the catalysis of the condensation reactions [step v)]. Overall p-toluenesulphonic acid was found to be the most satisfactory acid catalyst.
A range of spin labelled compounds for use as probes, or as a part of such probes, can be derived from the precursor compound (1) of the present invention. A particular benefit is that the spin label is rigidly attached to nucleotide or nucleoside sugar residues and thereby, for example, rigidly oriented on proteins, This tight coupling of the spin label avoids the previously mentioned disadvantage of mobile nucleotide labels and thus opens up the possibility of new and improved orientation or structure studies of certain proteins. For instance, the use of such probes should enable betterinvestigation of how muscle cross-bridges move during contraction and relaxation, or how DNA interacts with DNA-binding proteins that control gene expression.
The spin label precursor compounds (1) of this
invention are suitable for use with all ribonucleotide and ribonucleoside types and thus, for example, could be incorporated into 5'-0-acylated derivatives of
adenosine, cytidine, guanosine, or uridine, or with AMP, CMP, GMP or UMP. It is further envisaged that the spin label precursor compounds (1) could be used to produce other types of labelled probes based on, for example, the common ribonucleoside 5' di- and 5'-triphosphates, caged ATP compounds (i.e. photo-labile derivatives of ATP from which ATP can be readily regenerated), pyridine nucleotides (e.g. NAD+, NADH), ribonucleotide analogues and ol igonucleotides.
The compounds and methods of this invention will now be further illustrated by reference to the following Examples. Example 1 relates to the preparation of a spin label precursor compound; Example 2 relates to the introduction of that molecule into AMP and further elaboration to produce a labelled compound suitable for use as a probe. Example 3 relates to the
introduction of a spin label precursor into a typical ribonucleoside derivative, 5'-0-benzoyluridine.
EXAMPLE 1
N-Acetoxy-2,2,6,6-tetramethyl-4-piperidone (6), 4-Oxo-2,2,6,6-tetramethyl piperidin-1-oxyl (20g, 188 mmol) was melted by gentle warming and treated with a solution of L-ascorbic acid (37.6g, 190 mmol) in water (320 ml). The solution was stirred vigorously at ambient temperature for 5 min, during which its colour changed rapidly from dark red to pale yellow. It was then diluted with saturated aqueous NaHCO3 (800 ml) in a 5 litre conical flask and cooled in ice. Acetic anhydride (64 ml, 679 rrmol) was added over 2 min to the stirred mixture ( pH 8) . Portions of sol id NaHCO3 were added carefully to maintain the mixture at pH 8 until no further pH chang occurred (ca. 1 h) and the mixture was then extracted with CHCl3 (3 × 200 ml). The combined CHCI3 extract was washed with saturated aqueous NaHCO3 (2 × 200 ml), dried (Na2SO4) and evaporated under reduced pressure, to leave the ketoacetate (6) as a. pale solid (23.3g, 94%). A sample recrystallised from petroleum ether gave pale needles, m.p. 95-95.5°C. Anal: Calc. for C11H19NO3: C, 61.9; H, 9.0; N, 6.6; M.W 213.1365. Found: C, 62.0; H, 8.6; N, 6.6; M+ 213.1362. N-Acetoxy-4.4-dimethoxy-2.2.6.6- tetramethylpiperidine (7) . The crude ketoacetate (6) (23.5g, 110 mnol) and toluenesulphonic acid monohydrate (2.1g, 11 mmol) were dissolved in a mixture of methanol (250 ml) and trimethyl orthoformate (250 ml) and the solution was heated under reflux for 2 h, then cooled to room temperature. 3% Aqueous NaHCO3 (750 ml) was added and the mixture was extracted with CHCI3 (3 × 200 ml). The combined CHCl3 extract was dried (Na2SO4) and evaporated under reduced pressure. Distillation of the residual oil afforded the ketal (7) as a pale liquid (25.9g; 91%), b.p. 84°C (0.5 mm Hg) . Anal: Calc. for C13H25NO4 : M.W. 259.1784. Found: M+ 259.1770.
N-Acetoxy-4-methoxy-2,2,6,6-tetramethyl-3-piperideine ( 1 ). A solution toluenesulphonic acid monohydrate (684 mg, 3.6 mmol) in benzene (400 ml) was heated under reflux in a flask fitted with a Dean and Stark trap until no further water separated (ca. 30 min). The water was run off and a solution of the ketal (7) (14.4g, 56 mmol) in benzene (25 ml) was added to the toluenesulphonic acid solution. The mixture was heated under reflux for 30 min and the contents of the trap were run off twice during this period to remove entrained methanol. The mixture was then cooled to room temperature, washed with saturated aqueous NaHCO3 (2 × 200 ml), dried (Na2SO4) and the solvent removed under reduced pressure. The residual oil was purified in batches by short-path distillation at 0.8 mm Hg (Kugelrohr, oven temperature 150°C) to give the enol ether (1) as a pale oil (11.5g; 91%) which solidified to a waxy solid, m.p. 37-40°C on standing at 4°C Anal: Calc. for C12H21NO3 : M.W. 227.1522. Found: M+ 227.1519.
EXAMPLE 2
2'-0.3'-0-(N-Acetoxy-2,2,6,6-tetramethylpiperidinylidene')adenosine
monophosphate (10). AMP monohydrate (free acid form) (2.2g, 6 mmol) was suspended in dry dimethylformamide (50 ml) and the solvent was removed under vacuum (< 1 mm Hg) at a bath temperature of 35°C. The procedure was repeated a further three times in order to remove traces of water from the nucleotide, and at the end of the final cycle the residue was thoroughly pumped under vacuum to ensure complete removal of the dimethylformamide.
In a separate flask, toluenesulphonic acid monohydrate (5.7g; 30 mmol) was similarly dried by repeated vacuum evaporation from anhydrous acetonitrile (4 × 50 ml), then dissolved in anhydrous acetonitrile (440 ml) together with the enol ether (1) (20g, 88 mmol). This solution was adde rapidly to the dried nucleotide and the resulting suspension was stirre for 7 days at room temperature. The undissolved fraction of the AMP was removed by filtration, the filtrate was diluted with 10 mM triethylammonium
bicarbonate (TEAB) buffer, pH 7.4 (2 1) and the mixture was extracted with petroleum ether (3 × 400 ml). The organic extracts were discarded and the aqueous solution was adjusted to pH 7.0 and applied to a DEAE-cellulose column (500 ml void volume) at a flow rate of 80 ml/h. When fully loaded, the column was washed with 10 mM TEAB, pH 7.4 until the absorbance (260 nm) of the effluent returned to zero, then eluted with a linear gradient formed from 10 and 250 mM TEAB, pH 7.4 (each 1.5 1). Fractions were collected in
13 ml aliquots and monitored by u.v. and analytical h.p.l.c. (Merck Lichrocart C8 column, mobile phase 45% MeOH - 55% 10 mM sodium acetate pH
6.5, flow rate 1.5 ml/min. Retention times for AMP and the spiroketal ( 10) were 1.5 and 3.3 min respectively). All fractions containing the spiroketal (10) were combined and concentrated under reduced pressure, then evaporated under vacuum with methanol several times to remove buffer salts. The product was further purified by reverse -phase preparative h.p.l.c. (Waters C18, mobile phase 35% methanol - 65% 10 mM sodium acetate pH 6.5, flow rate 4 ml/min). Fractions of 16 ml were collected and assayed by analytical h.p.l.c. (see above) and those containing pure spiroketal (10) were combined and concentrated under reduced pressure to remove most of the methanol and finally desalted on a DEAE cellulose column as described above. The eluted material was freed of buffer salts as described to give the pure spiroketal (10)as its triethylarnmonium salt (1.2 mmol; 204).
2'-0,3'-0-(N-Acetoxy-2,2,6,6-tetrarnethylpiperidinylidene)adenosine triphosphate (11). The AMP spiroketal (10) (120 umol) was pyrophosphorylated by known procedures 2'3 and purified by ion-exchange chromatography on DEAE-cellulose as described above, using a column of void volume 150 ml, a linear gradient formed from 10 and 350 mM TEAB (each 600 ml) and a flow rate of 50 ml/h. Fractions were analysed by reverse-phase h.p.l.c. (Conditions as above). The retention time for the ATP spiroketal
(11) was 1.83 min). Fractions containing pure product were combined and freed from buffer salts as above to afford the ATP spiroketal acetate (11) as its
triethylammonium salt (57 μmol; 47%).
2'-0,3'-0-(N-0xyl-2,2,6,6-tetramethylpiperidinylidene) adenos i ne tr i pho s ph ate ( 1 2 ) . To a solution of the spiroketal (11) (400 μmol) in 50% aqueous methanol (62 ml) was added a solution of KOH (1.34g) in
methanol (21 ml), and the mixture was left at room temperature and open to the atmosphere. The reaction was monitored by h.p.l.c. (Merck Lichrocart C8, mobile phase 12% MeCN - 88% 50 mM KH2PO4 pH 5.5, flow rate 1.5 ml/min. Retention times for the spiroketal (11) and the ATP spin label (12) were 4.0 and 13.0 min respectively). When the hydrolysis/oxidation reaction was complete (48 h), the reaction mixture was
neutralised with 1M HCl (24 ml) and diluted with 10 mM TEAB (600 ml). The pH was adjusted to 7.0 and the solution was applied to a DEAE-cellulose column (void volume 400 ml) at a flow rate of 80 ml/h. The column was eluted with a linear gradient formed from 10 and 350 mM TEAB (each 2 1) and fractions were analysed by h.p.l.c. Fractions containing the product were seen to contain an impurity (approx. 3%; h.p.l.c. retention time 1.2 min) which was identified as free ATP. These fractions were combined and freed from buffer salts as above. A portion of the contaminated spin-labelled product (100 μmol) was further purified by preparative h.p.l.c. (Waters C18, flow rate 4 ml/min). The preparative column was eluted first with 10 mM sodium acetate, pH 6.5 until all the free ATP had eluted, then with 5% MeCN - 95% 10 mM sodium acetate, pH 6.5. Fractions containing the pure spin label (12) (90 μmol)
were pooled, desalted on DEAE-cellulose as described above and stored at -20°C either as the
triethylammonium salt or after treatment with Dowex 50 (Na+ form) as the sodium salt.
EXAMPLE 3
2'-0,3'-0-(N-Acetoxy-2,2,6,6-tetramethylpiperidinylidene)
-5'-O-benzoyluridine (13). 5'-0-benzoyluridine (350 mg,
1 mmol) was added to a solution of toluenesulphonic acid monohydrate (190 mg, 1 mmol) in dry
tetrahydrofuran (5.6 ml), followed by addition of N-acetoxy-2,2,6,6-tetramethyl-3-piperideine (2.2 g, 9.7 mmol). The solution was left at room temperature for 7 days, then diluted with ethyl acetate (100 ml) and washed with saturated NaHCO3, dried (Na2SO4) and
evaporated. The oily residue was purified by flash chromatography (Merck 9385 silica gel) using ethyl acetate - petroleum ether (3:2) as the eluting solvent to give the title product (13) as a pale gum (311 mg; 57%). The material was homogeneous by thin layer
chromatography on silica gel (ethyl acetate-light
petroleum 3 :2;Rf0.35) and spectroscopic properties
(IR, 1H and 13C NMR,UV) were in conformity with its structure.
References
CM. Paleos and P. Dais, J. Chem. Soc. Chem, Commun., 1977, 345-346.
D.E. Hoard and D.G. Ott, J. Am. Chem. Soc, 1965, 87, 1785-1788.
M. Maeda, A.D. Patel and A. Hampton, Nucleic Acids Res., 1977, 4, 2843-2853.
Claims
1. A compound of the formula:-
wherein R represents a CH3 or CD3 group;
R1 represents an alkyl group;
R2 represents an alkyl or aryl group; and N may be either an 14N or 15N atom,
with the proviso that when R represents CD3, the methylene and vinyl hydrogen atoms of the six-membered ring are deuterium.
2. A compound as claimed in claim 1, wherein each of R, R1 and R2 is methyl.
wherein R represents a CH3 or CD3 group;
N may be either an 14N or 15N atom;
X represents hydrogen or a mono-, di- or triphosphate group, or a phosphate ester derivative; and
Y represents a purine or pyrimidine base, with the proviso that when R represents CD3, the methylene hydrogen atoms of the six-membered piperidine ring are hydrogen or deuterium.
4. A compound as claimed in claim 3 of the formula:-
5. A compound as claimed in claim 4, wherein R is methyl.
6. A method for preparing a compound as claimed in claim 1, which comprises the following sequence of reactions:-
7. A method for preparing a compound as claimed in claim 3, which comprises the following sequence of reactions:-
wherein Z is a protecting group for the 5'-hydroxyl position and which may either equate to group X (as hereinbefore defined, except when X represents hydrogen) or be capable of conversion or removal to leave a group X in that position; and
R, R1 , R2 and Y are as previously defined, with the further proviso that when R represents CD3, the 2'- and 3'-hydroxyl hydrogen atoms in compound (8) may be deuterium in order that all four methylene hydrogen atoms of the six-membered piperidine ring of compound (9) are deuterium; vi) either a) where group Z equates to group X, optionally further phosphorylating the compound (9) at that position, or b) treating the compound (9) such that Z is removed, and optionally phosphorylating, to leave a group X at that position; and
8. A method as claimed in claim 7, wherein step v) comprises reacting compound (1) with adenosine
5'-monophosphate to produce a compound of the formula:-
step vi) comprises pyrophosphorylating the product of step v) to produce a compound of the formula:-
oxidation of the product of step vi) to produce a compound of the formula:-
9. Use of a compound as claimed in claim 3 as a spin labelled probe or as a part of such a probe.
10. A method of investigating or determining protein orientation or structure which comprises the use of a compound as claimed in claim 3 or claim 4.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB909015684A GB9015684D0 (en) | 1990-07-17 | 1990-07-17 | Synthesis and uses of spin labelled ribonucleosides and ribonucleotides |
GB9015684.5 | 1990-07-17 |
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WO1992001673A1 true WO1992001673A1 (en) | 1992-02-06 |
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ID=10679207
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Application Number | Title | Priority Date | Filing Date |
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PCT/GB1991/001146 WO1992001673A1 (en) | 1990-07-17 | 1991-07-11 | Synthesis and uses of spin labelled ribonucleosides and ribonucleotides |
Country Status (3)
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AU (1) | AU8193991A (en) |
GB (1) | GB9015684D0 (en) |
WO (1) | WO1992001673A1 (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7018985B1 (en) | 2000-08-21 | 2006-03-28 | Inspire Pharmaceuticals, Inc. | Composition and method for inhibiting platelet aggregation |
US7132408B2 (en) | 2000-08-21 | 2006-11-07 | Inspire Pharmaceuticals, Inc. | Composition and method for inhibiting platelet aggregation |
US7452870B2 (en) | 2000-08-21 | 2008-11-18 | Inspire Pharmaceuticals, Inc. | Drug-eluting stents coated with P2Y12 receptor antagonist compound |
US11566038B2 (en) | 2020-11-11 | 2023-01-31 | deutraMed Solutions Ltd. | Deuterium-stabilised ribonucleic acid (RNA) molecules displaying increased resistance to thermal and enzymatic hydrolysis, aqueous compositions comprising stabilised RNA molecules and methods for making same |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2235103A1 (en) * | 1973-06-29 | 1975-01-24 | Commissariat Energie Atomique | Marked nitroxide derivatives of saccharides - by reaction of acid nitroxide with halogenated saccharides |
EP0133674A1 (en) * | 1983-08-03 | 1985-03-06 | Schering Aktiengesellschaft | Nitroxyl compounds, process for their preparation and diagnostic agents containing them |
-
1990
- 1990-07-17 GB GB909015684A patent/GB9015684D0/en active Pending
-
1991
- 1991-07-11 AU AU81939/91A patent/AU8193991A/en not_active Abandoned
- 1991-07-11 WO PCT/GB1991/001146 patent/WO1992001673A1/en unknown
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR2235103A1 (en) * | 1973-06-29 | 1975-01-24 | Commissariat Energie Atomique | Marked nitroxide derivatives of saccharides - by reaction of acid nitroxide with halogenated saccharides |
EP0133674A1 (en) * | 1983-08-03 | 1985-03-06 | Schering Aktiengesellschaft | Nitroxyl compounds, process for their preparation and diagnostic agents containing them |
Cited By (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7018985B1 (en) | 2000-08-21 | 2006-03-28 | Inspire Pharmaceuticals, Inc. | Composition and method for inhibiting platelet aggregation |
US7101860B2 (en) | 2000-08-21 | 2006-09-05 | Inspire Pharmaceuticals, Inc. | Composition and method for inhibiting platelet aggregation |
US7132408B2 (en) | 2000-08-21 | 2006-11-07 | Inspire Pharmaceuticals, Inc. | Composition and method for inhibiting platelet aggregation |
US7452870B2 (en) | 2000-08-21 | 2008-11-18 | Inspire Pharmaceuticals, Inc. | Drug-eluting stents coated with P2Y12 receptor antagonist compound |
US7618949B2 (en) | 2000-08-21 | 2009-11-17 | Inspire Pharmaceuticals, Inc. | Drug-eluting stents coated with P2Y12 receptor antagonist compound |
US11566038B2 (en) | 2020-11-11 | 2023-01-31 | deutraMed Solutions Ltd. | Deuterium-stabilised ribonucleic acid (RNA) molecules displaying increased resistance to thermal and enzymatic hydrolysis, aqueous compositions comprising stabilised RNA molecules and methods for making same |
US11780869B2 (en) | 2020-11-11 | 2023-10-10 | deutraMed Solutions Ltd. | Deuterium-stabilised ribonucleic acid (RNA) molecules displaying increased resistance to thermal and enzymatic hydrolysis, aqueous compositions comprising stabilised RNA molecules and methods for making same |
Also Published As
Publication number | Publication date |
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AU8193991A (en) | 1992-02-18 |
GB9015684D0 (en) | 1990-09-05 |
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