JPH0411554B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to chromogenic and/or fluorescent mononucleotide 3'-phosphodiesters, and more particularly to a novel method for the synthesis of such mononucleotide phosphodiesters. These substances are used, for example, to perform various non-isotopic immunoassays. Farina and Gohlke, filed on the same day as the present application, Methods for conducting isotope-free immunoassays, labeled analytes and kits for use in such assays, filed on the same day as the present application. Gohlke, Hedaya, Kang and Mier, Novel Chromogenic and/or Fluorogenic Substrates for Tracking Catalytic or Enzymatic Activity, filed on the same day as the present application. Kang Methods for making chromogenic and/or fluorogenic substrates for use in tracking catalytic or enzymatic activity are related inventions. For various clinical purposes, such as monitoring dosage schedules, tracking hormone levels, checking recent intake or bioavailability, following pharmacokinetics of absorption, degradation or excretion, etc. It is highly advantageous to measure drug concentrations to nanomolar or even picomolar levels. As is known, radioimmunoassays can accomplish this type of analysis. In order to perform the analysis,
Available kits or systems must contain an antiserum, a standard compound to be measured (e.g., an analyte analyte), a radiolabeled derivative of the compound to be measured, one or more buffers, and often a substituent, etc. No. Antiserum can be obtained, for example, by collecting blood from an animal that has been immunized by inoculation with a hapten-protein complex (immunogen) corresponding to the compound to be measured. As is well known, radioimmunoassay techniques generally measure the competition between radiolabeled and unlabeled analytes for the binding sites of antibodies in antiserum. Adding a known amount of the analyte to be measured and a radiolabeled analog to the antiserum provides a dose response curve for bound or free analyte versus analyte concentration. After immunocorrection is performed, the unknown concentration is compared to a standard dose-response curve for assay. Important for this type of analysis is the presence of a radioactive analyte that effectively competes with the non-radioactive analyte. Therefore, the highest precision, accuracy, sensitivity,
Purified and well-characterized synthetic radioactive analytes are required to achieve specificity and reproducibility. Several deficiencies in radioimmunoassay methods have been identified. First, the radiolabeled analyte bound to the antibody must be physically separated from the free radiolabeled analyte. Furthermore, this method is considered labor-intensive, the necessary equipment is likewise relatively expensive, and uniformity cannot be obtained;
Furthermore, accurate performance of such tests requires the use of highly trained and skilled technicians. Radioisotope-labeled analytes are also relatively unstable and expensive, and present a significant and growing waste disposal problem due to the radiation exposure risks associated with commonly used radioisotope labels. Despite these drawbacks, the use of radioimmunoassays has expanded considerably. The recent substantial increase in the use of radioimmunoassays in clinical laboratories has spurred the development of alternative methods that overcome the deficiencies of radioimmunoassays described above. Methods that have been developed to overcome these deficiencies include:
The use of enzymatic or fluorophore labels instead of primarily radioisotope labels, preferably combined with conditions that allow the binding of labeled analytes and the measurement of chemical differences between the free fractions, allows for physical separation. Eliminating the need. Immunoassays with the latter simplified and advantageous characteristics are referred to as homogeneous immunoassays as opposed to heterogeneous immunoassays that require physical separation. Thus, a uniform immunoassay system was developed,
It is based on the use of enzyme-labeled analytes in which the enzymatic activity of the label decreases when complex formation with the antibody occurs. The unlabeled analyte, the concentration of which is measured, displaces the enzyme-labeled analyte bound to the antibody, thus resulting in an increase in enzyme activity. Standard substitution or dose-response curves are followed spectroscopically using increasing enzyme activity (referred to as the "substrate", which ultimately produces a unique chromophore as a result of enzymatic action). ) is plotted against increasing analyte concentration. These are then used to measure unknown analyte concentrations. The following US patents were disclosed in the field of homogeneous enzyme immunoassays:
3817837; 3852157; 3875011; 3966556;
3905871; 4065354; 4043872; 4040907;
4039385; 4046636; 4067774; 4191613; and
4171244. These patents state that the label for the analyte is essentially an enzyme having a molecular weight of 5000 or more. Furthermore, commercialization of this technique is limited to applications where the liquid concentration of the analyte is greater than 10 ^-10 M and the molecular size of the analyte is relatively small. As a result of the limitations of the homogeneous enzyme immunoassay described above,
Considerable effort has been directed toward the development of more photosensitive homogeneous immunoassays using fluorescence. This was primarily directed to the assay of larger molecules, such as immunoglobulins or polypeptide hormones such as insulin. The following US patents are disclosed for this type of assay: 3998943;
3996345; 4174384; 4161515; 4208479; 4160016.
The label in most of these patents is an aromatic, fluorescent molecule that is attached to either the analyte or the antibody.
Similarly, all of the patents describe various methods of quenching fluorescence through antibodies or other fluorescence quenchers in which the degree of quenching is related to the amount of analyte present in the sample. Another type of method, described as a reactant-labeled fluorescent immunoassay, uses a fluorescently labeled analyte in which the fluorescent product is designed to be released upon enzymatic hydrolysis. However, antibodies inhibit enzymatic hydrolysis of the analyte portion of the molecule. the result,
Due to the law of mass action, fluorescence is enhanced in the presence of increased analyte due to enzymatic hydrolysis of the displaced fluorescently labeled analyte. An example of a labeled analyte is β-galactosyl-umbelliferon-sisomicin. The enzyme β-galactosidase cleaves the sugar from the umbelliferon moiety, which allows umbelliferon to emit fluorescence. Literature describing this method includes: JF Burd, R. C. RCWong, J. E. Feeney (J.
E.Feeney), R.J., Kariko (RJ
Carrico) and R.C. Boguolaski, Clin.Chem. 23 1402
(1977); Burd, Carrico, M. C. Fetter (MCFetter)Other
Anal.Biochem. 77 , 56 (1977) and F. F.Kohen, Zett. Holland (Z.Holland)
and Jour.of Boguolaski.
Steroid Biochem. 11 161 (1979), etc. The previously mentioned co-pending Farina et al. applications provide methods for performing non-radioactive immunoassays that circumvent the deficiencies of this common type of prior analysis. Specifically, this method is
It utilizes a labeled analyte-polypeptide complex that exhibits ribonuclease-type activity that catalytically converts a substrate into a chromogenic or fluorescent reporter molecule. Many organic compounds have been used to detect the catalytic activity of ribonucleases. Such organic compounds or substrates include ribonucleic acids themselves, as they are commonly referred to, as cyclic phosphodiester and monoribonucleotide compounds that exhibit the same or similar structural constraints exhibited by natural substrates. . Thus, for example, one method of tracking the catalytic activity of ribonucleases is to use ribonucleic acid solutions. The method tracks the decrease in absorbance at 300 nm of a ribonucleic acid solution as a function of time.
M. M.Kunitz J.Biol.Chem., 164
563 (1946). Although this method is relatively simple to perform, it has several drawbacks; in particular, the rate of absorbance decrease is not linear, correction of each substrate solution is required, and direct detection of absorbance decrease at 300 nm is not possible. It is not practical for clinical samples. Other methods used to detect ribonuclease activity are endpoint variations of the methods described above. The endpoint variation technique incubates yeast ribonucleic acid with an enzyme sample for a period of time. Residual RNA is precipitated with perchloric acid or uranyl acetate/trifluoroacetic acid, and after centrifugation, the absorbance of the supernatant is measured. S. B. An Huinsen (SB
Anfinsen), RR Redfield, Dub. L.
WLChoate, A.Page and Dubrill.
R. Karol (WRCarrol), Jour.Biol.
Chem., 207 , 201 (1954). However, since this method mainly involves a precipitation step, the
Too cumbersome compared to the homogeneous immunoassay described in Farina et al. This is a further modification of the above method. C. Cam (RCKamm), A. G. AG Smith and H. Lions (H.
Lyons), Analyt.Biochem. 37 333 (1970). The method described herein is based on the formation of a fluorescent reaction product resulting from the reaction of the dye ethidium bromide with intact yeast ribonucleic acid (but not with the hydrolyzate). In this method, the tracked fluorescent signal decreases over time. However, tracking a fluorescent signal that decreases over time is disadvantageous since the method lacks sensitivity unless the difference in enzyme concentration is too large. Yet another drawback is that the rate of decrease in absorption is not linear and requires correction for each substrate solution. Another known substrate for tracking ribonuclease activity is the mononucleotide substrate cytidine 2′,
There are 3'-phosphate diesters. E. M. EMCrook, A. P. APMathias and B. R. BRRabin, Biochem.J., 74 234 (1960). In this method, the increase in absorbance at 286 nm, which corresponds to hydrolysis of the cyclic phosphate ring, is followed over a period of 2 hours to determine the ribonuclease activity of the sample. However, this method cannot be used in homogeneous immunoassays of the type described in applications by Farina et al.
This is because interference with the specimen sample occurs at 286 nm. Furthermore, the difference between the absorbance spectra of the substrate and product is small, and the ratio of extinction coefficients is only 286 nm.
It's only 1.495. Furthermore, 3'-uridylic acid, 3'-inosinic acid and 3'-
Certain mononucleotide-3'-phosphodiesters, including 1-naphthyl esters such as adenylic acid, have been used as substrates for ribonucleases. These naphthyl esters have been used to distinguish between the substrate specificities of ribonucleases of various origins. It is being H. Sierrakowska (H.
Sierakowska), M. M. Zan-Kowalczewska and D. D. Shugar, Biochem.Biophys.Res.
Comm., 19 138 (1965); M. M. Zan-Kowalczewska, A. A. Sierakowska and D. D. Shugar Acta.Biochem.Polon. 13 ,
237 (1966); H. Sierrakowska (H.
Sierakowska) and Day. Shugar (D.
Shugar), Acta.Biochem.Polon 18 , 143 (1971);
H. H. Sierakowska, H. H.Szemplinska D. D. Shugar, Biochem.
Biophys.Res.Comm. 11 , 70 (1963). As a result of hydrolysis caused by ribonucleases,
The use of such substances liberates 1-naphthol,
It forms azo compounds with strong visible absorption when reacted with diazonium salts. This method requires that the assay kit contain individually packaged dye formers (ie, diazonium salts). Also, this substrate cannot be used in fluorescence analysis methods. Various synthetic methods have been developed for the production of mononucleotide-3'-phosphodiesters. One method for the preparation of uridine-3'-(1-naphthyl) phosphate is described by R. R.Kole and H. Sierrakowska (H.
Sierakowska), Acta Biochem. Polon, 18 187
(1971). In the method presented therein, uridine is acetylated at the 3'-hydroxyl group: Next, 2′- and 5′- of 3′-O-acetyluridine
Protection of the hydroxyl group with dihydropyran followed by hydrolysis of the 3′-O-acetyl to form 2′-5′-bis-O-(tetrahydropyranyl)uridine: 2',5'-bis-O-(tetrahydropyranyl)
Condensation of uridine with naphthyl phosphate dicyclohexyl carbodiimide or naphthyl phosphoryl dichloride results in 1-naphthyl phosphorylation of the 3'-hydroxyl group, resulting in a protected form of the substrate.
2′,5′-di-O-(tetrahydropyranyl)uridine-3′-(1-naphthyl)phosphate is formed: The tetrahydropyranyl protecting group is acid labile and is removed without competitive phosphate hydrolysis to form the substrate uridine-3'-(1-naphthyl) phosphate: A modification of the formula described in the paper by Sierakowska and Shugar cited above was published by Rubsamen, Khandlar and Witzel in Hoppe-Seyler. ) of Z.Physiol.Chem. 355 , 687
(1974). There, 2',5'-bis-O-(tetrahydropyranyl)-3'-uridine phosphate was combined with dihydropyran and uridine-3'-
Produced by reaction with phosphates. 2′,
5'-bis-O-(tetrahydropyranyl)-3'-uridine phosphate can be dephosphorylated, e.g. by phosphatase or lead hydroxide ().
2',5'-di-O-(tetrahydropyranyl)uridine is formed. The 3'-hydroxyl group of this compound is then phosphorylated to give the desired mononucleotide-3'-phosphodiester, such as uridine-3'-( 1-naphthyl) phosphate is formed. However, Sierakowska
The synthetic methods described by Rubsamen et al. have several serious deficiencies. For example, in each synthetic method, key intermediates
The preparation of 2',5'-bis-O-(tetrahydropyranyl)uridine involves undesirably lengthy chromatography. Furthermore, the resulting product is a mixture of diastereomeric pairs with low yields, which complicates subsequent synthesis. Finally, total synthesis is labor intensive. A synthetic method very similar to that of Sierakowska et al. and Rubsamen et al. is disclosed in Polish Patent No. 81969. In one of the synthetic methods described therein, 2',5'-di-O-(tetrahydropyrano-3'-(1-naphthyl)phosphate is synthesized in dicyclohexylcarbodiimide and pyridine.
Formed by the reaction of a salt of 1-naphthylphosphoric acid (e.g. a salt of this acid such as pyridine, aniline, lutidine or tri-n-butylamine) with 2',5'-di-O-(tetrahydropyranyl)uridine be done.
In another synthetic method described in that paper, uridine 2'-O-tetrahydropyranyl-5'-O-
Methyl-3'-(1-naphthyl) phosphate is obtained by reaction of 1-naphthyl phosphate with 5'-O-methyl-2'-O-(tetrahydropyranyl)uridine in pyridine. These synthetic methods similarly have the deficiencies of the Sierakowska et al. and Rubsamen et al. methods. In addition, methods for the synthesis of oligoribonucleotides are known that involve the synthesis of 2',5'-diprotected nucleotides as intermediates. Thus, J. Smilt (J.Smrt) and F. Solm (F.
Sorm), Collection Czechosloz.chem.Commun.
27 73 (1962), uridylic acid is 5'-O-
Acetyluridine 2′,3′-cyclic phosphate is converted to 5′-O-acetyluridine 3′-phosphate after enzymatic cleavage of the cyclic phosphate by pancreatic ribonuclease, and then by reaction with dihydropyran. It is converted to 2'-O-tetrahydropyranyl-5'-O-acetyluridine 3'-phosphate. In this method, the 5′-
Acetylation at hydroxyl groups has been used as a synthetic convenience to produce intermediates in the synthesis of oligoribonucleotides. 5′−
Deprotection of the acetyl occurs upon final formation of the desired oligoribonucletide. However, this chromogenic and/or fluorescent mononucleotide
No suitable method for the synthesis of 3'-phosphodiesters is given. Moreover, to the best of our knowledge, the method of Smrt et al. has so far not been used for the production of chromogenic and/or fluorescent mononucleotide 3'-phosphodiesters, despite the deficiencies of previous methods. do not have. Thus, although a considerable number of methods have been developed and used to synthesize various substrates suitable for use in tracking enzyme or catalytic activity, the variety of currently known synthetic methods is limited. There remains a need for further developments that can overcome these shortcomings. To the best of our knowledge, none of the synthetic methods reported so far are currently used commercially for the production of mononucleotide-3'-phosphodiesters. It is therefore an object of the present invention to provide a new method for synthesizing mononucleotide-3'-phosphodiesters having chromogenic and/or fluorescent functional groups in the 3' phosphate moiety of the furanoside ring. A related object is to provide a method for synthesizing such mononucleotides in a manner that eliminates the formation of undesired diastereomeric pairs. Another object of the present invention is to provide a new method for the synthesis of chromogenic and/or fluorescent mononucleotide 3'-phosphodiesters which is less labor intensive than previously known synthetic methods. Yet another object of the present invention is to provide a new method for the synthesis of chromogenic and/or fluorogenic mononucleotide-3'-phosphodiesters that improves the overall yield of the desired substrate. Yet another object is to provide a new method for the synthesis of chromogenic and/or fluorescent mononucleotide-3'-phosphodiesters that can be carried out in various amounts sufficient for commercial use. These and other objects and advantages of the invention will become apparent from the detailed description below. Since many modifications and alternative forms of the invention are possible, preferred embodiments will now be described in detail. However, it should be understood that the invention is not intended to be limited to the particular forms disclosed. On the contrary, the intention is to cover all modifications and alternative forms falling within the spirit and scope of the invention as defined by the appended claims. For example, although the present invention has been described primarily in connection with the preparation of uridine-3'-phosphodiester, it should be recognized that bases other than uracil may be used as described herein. Generally, the present invention provides color development and/or
or a mononucleotide having a fluorescent functional group.
A 3'-phosphodiester substrate can be easily synthesized from a 2',5'-diprotected mononucleotide by reaction with a compound having a desired chromogenic and/or fluorescent moiety; It is based on the discovery that protective colored and/or fluorescent matrices are formed. Generally, 2',3'-cyclic phosphates are first protected at the 5'-hydroxyl to form an intermediate, which is then 2'-
undergoes enzymatic cleavage of the ester bond, thereby creating the 5′-
Generates a protected-3'-mononucleotide. The 5'-protected-3'-mononucleotide is then reacted with a suitable protecting group to form a 2',5'-diprotected-3'-mononucleotide, which is further labeled with the desired color and/or fluorophore. to form a protected substrate. Another procedure is to dephosphorylate a 2',5'-diprotected-3'-mononucleotide to form a 2',5'-diprotected mononucleoside, which is then combined with the desired chromophore and/or fluorophore. can be reacted with phosphorylated derivatives of Chromogenic and/or fluorescent mononucleotides
The 3'-phosphodiester substrates can be used to monitor the catalytic activity of various enzymes, such as ribonuclease A, _T2, etc., and/or pairs of polypeptides with catalytic activity of such enzymes. The chromogenic and/or fluorescent mononucleotide substrates formed by the methods of the present invention are particularly useful in the immunoassays disclosed by the aforementioned co-pending application Farina et al. . According to the method of the invention, suitable starting materials include 2'- and 3'-mononucleotides having the following structural formula:
Consists of a mixture of phosphate isomers. [In the formula, B is a nucleotide base] In this structure, there are certain steric constraints,
It must be filled in order to ultimately provide a suitable substrate for monitoring the catalytic activity of hydrolysis induced by eg ribonuclease A or related polypeptide pairs. Thus, base B
and the trans and cis orientations of substituents at the 1'-, 2'-, and 3'-positions, respectively, appear to have fixed structural constraints to provide suitable substrates.
However, the substituent at the 4′-position, i.e., CH ₂ OH, can be added to the 2′-position without affecting the desired properties of the substrate.
It clearly has a configuration in which the CH ₂ OH group is cis for both the and 3′-functional groups. A.Hoyl and F.Sorn,
Biochemica.Biophysica.Acta. 161 26 (1968).
Therefore, even though the process of the invention is described for the production of substrates in which the 4'- _CH2OH substituent is trans to the 2'-,3'-substituent, the process
It should be understood that it is equally applicable to the preparation of substrates in which the 4'-CH ₂ OH substituent is cis to the 2'-,3'-substituent. From a functionality point of view, as well as, of course, the effect on the stability of the product, the selection of the base should take into account the following factors: (1) Any adjustment (increase or decrease),
(2) difficulty of synthesis; (3) effects on endogenous enzyme activity; and (4) solubility in aqueous or other relevant media should not be adversely affected to any significant degree. Other factors to consider include possible effects on hydrolysis and hydrolysis caused by non-specific media. A wide variety of pyrimidine analogs are useful bases, including uracil, dihydrouracil, cytosine, dihydrocytosine, and lauryl halides. In addition, based on data inferred from the results of ribonuclease-induced hydrolysis of both natural substrates, RNA, and synthetic substrates, such as nucleotide homopolymers [FM
Richards and WWWykoff, The Enzyme (P.
Edited by D. Boyer), Academic Press, 3rd edition, 4th edition.
Volume 647-806, London and New York (1978)]
The following pyrimidine analogues are suitable bases: [Formula] [Formula] [Formula] [Formula] [Formula] [Formula] [Formula] [Formula] [Formula] [Formula] [Formula] [Formula] As a base, for example adenosine Although the use of purine analogs such as and guanosine do not provide active substrates for tracking the catalytic activity of ribonuclease A, these bases
It should prove useful in cases involving _T2 activity. Additionally, any other pyrimidine, purine or such analog may be used consistent with the functional considerations set forth herein. In carrying out the first step of the method, 2'-
and 3' phosphate mononucleotide isomers are reacted with a condensing reagent to form a mononucleotide-2',3'-cyclic phosphate. Suitable condensation reagents are N,
N′-dicyclohexylcarbodiimide (DCC)
It is. Other condensing reagents found to be useful include 1,1'-carbonyldiimidazole, 1-hydroxybenzotriazole monohydrate (HBT), 1-cyclohexyl-3-(2-morpholino-ethyl)-carbodiimidemeth -p-Toluenesulfonate (morpho-CDI), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC), N-ethoxycarbonyl-
Included are 2-ethoxy-1,2-dihydroquinoline and ethyl 1,2-dihydro-2-ethoxy-1-quinoline carboxylate (EEDQ).
The use of adjuvants such as tertiary-butyl alcohol is also helpful. Alternatively, N-hydroxysuccinimide, N-hydroxypiperidine, and N-hydroxyphthanolimide may also be used in place of tertiary butyl alcohol. Method parameters useful for carrying out the condensation step can vary widely. A molar ratio range of approximately 1:2 has been found to be suitable regarding the relative proportions of mononucleotide and condensing reagent. Molar ratios of about 1:1 to about 1:5 or perhaps even higher could be used as well. N, given in ammonia solution
A basic medium in a polar solvent such as N-dimethylformamide should be used. Other polar solvents such as pyridine, tetrahydrofuran, and dioxane and reagents other than ammonia to provide the resulting basic medium could be used. The particular proportions of these components are not believed to be particularly critical, and examples of suitable proportions are set forth in the Examples as described below. The reaction is about 110℃
It can be carried out for about 3 hours at a temperature of . A temperature range of about 30°C to about 130°C and a reaction time of about 1 to about 5 hours can be used. The second step is formed by the reaction of uridine 2',3'-cyclic phosphate with a suitable blocking reagent.
Involves the formation of a 5'-O-blocked uridine-2',3'-cyclic phosphate. The reaction is depicted below. The protecting groups and the manner in which protection is carried out should be chosen to achieve the following objectives: (1) Introduction of a protecting group should be easy and should not adversely affect the integrity of other important functional groups.
For example, avoid ring opening. (2) Once introduced, the protecting group should be able to be retained in subsequent synthetic steps until deprotection is desired, if so desired, and furthermore, in such later steps. Should not interfere to a significant extent. and (3) if retained, the protecting group should not adversely affect the performance of the resulting material in its intended use. For example, if the protecting group is too large it may reduce the reactivity of the protected mononucleotide during the phosphodiester synthesis step described below. Furthermore, if retained in the end-product, it may interfere with the action of the enzyme or catalyst on the substrate. Functional groups that could modulate (i.e. either increase or decrease) the enzyme or catalytic activity in any way or that could interact with the phosphodiester and destabilize it, e.g. by hydrolysis, should be avoided. It is. Other considerations include (1) that the solubility in the aqueous or other medium in question should not be adversely affected to any significant degree, and (2) that if the protecting group is retained, the endogenous Includes effects on enzyme activity. In general, the protecting group R can be any acid- or base-labile moiety. Suitable protecting groups thus include alkyl, alkenyl, cycloalkyl, aryl, aralkyl, acyl, oxaalkyl, thioalkyl, oxacycloalkyl, and thiocycloalkyl. More specifically, methyl, ethyl, allyl, cyclohexyl, phenyl, benzyl, nitrobenzyl, acetyl, 1-methoxyethyl,
1-ethoxyethyl, 1-ethoxythioethyl,
Tetrahydropyranyl, tetrahydrothiofuranyl, tetrahydrothiopyranyl, and 4-methoxytetrahydropyran-4-yl are probably suitable for use. When R is acetyl, the furanoside ring shown is
Protection of the 5'-hydroxyl substituent is particularly advantageous since it eliminates the formation of undesired diastereomeric pairs. Furthermore, the acetyl group is small enough that it does not reduce the reactivity of the protected mononucleotide during the phosphodiester synthesis step. A further advantage of the acetyl group as an additional 5'-protecting group is that the overall yield of suitable substrate is significantly improved. Further performance considerations indicate that the presence of a 5'-acetyl group in the final product is useful for various enzymes such as ribonuclease A or _T2 or for catalytic polypeptides as described in the co-pending Farina et al. S-peptide/
There is no appreciable effect on the activity of the substrate towards the S-protein polypeptide pair. Also, its presence does not adversely affect the stability of the resulting substrate. For these two reasons, removing the acetyl group is unnecessary. Indeed, the presence of an acetyl group in the resulting substrate as a 5'-protecting group is sufficient to minimize and Can be removed. Protection of the 5'-hydroxyl of the 2',3'-cyclic phosphate can be suitably carried out in a neutral polar solvent as described above. When an acetyl group is used for protection, the reaction may be carried out using the following parameters. (1) a concentration of the product in unprotected pyridine of about 0.2 M to about 0.5 M; (2) a substoichiometric excess of about 60 to about 80 equivalents of acetic anhydride in the unprotected product; 3) Temperature of approximately 15℃ to approximately 30℃, (4) Approximately 5
reaction time of approximately 15 hours. These parameters may vary widely and the following may be useful as well. (1)
(2) a concentration of about 0.1 to about 1.0M; (2) about 10 to about 100M;
the use of an equivalent excess of acetic anhydride or other acylating reagents such as acetyl chloride or other acetyl halides; (3) a temperature of about 10°C to 50°C; and (4) a temperature of about 2 to about
20 hours reaction time. alkyl, cycloalkyl,
Other protecting groups such as and aralkyl can be introduced by known substitution reactions. Introduction of oxaalkyls, oxacycloalkyls, thioalkyls and thiocycloalkyls can be carried out by acid-catalyzed addition to the corresponding orphin ethers,
and this will be more fully described in the following discussion of 2'-protecting groups. The third step in the synthesis is the formation of a specific 3'-
Includes specific phosphate ring opening to provide the phosphate moiety. The reaction requires a specific and efficient catalyst so that essentially only 3'-uridine phosphate is formed. A suitable catalyst for this purpose is pancreatic ribonuclease. Other synthetic or natural catalysts having the requirements defined herein can be used as well. The reaction formula is drawn as below. The reaction can be carried out in an aqueous polar solvent (eg pyridine), eg containing about 20% pyridine, using a sufficient amount of catalyst to provide convenient reaction times. By way of example, a catalyst concentration of about 1% by weight based on the weight of cyclic phosphate is satisfactory. The resulting decyclization product must then be isolated by removal of the catalyst and concentration of the liquid product. This can be done, for example, by removing the catalyst using an ion exchange column and then concentrating the reaction solution. If pyridine is used, the resulting product is a pyridinium salt. If desired, the concentrated pyridinium salt can be converted to ammonium, tert-butylammonium, calcium, sodium, lithium, etc. salts. This may be accomplished by precipitation from a suitable aqueous solution (eg aqueous tetrahydrofuran). The use of ammonium salts offers a particular advantage in that no further modification is required. Calcium salts, on the other hand, give products that are more crystalline, easily isolated and easier to handle. The fourth step of this procedure involves protection of the 2'-hydroxyl group. In the formula, R' is a 2'-O-protecting group. A suitable 2'-protecting group should meet the following criteria: (1) can be easily introduced without affecting other key functional groups; (2) is compatible with subsequent phosphodiester formation steps; in particular, avoids unwanted side reactions during such steps; that should be minimized or eliminated;
(3) be sufficiently stable to be stored for long periods of time without any adverse effects; and (4) be easily removed without disturbing the phosphodiester bonds. These criteria, and especially the last one, can be most easily met by the use of protecting groups that can be introduced and removed by acid-catalyzed reactions. Thus, suitable protecting groups include oxaalkyl, thioalkyl, oxacycloalkyl, silyl derivatives and thiocycloalkyl. More specifically, tetrahydropyranyl, 4
-methoxytetrahydropyran-4-yl, and tert-butyldimethylsilyl can be used. The protection reaction can be accomplished in a polar neutral solvent such as N,N-dimethylformamide or dioxane.
Diglyme (diethylene glycol dimethyl ether), tetrahydrofuran or acetone can be used. As a specific example, when the protecting group is tetrahydropyranyl, the protection is dihydropyran or 4-methoxy-5,6-dihydro-
2-H-pyran is combined with a catalytic amount of an acid catalyst, such as dry hydrogen chloride in dioxane, in an amount generally in sufficient excess of the stoichiometric amount to ensure that the reaction is completed in a convenient time. It is done by doing things together. The reaction is generally complete in about 2 to about 15 hours. The temperature can vary from about -20°C to about 25°C. Other useful acid catalysts include p-toluenesulfonic acid and trifluoroacetic acid. Similarly, reaction times can vary from as short as 1 hour to as long as 20 hours, and temperatures can vary from about -30°C to about 50°C.
This general reaction scheme is equally applicable to the introduction of other protecting groups described herein by acid-catalyzed reactions. Alternatively, protecting groups can be introduced into known substitution reactions and removed by photochemical means. An example of a protecting group of this type is o-nitrobenzyl. In the embodiment of the present invention, the fifth step is to prepare the intermediate
Esterification reaction at the chromophore or fluorophore moiety R'' of the 2',5'-O-diprotected mononucleotide to form a chromogenic and/or fluorescent mononucleotide-3'-phosphodiester substrate .The reaction is depicted as follows. As a functional group, R'' can be defined as any moiety that provides a substrate with fluorescent and/or chromogenic properties. The R'' group can be aryl, aralkyl, heteroaryl or heterocyclic. In preferred embodiments R'' is umbelliferonyl, 4-methylumbelliferonyl or 3-flavonyl.
Other suitable R'' groups include aryls such as, for example, 1-naphthyl. Further suitable R groups include aryl groups having electron withdrawing and conjugated substituents that increase the acidity of ortho and parabenzoic acid. Such groups include ortho-, meta-, and para-nitrophenyl, 2,4-dinitrophenyl, cyanophenyl, acylphenyl, carboxyphenyl, phenylsulfonate, phenylsulfonyl, and phenylsulfoxide. Generally Mixtures of mono- and bi-substituted derivatives are suitable as well. It is typical and preferred to use the alcoholic form of the chromogenic and/or fluorophore moieties to produce the protected substrate product. Selected Esters The reagent should not result in deprotection of the 2'-blocking group and should be easily removed from the phosphodiester formed. Furthermore, and importantly, the reagent selected should be able to produce high yields under mild reaction conditions. Appropriately, the reagent should be given a rate of 2,
4,6-triisopropylbenzenesulfonyl chloride, N,N'-dicyclohexylcarbodiimide, and mesitylenesulfonyl chloride,
Contains toluenesulfonyl chloride, mesitylenesulfonylimidazolide, p-toluenesulfonylimidazolide, picryl chloride, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, N-hydroxy Other carbodiimide analogs with or without additives such as phthalimide and the like are also suitable. For 2',5'-diprotected uridine-3'-monophosphates, it has been found necessary to use either pyridinium or ammonium salts under the reaction conditions used. It has therefore been found that if calcium or metal ion salts have been utilized in previous synthesis steps, it is necessary to convert them to the desired salts by ion exchange. An excess of chromogenic and/or fluorescent alcohol with respect to the esterification reaction conditions is used to maximize yield, but no excess is required. It has been found satisfactory to use a molar ratio of about 2:1 to maximize yield.
The reaction is carried out in a neutral polar solvent such as N,N-dimethylformamide, dioxane, or tetrahydrofuran in the presence of a base such as pyridine. It has been found suitable to use dry pyridine as a solvent at a temperature in the range of about -20°C to about 25°C. Additionally, reaction times can range from about 5 to 18 hours. These reaction conditions can be varied if desired. Therefore, the temperature is approximately -20℃
to 50° C. and times from 2 to 72 hours. An important aspect of the present synthetic method is that it provides a suitable substrate for use in, e.g., immunoassays, without requiring purification, e.g., by chromatography, before use. be. In another embodiment of the invention, the fifth step of the procedure is first performed using methods known in the art, such as by the use of phosphatases or Pb() hydroxide, to effect the cleavage of the 3'-phosphate. The method is utilized to dephosphorylate a 2',5'-diprotected mononucleotide resulting in the formation of a 2',5'-diprotected mononucleoside. generated in this way
The 2',5'-diprotected mononucleoside is then reacted with a phosphorylated derivative of the desired chromophore and/or fluorophore moiety to form the 2',5'-diprotected mononucleotide-3'-
Forms a phosphodiester substrate. The chromophore and/or fluorophore moiety R'' suitable for use in this embodiment of the invention is the same as the R'' moiety described above. The choice of specific embodiments for use in phosphodiester formation will depend on the particular chromophore and/or fluorophore moiety used. For example, in the case of naphthyl chromophores, it is highly desirable to utilize the alternative embodiments described above. 2′, 5′− manufactured by either of the two options
Diprotected substrates are stable compounds that can be stored for long periods of time. However, deprotection of the 2'-blocking group is necessary to provide the appropriate enzyme, substrate. The deprotection reaction is expressed as follows. Acid-catalyzed deprotection is performed briefly under mild conditions with dilute acids in a protic solvent such as water. Commonly used acid catalysts include hydrochloric acid, trifluoroacetic acid,
Includes p-toluenesulfonic acid and acetic acid.
Preferred reagents are about 0.01 to about 1, more usually
Dilute hydrochloric acid with a molar concentration of 0.01 to 0.1 and preferably 0.01 to 0.05. Acid forms of ion exchange resins can also be used. When the protecting group is silyl, deprotection is carried out with a nucleophilic reagent such as, for example, tetrabutylammonium fluoride. Regarding deprotection conditions, ambient temperature is suitable.
The deprotection time can vary over a relatively wide period of time and depends on the concentration of the deprotection reagent and the temperature at which the deprotection reaction is carried out. Generally, the higher the temperature and concentration, the shorter the reaction time. Therefore, the reaction takes about 5 minutes to about 24 hours,
More usually it may be carried out for about 10 minutes to about 120 minutes, and preferably about 20 minutes to about 60 minutes. Too harsh reaction conditions should be avoided as they can lead to unfavorable hydrolysis of the deprotected substrate. The following examples are merely illustrative of the invention and are not intended as limitations on the scope. EXAMPLE The present invention illustrates the preparation of uridine 2',3'-cyclic phosphate. 74 ml 3N of uridine 2'- and 3'-phosphate
- A 10 g, 0.031 mol solution of the mixture in ammonia was mixed successively with 60 ml of N,N-dimethylformamide (DMF) and a solution of 15 g, 0.075 mol dicyclohexylcarbodiimide in 60 ml of tertiary butyl alcohol. The resulting reaction mixture was refluxed at 120° C. in an oil bath for 3 hours. High pressure liquid chromatography analysis (HPLC) was performed on a portion of the reaction mixture to determine whether starting material was converted to product. A portion of the reaction product mixture was concentrated under vacuum (35° C. bath), the residue was dissolved in water and the solution was filtered through a 5 μm Millipore filter. The sample was then passed through Whatman Partisil 10/
25 with a buffer consisting of 20% phosphate, 0.05M of approximately pH 6.25, and 80% water.
Elution was performed at a flow rate of ml/min. HPLC analysis showed quantitative conversion of starting uridine to product. The entire product mixture was then allowed to cool to room temperature, the dicyclohexyl urea precipitate was separated by filtration, and 20 ml of
I washed it with DMF. The filtrate was then evaporated in vacuo at 12-15 torr with a bath temperature of 35°C and the residue was shaken with 100ml of water and filtered to remove dicyclohexylurea.
Wash the solid with an additional 20 ml of water and dilute the combined solution with 2
Extraction was carried out with 150 ml of ether twice and evaporation to dryness was carried out in vacuo at a bath temperature of 35°C. The residue was coevaporated with two 100 ml portions of pyridine using a liquid nitrogen trap at 0.01 torr to give the glass-like product uridine 2',3'-cyclic phosphate. Example This example shows 5'-O-acetyluridine 2',
The production of 3'-cyclic phosphate is illustrated. The uridine 2',3'-cyclic phosphate prepared in the examples is dissolved in 100 ml of anhydrous pyridine and 200 ml of acetic anhydride. Keep this solution in the dark at room temperature overnight. At this point a portion of the reaction product is analyzed by HPLC under the conditions given in the example. HPLC showed a major peak at 1.7 minutes, representing the product 5'-O-acetyluridine 2',3'-cyclic phosphate. The entire product mixture was evaporated to dryness using a liquid nitrogen trap at 0.1-1 torr with a bath temperature of about 35°C. The residue was co-distilled twice with 50 ml portions of pyridine to remove residual acetic anhydride and then dissolved in 100 ml of 50% aqueous pyridine. After stirring for 1 hour at room temperature, the solution was evaporated to dryness at room temperature and 0.05 mm Hg to yield the product 5'-O-acetyluridine 2',3'-cyclic phosphate. EXAMPLE This example illustrates the preparation of the pyridinium salt of 5'-O-acetyluridine 3'-phosphate. 5'-O-acetyl uridine produced in Examples
Glass-like products of 2′,3′-cyclic phosphates
Dissolved in 200ml of 20% pyridine aqueous solution. To this solution was added 50 mg of pancreatic ribonuclease in 5 ml of water. The mixture was kept at room temperature overnight (about 5 hours with stirring in the dark. At this point a portion of the reaction mixture was analyzed by HPLC under the conditions given in the example. HPLC showed a major peak at 4.5 minutes; This represents the product 5'-O-acetyluridine-3'-phosphate.The product mixture is then
Pass through a 2.2 x 4 cm ion exchange resin column, Dowex ^R 50W-X8, and remove the contents before use.
100-200 mesh of hydrogen ion type resin was converted to pyridinium type. Resin 300ml 20
% pyridine aqueous solution. Eluate is 0.1-1
Concentrate to an oily residue at approximately 35° C. with torr. The oily residue was dissolved in 5 ml of water and 200 ml of tetrahydrofuran (THF). 27% while stirring into the solution
Further NH ₄ OH was added dropwise until no more precipitate formed. Approximately 3 ml of NH ₄ OH was added. The mixture was kept in the cold overnight, filtered and the residue co-evaporated twice with a little dimethylformamide. The resulting residue is
It contained the pyridinium salt of 5'-O-acetyluridine-3'-phosphate. EXAMPLE This example illustrates the preparation of 5'-O-acetyl-2'-O-(tetrahydropyran-2-yl)uridine 3'-pyridinium phosphate. 12 g, 0.026 mol of 5'- produced in the example
Finely ground pyridinium salt of O-acetyluridine-3'-phosphate, 160 ml anhydrous N,N
A stirred suspension of -dimethylformamide and 70 ml of dihydropyran was cooled to -20 DEG C. and treated dropwise with 14.2 ml of 5M hydrogen chloride in dioxane over 15 minutes free of atmospheric moisture. The cooling bath was then removed and stirring continued until a clear solution was obtained, ie for 2 hours. After storage overnight at room temperature, the mixture was cooled to -20°C, treated with 12 ml triethylamine and 3 ml ammonium hydroxide, and the resulting suspension was poured into 500 ml THF and 500 ml ether. The precipitate was collected and removed on a medium porosity sintered glass funnel. The filtrate was evaporated under vacuum to evaporate the solvent and unreacted dehydropyran. The residue was taken up in 50 ml of tetrahydrofuran and stirred. The product was removed from the tetrahydrofuran polymer by adding 100 ml of ether to the suspension.
The ether layer was separated and washed once more with 50 ml of ether. Dry the residue in an aspirator vacuum;
The ammonium salt product was then dried under high vacuum using a liquid nitrogen trap. Example This example shows 5'-O-acetyl-2'-O-(tetrahydropyran-2-yl)uridine 3'-(4
-Methylumbelliferon-7-yl)pyridinium phosphate is illustrated. 1.00 g (2.01 mmol) produced in the example
5'-O-acetyl-2'-O-(tetrahydropyran-2-yl)uridinepyridinium phosphate, 0.531 g (3.00 mmol) of 4-methylumbelliferone and 1.52 g (5.02 mmol) of 2,
A mixture of 4,6-triisopropylbenzenesulfonyl chloride in 6 ml of dry pyridine was stirred free of atmospheric moisture. The mixture gradually becomes a homogeneous yellow solution after about 30 minutes at room temperature. After about 1 hour the pyridine HCl salt precipitates. After stirring overnight, 6 ml of water was added and stirring continued for a further 2 hours. The mixture is concentrated at room temperature under vacuum using a liquid nitrogen trap and the solid product mixture is dissolved in 15 ml of water. The solution is extracted 5 times with 50 ml ether/extraction, where the unreacted 4-methylumbelliferone is excited at 450 nm when the solution is excited at 325 nm.
Extract until most of the fluorescence is removed, as indicated by a decrease in the fluorescence emission at . The aqueous solution was lyophilized under vacuum to produce 5′-O-acetyl-
2'-O-(tetrahydropyran-2-yl)-uridine-3'-(4-methylumbelliferone-7-
yl) to give a product containing pyridinium phosphate. Example This example shows 5'-O-acetyluridine-3'-
The production of (4-methylumbelliferon-7-yl)pyridinium phosphate is illustrated. Prior to use, the 5'-O-
Acetyl-2'-O-(tetrahydropyran-2-
yl)-uridine-3'-(4-methylumbelliferon-7-yl)pyridinium phosphate is easily deprotected with hydrochloric acid. Add 15 mg of 2',5'-diprotected-phosphodiester to 1 ml of 0.01N HCl to give a clear solution. After 45 minutes, the product solution was extracted six times with 1 ml of ether to remove residual 4-methylumbelliferone. Nitrogen is then bubbled through the aqueous solution to remove the last traces of ether. 0.1N in 100ml
A working solution was made by diluting with sodium acetate buffer at approximately pH 5.0. The substrate was stable at 4°C for at least 2 days in working buffer. Example This example shows 5'-O-acetyluridine-3'-
The production of calcium salts of phosphoric acid is illustrated. 5′-O prepared as described in Examples and
-Acetyl uridine-2',3'-cyclic phosphate (using 4 g of a mixture of 2'- and 3'-phosphoric acids of uridine)
was dissolved in 100 ml of 20% pyridine aqueous solution. 50 mg of pancreatic ribonuclease A was added to this solution.
The solution was stirred at room temperature in the dark for 15 hours. After removing ribonuclease A through a Dowex 50 column, under the conditions given in the example.
A portion of the solution was analyzed by HPLC. The analysis is
It showed a very small amount of starting cyclic phosphate at 1.7 minutes and a major product peak at 4.5 minutes. An additional 20 ml of ribonuclease A was added to the remaining product mixture and the mixture was stirred for a further 3 hours at room temperature. The product solution was passed through a column of Dowex-50 (1 x 5 cm) eluting with 160 ml of 20% aqueous pyridine. Concentrate the solution to approximately 50ml;
Pour into 1000 ml of absolute ethanol containing 5 g of calcium chloride. The mixture was stirred at room temperature for 2 hours and then left to precipitate the calcium salts. The precipitate was collected by centrifugation at 3000 rpm for about 5-10 minutes, washed with ethanol (7 x 150 ml) and centrifuged repeatedly. The calcium salt cake was washed twice with 150 ml of ether and air dried. After further drying in vacuo, the product contains the calcium salt of 5'-O-acetyluridine 3'-phosphate, as confirmed by HPLC analysis (under the above conditions) showing a major peak at 4.5 min.
13.1g was obtained. EXAMPLE This example uses 5,6-dihydro-4-methoxy-2H-pyran as the 2'-protecting reagent.
The preparation of 5'-O-acetyl-2'-O-(4-methoxytetrahydropyran-4-yl)uridine 3'-calcium phosphate is illustrated. 5′-O-acetyluridine produced in the examples
1 g of 3'-calcium phosphate was dissolved in 8 ml of dry N,N-dimethylformamide. To this solution was added 5.0 g of 5,6-dihydro-4-methoxy-2H-pyran. Solution in acetone
Cooled to below 0°C in an ice bath. N to the stirred mixture,
1.4 ml of 5M hydrogen chloride in N-dimethylformamide
was added dropwise in an atmosphere excluding moisture. After about 20 minutes,
The cooling bath was removed and the reaction mixture was stirred at room temperature overnight for 15 hours. The mixture was cooled again in an acetone-ice bath and 25 ml of triethylamine was added dropwise with stirring. Pour the resulting mixture into 100 ml of ether,
A white powder was collected by filtration. Powder 1% triethylamine in 100ml ether, chloroform
I washed it with 100ml. The solid was first air-dried and then further dried in a vacuum.
1.398 g of product containing 5'-O-acetyl-2'-O-(4-methoxytetrahydropyran-4-yl)uridine 3'-calcium phosphate was given. Whatman Partisil RXS10/eluted with 0.01M phosphate buffer PH6.3
HPLC on a 25SAX column (flow rate 1 ml/min) (UV
Detection at 253 nm) shows the product at 3.4 minutes, while the starting material has a residence time of 4.7 minutes. Example This example shows 5'-O-acetyl-2'-O-(4
-methoxytetrahydropyran-4-yl)uridine-3'-(4-methylumbelliferone-7-
Figure 2 illustrates the production of yl) phosphate. 1.1 g of the hydrogen ion form of Bio-Rad AG 50W-X8 cation exchange resin was exchanged to the pyridinium form. The column contained 5'-O-acetyl-2'-O-(4-methoxytetrahydropyran-4-yl)uridine 3'-calcium phosphate made in the example dissolved in a cold 50% pyridine solution. Add 100mg of product and run the column.
Elution was performed with 270 ml of 50% pyridine solution. The eluate solution was collected in a flask chilled in ice water. The eluate solution was concentrated to 15 ml on a rotary evaporator with a bath temperature of approximately 25°C using a dry ice trap. The remaining solution was further concentrated under vacuum at room temperature using a liquid nitrogen trap (0.05 mmHg) to give a glassy residue. The residue was further evaporated twice with dry pyridine to dryness. Finally, the residue was dissolved in 1 ml of dry pyridine and
52.75 mg of 4-methyl-umbelliferone and 102.7 mg of 2,4,6-triisopropylbenzenesulfonyl chloride were added to the mixture. The mixture was cooled in an ice water bath with stirring for 15 minutes. The resulting yellow solution was stirred for an additional 2 hours at room temperature, overnight,
That is, the mixture was stirred for 15 hours at about 4-8°C. The entire product mixture was then stirred with 3 ml of a saturated solution of tetraethylammonium bromide for 5 minutes and then extracted five times with chloroform. The chloroform layer was concentrated in vacuo to yield 635 mg of light gray solid crude product. The phosphodiester was further purified by anion exchange column chromatography eluting with ammonium bicarbonate buffer. Appropriate fractions were deprotected using the method given in the Examples and then identified by ribonuclease (RNase) assay. The fractions so fixed were collected and concentrated to give 137 ml of a solid, which was then dissolved in methanol and repeatedly evaporated in vacuo to remove the ammonium bicarbonate. the result
5'-O-acetyl-2'-O-(4-methoxytetrahydropyran-4-yl)-uridine-3'-(4
59 mg of product containing methylumbelliferon-7-yl) phosphate were obtained. Example This example shows 5'-O-acetyl-2'-O-(4
-Methoxytetrahydropyran-4-yl)uridine-3'-flavonyl phosphate is illustrated. 50 mg of the product containing 2'-O-(4-methoxytetrahydropyran-4-yl)-5'-O-acetyl-3'-uridine calcium phosphate prepared in the examples was added to BioRad AG (Bio- Rad
AG ^R ) was converted to the pyridinium salt by passing through the pyridinium form on a 50W-X8 cation exchange column. The pyridine solution was concentrated in vacuo and dried by repeated evaporation with dry pyridine to give a glassy residue. The glassy residue was dissolved in 1 ml of dry pyridine and the solution was added with 35.6 mg of 3-hydroxyflavone and
51.4 mg of 2,4,6-triisopropylbenzenesulfonyl chloride are added with stirring in an ice-water bath under a nitrogen atmosphere. After 15 minutes, the mixture was allowed to warm to room temperature and stirred over the weekend for approximately 3 days. The reaction mixture was then followed for product formation. A 0.3 ml portion of the reaction mixture is stirred with 1 ml of saturated tetraethylammonium bromide and extracted four times with chloroform. The chloroform was evaporated and the resulting yellow solid was treated with 0.01N HCl for 40 minutes. The solution was then at pH 5 containing 4 x 10 ^-3 aluminum chloride and 1% dimethyl sulfoxide.
Buffered with 0.1M acetate buffer. The resulting buffered solution, in the presence of ribonuclease (RNase) _T2 enzyme, produced the fluorescent properties of the aluminum chelated 3-hydroxyflavone, thereby indicating the formation of the desired product. The remainder of the reaction mixture was stirred for 5 minutes with 2 ml of a saturated solution of tetraethylammonium bromide. The mixture was then extracted four times with chloroform. The chloroform layer was dried over anhydrous sodium sulfate and concentrated to give 0.355 g of yellow solid product. The product was further purified by chromatography on a silica gel column 2.5 x 6.5 cm eluting with 10% methanol in chloroform. Fractions of 100 milliliters each were collected and fractions 9, 10 and 11 were shown to have positive substrate when deprotected in acid and assayed with ribonuclease. Fractions 9, 10 and 11 were combined, concentrated and 5′-O-
160 mg of product containing acetyl-2'-O-(4-methoxytetrahydropyran-4-yl)-uridine-3'-flavonylphosphate were obtained.

Claims (1)

Claims: A method for producing a substrate capable of producing spectrophotometrically or fluorometrically traceable species by catalyst-induced hydrolysis of a phosphate ester at the 1 3'-position, comprising: is (a) Eq. [In the formula, B is a nucleotide base, and the 4′-position
the CH ₂ OH group is either cis or trans to the cyclic phosphate ester]; (b) forming the mononucleotide 2',3'-cyclic phosphate; At 5′-hydroxyl, alkyl, alkenyl, cycloalkyl, aryl, aralkyl, acyl, oxaalkyl, thioalkyl,
protected with a 5'-protecting group selected from the group consisting of oxacycloalkyl and thiocycloalkyl to form a mononucleotide 5'-O-protected-2',3'-cyclic phosphate; (c) 5'- The cyclic phosphate is ring-opened to form essentially only the mononucleotide 5'-O-protected-2'-hydroxyl 3'-phosphate under conditions which leave the position substantially protected, (d ) The above mononucleotide 5'-O-protected-3'-phosphate is combined at the 2'-hydroxyl with a 2'-O- Mononucleotide 2'-O-protected-5'-O-protected-3'- by protection with a protecting group
phosphate, and (e)(1) said mononucleotide 2'-O-protected-5'-
or (2) the above mononucleotide 2'-O-protected-5'-
dephosphorylation of the O-protected 3'-phosphate to produce the 2'-O-protected-5'-O-protected mononucleoside, and the 2'-O-protected-
A 2'-O-protected-5'-O-protected phosphodiester is produced by reacting a 5'-O-protected mononucleoside with a phosphorylated derivative of a group selected from the group consisting of chromophores or fluorophores. the 2'-protecting group is capable of at least substantially protecting the phosphate ester from media-induced hydrolysis at the 3'-position, and the 2'-protecting group is removable. , characterized in that the catalyst-induced hydrolysis of the phosphate ester at the 3'-position provides a substrate capable of producing spectrophotometrically or fluorometrically traceable species. A method of producing a chromogenic or/fluorogenic substrate. 2 Chromogenic and/or chromogenic, capable of producing spectrophotometrically or fluorometrically traceable species by catalyst-induced hydrolysis of the phosphate ester at the 2 3′-position.
or in a method of producing a fluorogenic substrate,
This method uses (a) Eq. [In the formula, B is a nucleotide base, and the 4' position
the CH ₂ OH group is either cis or trans to the cyclic phosphate ester]; (b) forming the mononucleotide 2',3'-cyclic phosphate; At 5′-hydroxyl, alkyl, alkenyl, cycloalkyl, aryl, aralkyl, acyl, oxaalkyl, thioalkyl,
protected with a 5'-protecting group selected from the group consisting of oxacycloalkyl and thiocycloalkyl to form a mononucleotide 5'-O-protected-2',3'-cyclic phosphate; (c) 5'- opening the cyclic phosphate to form substantially only the mononucleotide 5'-O-protected 2'-hydroxyl 3'-phosphate under conditions which leave the position substantially protected; (d) The mononucleotide 5'-O-protected 3'-phosphate is modified at the 2'-hydroxyl with a 2'-O-protecting group selected from the group consisting of oxaalkyl, thioalkyl, oxacycloalkyl, silyl and thiocycloalkyl. The mononucleotide 2'-O-protected-5'-O-protected-3'-
(e)(1) the mononucleotide 2'-O-protected-5'-
or (2) the above mononucleotide 2'-O-protected-5'-
Dephosphorylation of the O-protected-3'-phosphate to produce the 2'-O-protected-5'-O-protected mononucleoside and the 2'-O-protected-5'-O-protected mononucleoside 2'-O-protected-5'-O-protected phosphodiester is produced by reacting with a phosphorylated derivative of a group selected from the group consisting of chromophores or fluorophores, can at least substantially protect the phosphate ester from media-induced hydrolysis at the 3′-position, and
The 2'-protecting group is removable, and (f) the 2'-protecting group is removed from the 2'-O-protected-5'-O-protected phosphodiester to catalyze the phosphodiester. chromogenic and/or fluorogenic substrates, characterized in that they consist in providing a substrate capable of giving rise to species that can be tracked spectrophotometrically or fluorometrically by induced hydrolysis. How to manufacture. 3. The method of claim 1, wherein the base is a pyrimidine analog. 4. The method of claim 1, wherein the base is a purine analog. 5. The method of claim 1, wherein the base is selected from the group consisting of uracil, dihydrouracil, cytosine, dihydrocytosine and uracil halides. 6. The method of claim 1, wherein the base is laucil. 7 The above 5'-protecting group is methyl, ethyl, allyl,
Cyclohexyl, phenyl, benzyl, nitrobenzyl, acetyl, 1-methoxyethyl, 1-ethoxyethyl, 1-ethylthioethyl, tetrahydropyranyl, tetrahydrothiofuranyl, tetrahydrothiopyranyl, and 4-methoxytetrahydropyran-4- 2. The method of claim 1, wherein the method is selected from the group consisting of: 8. The method of claim 7, wherein said protecting group is acetyl. 9 The above 2'-protecting group is tetrahydropyranyl,
2. The method of claim 1, wherein the compound is selected from the group consisting of 4-methoxytetrahydropyran-4-yl, and tertiary-butyldiethylsilyl. 10. The method of claim 9, wherein said 2'-protecting group is tetrahydropyranyl. 11. Claim 9, wherein the 2'-protecting group is 4-methoxytetrahydropyran-4-yl.
Section method. 12. The method of claim 1, wherein said group (selected from the group consisting of chromophores or fluorophores) is selected from the group consisting of aryl, aralkyl, heteroaryl or heterocycle. 13 The above groups include umbelliferonyl, 4-methylumbelliferonyl, 3-flavonyl, 1-naphthyl, o-nitrophenyl, m-nitrophenyl, p-nitrophenyl, 2,4-dinitrophenyl, cyanophenyl, acylphenyl, carboxyphenyl. Claim 12 is selected from the group consisting of phenyl, phenyl sulfonate, phenyl sulfonyl and phenyl sulfoxide.
Section method. 14. The method of claim 13, wherein said group is 1-naphthyl. 15. The method of claim 13, wherein said group is 4-methylumbelliferonyl. 16. The method of claim 13, wherein said group is 3-flavonyl. 17 The above 2'-O-protected-5'-O-protected-3'-phosphate is esterified with the alcohol form of the above group to form the above 2'-O-protected-5'-O-protected phosphodiester. The method of claim 1 for forming. 18 The above esterification reaction is carried out using 2,4,6-triisopropylbenzenesulfonyl chloride,
From the group consisting of N,N'-dicyclohexylcarbodiimide, mesitylenesulfonyl chloride, toluenesulfonyl chloride, mesitylenesulfonylimidazole, p-toluenesulfonylimidazole, picryl chloride and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride 18. The method of claim 17, carried out in the presence of a selected esterification reagent. 19. The method of claim 18, wherein the esterification reaction is carried out in the presence of an additive selected from the group consisting of N-hydroxysuccinimide and N-hydroxyphthalimide. 20. The method according to claim 18 or 19, wherein the esterification reaction is carried out in the presence of pyridine. 21 The above esterification reaction is carried out at about -20°C to about 25°C.
Claim 20 carried out at a temperature in the range of
Section method. 22. The method of claim 21, wherein the esterification reaction is conducted for about 5 to about 18 hours. 23. The method of claim 2, wherein said removal of said 2'-protecting group is carried out in a protic solvent. 24. The method of claim 23, wherein the protic solvent is water. 25. The method of claim 24, wherein the removal of the 2'-protecting group is carried out in the presence of an acid selected from the group consisting of hydrochloric acid, trifluoroacetic acid, and p-toluenesulfonic acid. 26. The method of claim 25, wherein the acid is hydrochloric acid at a molar concentration of about 0.01 to about 1. 27. The method of claim 26, wherein the molar concentration of hydrochloric acid is from about 0.01 to about 0.05. 28. The method of claim 27, wherein the reaction is carried out at ambient temperature. 29. The method of claim 28, wherein the reaction is carried out for about 5 minutes to about 24 hours.