KR20160123677A

KR20160123677A - Enzymatic production of cytosinic nucleoside analogues

Info

Publication number: KR20160123677A
Application number: KR1020150054045A
Authority: KR
Inventors: 마르타 파스쿠알 길라베르트; 빅터 엠. 데론첼레 토마스; 라파엘 몬틸라 아레발로
Original assignee: 플라스미아 바이오텍 에스엘
Priority date: 2015-04-16
Filing date: 2015-04-16
Publication date: 2016-10-26

Abstract

Enzymatic production of cytosine nucleoside analogs
The present invention relates to the use of a nucleoside phosphorylase, particularly a pyrimidine nucleoside phosphorylase (PyNP) or a purine nucleoside phosphorylase (PNP) Lt; RTI ID = 0.0 > cytosine < / RTI > nucleoside analogs.

Description

ENZYMATIC PRODUCTION OF CYTOSINIC NUCLEOSIDE ANALOGUES < RTI ID = 0.0 >

Field of invention

The present invention relates to the production of cytosine nucleoside analogues (NAs), particularly to novel enzymatic processes for the industrial production of cytosine NA having activity as antiviral and anticancer drugs, intermediates or prodrugs thereof, do.

BACKGROUND OF THE INVENTION

Nucleoside analogs (NAs) are synthetic compounds that are structurally related to natural nucleosides. In terms of their structure, the nucleoside is composed of three core elements: (i) a hydroxymethyl group, (ii) a heterocyclic nitrogen base moiety, and (iii) a furanose ring, Hydroxymethyl groups and bases in the correct orientation.

NA is characterized by a large structural diversity because they exhibit various modified carbohydrates and / or aglycone fragments.

NA is widely used as an antiviral and anticancer agent. These molecules can be prepared by conventional chemical methods that often require a time-consuming multi-step process involving a protective-deprotection reaction on a heterocyclic base and / or a pentose moiety to allow modification of the naturally occurring nucleoside (Boryski J. 2008. Reactions of transglycosylation in the nucleoside chemistry. Curr Org Chem 12: 309-325). This time consuming multi-step process often results in low yield and increased cost. Indeed, chemical methods typically increase the difficulty of obtaining products with precise steric- and positional selectivity and produce by-products as impurities (Condezo, LA, et al. 2007. Enzymatic synthesis of modified nucleosides, p. Sinisterra, JV et al. 2010. Enzyme-catalyzed synthesis of nonnatural or modified nucleosides, p. 1-25, Encyclopedia of Biocatalysis, Biocatalysis in the pharmaceutical and biotechnology industries, CRC Press, Boca Raton, FL, Mikhailopulo, Industrial Biotechnology: Bioprocess, Bioseparation, and Cell Technology, John Wiley & In addition, chemical methods involve the use of expensive and environmentally hazardous chemical reagents and organic solvents.

Enzymatic synthesis of nucleosides catalyses the compression of heterocyclic bases and sugars, and thus involves the use of enzymes to form glycosidic bonds. In general, nucleoside analogs can be prepared by transglycosilation reactions with two different kinds of enzymes: nucleoside phosphorylase (NPs or NP) and N- 2'-deoxyribosyl transfer Enzymes (NDTs or NDT). NPs include: pyrimidine nucleoside phosphorylase (EC 2.4.2.1), purine nucleoside phosphorylase (EC 2.4.2.2), and phosphorylase phosphorylase, depending on their substrate specificity for pentafuranose donors and nuclear base receptors Uridine nucleoside phosphorylase (EC 2.4.2.3) and thymidine nucleoside phosphorylase (EC 2.4.2.4). However, cytosines and their nucleosides (cytidine, 2'-deoxycytidine) or modified cytosine analogs or derivatives and their corresponding nucleosides are not substrates for these NPs enzymes (Mikhailopoulo, I. A, meat al . 2010. New Trends in Nucleoside Biotechnology, Acta Naturae, 2 (5), 36-58).

Conversely, the N- 2'-deoxyribosyl transferase (EC 2.4.2.6.), Without intermediate formation of 2-deoxyribofuranosyl phosphate, has a deoxyribofuranosyl moiety between the nucleoside and the receptor base Lt; RTI ID = 0.0 > direct < / RTI > The NDTs substrate specificity is based on the stringent specificity for the 2-deoxyribofuranosyl moiety rather than the broad tolerance for the modified purine, and the excellent specificity of the cytosine as a receptor for the 2-deoxy- and 2,3-dideoxyribofuranose residues Lt; / RTI > activity (Mikhailopoulo, I. A, et al . 2010. New Trends in Nucleoside Biotechnology, Acta Naturae, 2 (5), 36-58). Fresco-Taboada et al (New insights on NDTs:. . a versatile Biocatalyst for one-pot one-step synthesis of nucleoside analogs, Appl Microbiol Biotechnol, 2013, 97, 3773-3785) is cytosine is 2'-deoxy ribonucleic Pura furnace in all cases Lt; RTI ID = 0.0 > osmo < / RTI >

For this reason, the choice of NPs or NDTs depends on the structure of the receptor-base and the donor nucleoside. NPs and NDTs complement each other and enable biocatalytic production of natural nucleosides and their modified analogues, and many of them are useful for the production of many anticancer and antiviral drugs.

However, neither NPs nor NDTs allow the production of cytosine ribonucleosides because cytosines are not the receptor bases for NPs, and at the same time ribofuranose nucleoside donors are not substrates for NDTs Because. For this reason, the production of cytosine nucleoside analogs requires not only substrate specificity as described above, but also certain enzymes normally present in biocatalyst products, such as cytosine and cytidine deaminase, or cytosine and cytidine deacetylase, Or decompose final products, and cause unproductive methodologies for industrial purposes.

Araki et al (EP 1254959, US 7629457) by using a bacterium having a reactive nucleoside kinases or the enzyme activity to cytosine pentose-1-phosphate and cytosine or a cytosine for the production of the nucleoside compounds and derivatives thereof from / RTI > In particular, the present inventors have found that purine nucleoside phosphorylase (PNP) from bacteria belonging to the genus Escherichia , which is required to catalyze a reaction required by itself as a substrate instead of a pyridine base, 1-phosphate can catalyze the production of cytosine nucleosides. However, the reaction of pentose-1-phosphate with cytosine or its derivative in the presence of a bacterium having the enzyme activity disclosed therein almost exclusively produces a deaminated product of cytosine, or a derivative thereof, It has been found that the efficient accumulation of the < RTI ID = 0.0 > chelocide < / RTI > In order to minimize the production of deaminated cytosine by-products, the mentioned patents disclose that by contacting the enzyme preparation with an organic solvent and heating the enzyme preparation at an effective temperature between 60 ° C and 90 ° C for a period of time, A method for specifically reducing the amino enzyme activity is additionally disclosed.

Araki et al (US 2005/0074857) is a microorganism, e.g., E. coli using a pyrimidine base derivatives by using the enzyme, also known as (E. coli) the purine nucleoside kinases (PNP) derived from the pyrimidine nucleoside A seed compound and a novel pyrimidine nucleoside compound, which enzyme also has cytosine nucleoside phosphorylase activity. The authors overcome certain deacetylation deactivation by heating bacterial cells or treated enzymes of microorganisms or by placing them in contact with suitable organic solvents.

These same authors have reported that by producing microorganisms lacking both cytosine deaminase and cytidine deaminase genes (JP2004344029) or zinc salts (JP2004024086), without applying complex deaminase-inactivation treatments Several methods for producing the compounds described above have been disclosed.

Ding Q. 2010. Enzymatic synthesis of nucleosides by nucleoside phosphorylase co-expressed in Escherichia coli . J. Zheijiang Univ-Sci B (Biomed & Biotechnol) 11 (11): 880-888 initiated the synthesis of many types of nucleosides. However, the above disclosures have failed to produce cytosine or arabinoside nucleosides and are therefore away from using NPs for the synthesis of this particular type of nucleoside.

Szeker, K. 2012. Nucleoside phosphorylases from thermophiles, Ph.D. Thesis, Berlin Institute of Biotechnology, Germany discloses that GtPyNP ( Geobacillus < RTI ID = 0.0 > thermoglucosidasius ), but only TtPyNP ( Thermus thermophilus ( Thermus) Thermophilus to MP) is determined for cytidine as a substrate (see above, p. 84), and cytidine is coupled to PNPs and ApMTAP ( Aeropyrum pernix ), 5'-methylthioadenosine phosphorylase, see above, p. 116). &Lt; / RTI >

Thus, biocatalytic synthesis of cytosine nucleoside analogs remains a challenge, and its application at an industrial scale is limited by product degradation due to competitive enzymes present in biocatalyst products, as described above.

Because many non-natural nucleosides that act as antiviral or anticancer agents contain cytosine as a nucleobase moiety, new and effective methods of overcoming the aforementioned drawbacks and improving the synthesis of such types of cytosine nucleoside analogs It is interesting to explore the development of industrial enzymatic processes.

Capecitabine, a well-known anticancer agent with a cytosine NA chemical structure, has been produced in the past according to the various processes described in the prior art for the multistage chemical synthesis of such NA (Founder, F. Hoffmann-La Roche AG, Arasaki meat al. , EP 0602454; Kamiya et al , EP0602478).

This process can be carried out in the presence of certain defects such as: toxic solvents or reagents used, the need for chromatographic purification steps unsuitable for large-scale production, the multi-step complex process involving the protection and deprotection of hydroxyl groups on glycosidic units , And low to intermediate overall yields of capecitabine synthesis. The order of protection and deprotection adds two separate steps in all these processes, which in turn reduces the overall yield and increases the time and cost of the process from a commercial production standpoint.

A one-step process for the preparation of capecitabine is disclosed in WO 2011/104540. This process comprises reacting a pentyloxycarbonylation reagent and a 5-fluoro-5'-deoxycytidine reagent to prevent the need for conventional protection of the hydroxyl group of the preformed nucleoside 5-fluoro-5'-deoxycytidine Lt; / RTI > The reaction takes place in an organic solvent and can be carried out in the presence of a chemical catalyst (such as a base or a caustic mineral acid such as HCl gas), a high temperature (up to 110 DEG C at reflux) and a long reaction time (typically 9 to 10 hours) , And capecitabine is made at an intermediate yield (50 to 67%).

We have demonstrated that caffeicitabine undergoes thermal instability at elevated temperature and that the product is lost to 40% of the product after heating the product at neutral pH for 10 hours at 90 ° C. For this reason, the process described in WO 2011/104540 suffers from a significant loss of final product.

In contrast, the enzymatic process of the present invention is carried out at milder conditions using water as a solvent and provides a yield of greater than 70%.

Kanan et al (WO 2010/061402) is in the presence of an organic solvent (CALB) Novozyme 435 (Candida not tar urticae (Candida antartica ) as a catalytic agent. This process requires at least four steps to supply caffeitabine.

Thereafter, depending on the importance of capecitabine in the treatment of the above mentioned disadvantages and cancers associated with the prior art, the use of relatively inexpensive reagents without involving a plurality of steps and the production of nucleoside analog products at high yields and purity It is highly necessary to develop improved processes for the production of these active principles (API).

The same applies to cytarabine synthesis. Cytarabine is another well-known anti-cancer agent that shares the cytosine NA chemical structure with capecitabine. There are initial patents under the name of Merck (NL 6511420) or Salt Lake Institute of Biological Studies (1969) in 1964, which have already described the chemical synthesis of the active compounds, Faults may be referred to equally.

Surprisingly, it has been found that the aforementioned disadvantages of biocatalytic synthesis can be avoided, and that cytosine NAs can be obtained at conversions higher than 70% and anomer purity higher than 95%. It consists of nucleoside phosphorylase (NPs), purine nucleoside phosphorylase (PNPs), or pyrimidine nucleoside phosphorylase (PyNP), or pyrimidine nucleoside phosphorylase (PyNP) and purine nucleoside phosphorylase Lt; RTI ID = 0.0 > (PNP) < / RTI > The mentioned enzymes can, surprisingly, recognize appropriate chemically modified cytosines and perform glycosyl transfer reactions on donor nucleoside analogs without any evidence of deamidation or deacetylation impurities. The present inventors have demonstrated that a biocatalytic reaction using the same but unmodified cytosine as a nucleoside receptor does not produce the expected final product. For the purposes of this specification, the preferred enzyme used in the process of the invention is a pyrimidine nucleoside phosphorylase. Another preferred embodiment of the invention relates to the use of a mixture of a pyrimidine nucleoside phosphorylase (PyNP) and a purine nucleoside phosphorylase (PNP) enzyme, especially when a purine nucleoside or derivative or analog thereof is used as the starting compound .

The process of the invention is applied, by way of example, to the production of capecitabine or cytarabine, since cytosine NAs is an example, for enzymatic synthesis for the chemical synthesis of these NAs and also using CALB Novozyme 435, Because it has several advantages over the process used in the technology:

(i) a reduced number of steps,

(ii) higher conversion and yield,

(iii) a protection / deprotection strategy for the hydroxyl groups in the sugar is not required,

(iv) Mild reaction conditions: Environmentally friendly techniques (water or aqueous medium, neutral pH)

(v) avoiding organic solvents in the enzymatic step,

(vi) Extremely selectivity: stereoselectivity - enantioselectivity, chemical position selectivity,

(vii) No side reactions: impurity profile (reduced by-product content),

(viii) reduction in overall waste generation,

(ix) process productivity,

(x) Overall lower production costs

In addition, there are other additional advantages of the inventive biocatalytic process over the chemical processes used in the prior art. In the process of the present invention, Arasaki et al . And Kamiya et al. In the organic solvents used in chemical processes, for example, pyridine, dichloromethane (DCM), acetonitrile (ACN), methanol, tetrahydrofuran (THF), or Kannan et Particularly relevant is the absence of organic solvents (pyridine, dichloromethane (DCM), etc.) used in the enzymatic process of al .

Particularly relevant in the process of the present invention is the absence of a metal catalyst, such as a second tin chloride, a toxic reagent, for example a silylating agent. All these process organic solvents and reagents must be removed or destroyed prior to any waste release into the environment. The disadvantage of the process described in the prior art with regard to the procedure of the invention is that the process described in the prior art comprises a multistep procedure involving a protection and deprotection step in contrast to the simpler enzymatic process of the invention This is its complexity.

DESCRIPTION OF THE INVENTION

Processes of the invention as described herein using PyNP or PyNP / PNP enzymes are set forth as follows:

[Reaction Scheme 1]

Enzymatic synthesis of cytosine nucleoside analogs

Applicants have surprisingly found that suitable N ⁴ -modified cytosine or N ⁴ -modified cytosine derivatives are produced by using nucleoside phosphorylase (native, recombinant or mutant proteins from bacteria or from high bacteria), and in particular by using pyrimidine nucleoside By using phosphorylating enzymes (innate, recombinant or mutant proteins from bacteria or from high bacteria), it is possible to produce / produce cytosine nucleoside analogs at high conversions (greater than 70%), But completely avoid side reactions, such as deamination or deacetylation.

No evidence has been found in the prior art to indicate that chemical modification / substitution / protection at this location or at any other location within the cytosine chemical center will alter the substrate specificity for the nucleoside phosphorylase. For purposes of this specification, the term N ⁴ -modified cytosine / cytosine derivatives are synonyms for N ⁴ -substituted cytosine / cytosine derivatives or N ⁴ -protected cytosine / cytosine derivatives, respectively.

For purposes of the present patent specification, the term cytosine or cytosine derivatives, nucleosides, intermediates, bases or nucleobases should all be understood to be chemical compounds derived from the cytosine backbone. Particularly for the purposes of the present invention, cytosine or cytosine derivatives are represented by formula II:

&Lt; RTI ID = 0.0 &

A suitable chemical modification for the purposes of the present invention provides a more convenient, efficient and easier process for the synthesis of nucleoside analogs that completely avoids the unstable problems associated with side reactions. Particularly preferred N ⁴ -straps are carbamate (-NHCOO-), more specifically an alkyl chain of 1 to 40 carbon atoms, an alkenyl chain of 1 to 40 carbon atoms or an alkynyl chain of 1 to 40 carbon atoms (These chains being linear in each case, branched, or substituted by any other functional group), carbamates linked to aryl or heterocycles. The N ⁴ -substitution of the amino group in the form of an amide or related acyl derivative (-NHCO-) is particularly preferred when the amido group is an alkyl chain of 4 to 40 carbon atoms, an alkenyl chain of 1 to 40 carbon atoms, Glycosyl transfer reactions are also allowed when the alkynyl chains of five carbon atoms, which chains are in each case straight, branched, or substituted by any other functional group, to an aryl or heterocycle. Surprisingly, longer alkyl chains are preferred over shorter alkyl chains (1 to 3 carbon atoms), because, according to the experiments performed, unlike US 2005/0074857 teaches that the deacetylation side reactions are short alkyl chains . According to the present invention, an acyl group having an alkyl group of one carbon atom does not prevent deacetylation.

More precisely, the chemical modification is introduced into the cytosine backbone at N ⁴ (nitrogen atom at position 4 in the cytosine heterocycle) according to the detailed description. More specifically, these chemical modifications that have been activated in the cytosine skeleton are those represented by the R < ₂ > and / or R < _{3 &}

&Lt; RTI ID = 0.0 &

here

R ¹ is O, CH ₂ , S, NH;

R ² is selected from hydrogen, optionally substituted C _4-40 alkyl chain, optionally optionally substituted alkenyl chain, optionally optionally substituted alkynyl chain, optionally optionally substituted alkyl, alkenyl or alkynyl chain Optionally substituted aryl, optionally substituted aryl, optionally substituted alkyl, alkenyl or alkynyl chain connected to N, COR ⁶ , CONR ⁶ R ⁷ , CO ₂ R ⁶ , C (S) OR ⁷ , CN, SR ⁶ , _{^{_{SO 2 R 6, SO 2 R}}} 6 R 7, CN, P (O) aryl, P (O) heterocycle, P (S) aryl, P (S) heterocycle, P (O) O ₂ R ^8, and;

R ³ is selected from hydrogen, optionally substituted C _4-40 alkyl chains, optionally optionally substituted alkenyl chains, optionally optionally substituted alkynyl chains, optionally optionally substituted alkyl, alkenyl or alkynyl chains Optionally substituted aryl, optionally substituted aryl, optionally substituted alkyl, alkenyl or alkynyl chain connected to N, COR ⁶ , CONR ⁶ R ⁷ , CO ₂ R ⁶ , C (S) OR ⁷ , CN, SR ⁶ , SO ₂ R ⁶ ; _{^{SO 2 R 6 R 7, CN}} , P (O) aryl, P (O) heterocycle, P (S) aryl, P (S) heterocycle, P (O) O _2, and R ^8; R ³ and R ² are independent of each other; And as a clue at least one of R ₂ or R ₃ is different from hydrogen;

R ⁴ is hydrogen, OH, NH ₂ , SH, halogen (preferably F or I); An optionally substituted alkyl chain; An optionally substituted alkenyl chain; Optionally substituted alkynyl, trihaloalkyl, OR ⁶ , NR ⁶ R ⁷ , CN, COR ⁶ , CONR ⁶ R ⁷ , CO ₂ R ⁶ , C (S) OR ⁶ , OCONR ⁶ R ⁷ , OCO by _{^{2 R 6, OC (S)}} oR 6, NHCONR 6 R 7, NHCO 2 R 6, NHC (S) oR 6, SO 2 NR 6 R 7, any optionally substituted alkyl, alkenyl or alkynyl chain Any optional substituted aryl, optionally substituted alkyl, alkenyl or alkynyl chain connected to Y by any optionally substituted heterocycle, and independently selected from R ¹ , R ^2, , Any of R ³ , R ⁴ or R ⁵ , and optionally substituted aryl;

As a clue, Y is a carbon or sulfur atom and, alternatively, when R ⁴ is absent, Y is a nitrogen atom as a cue;

here

X is O, S, NR ^B2 , Se; R ^B1 is H, OH, NH ₂ , SH, straight or branched C _1-10 alkyl, F, Cl, Br, I, XR ^B2 , -C≡CR ^B2 , CO ₂ R ^B2 ; R ^B2 is H, OH, NH ₂ , straight or branched C _1-5 alkyl, phenyl;

R ⁵ is selected from the group consisting of hydrogen, OH, NH ₂ , SH, halogen (preferably F or I), optionally substituted alkyl chains, optionally optionally substituted alkenyl chains, optionally optionally substituted alkynyl chains, roal ^{Kiel, OR 6, NR 6 R 7} , CN, COR 6, CONR 6 R 7, CO 2 R 6, C (S) OR 6, OCONR 6 R 7, OCO 2 R 6, OC (S) OR 6, NHCONR ⁶ R ⁷ , NHCO ₂ R ⁶ , NHC (S) OR ⁶ , SO ₂ NR ⁶ R ⁷ ; CH ₂ -heterocyclic ring, CN;

And independently any optional optionally substituted heterocycle of R ² , R ³ , R ⁴ or R ⁵ , or optionally substituted aryl,

here

R ⁶ and R ⁷ are independently of each other hydrogen, optionally substituted alkyl chain, optionally optionally substituted alkenyl chain, optionally optionally substituted alkynyl chain, heterocyclic or optionally substituted aryl;

R < ⁸ > is hydrogen, optionally substituted alkyl chain, optionally optionally substituted alkenyl chain, optionally optionally substituted alkynyl chain, optionally optionally substituted aryl, or optionally substituted heterocycle;

Y is C, N, S.

According to a comparative example carried out by the Applicant and shown in the detailed description, cytosine derivatives which do not contain a suitable modification in N ⁴ can be prepared using nucleoside phosphorylase (congenital, recombinant or mutant enzymes), in particular pyrimidine nucleobases Does not undergo glycosyl transfer using a seed phosphorylase, and for this reason does not produce the desired nucleoside end product.

In addition, unprotected cytosines (bases or equivalent nucleosides) may be produced by some authors by conventional methods, for example by heating or using organic solvents (EP1254959 and US2005 / 0074857), by using a microorganism which simultaneously lacks both a cytosine deminotase gene and a cytidine deaminase gene by adding a zinc salt (JP2004024086) or by using a more sophisticated method (JP2004344029), side reactions attempting to deactivate (e.g., deamidation or deacetylation). All of the foregoing technical solutions described in the prior art dramatically reduced the overall yield of the corresponding synthesis process. The present invention provides a method of synthesizing nucleoside analogs that completely and definitively solves the aforementioned technical problems associated with side reactions that reduce the overall yield of the synthesis process described herein.

The functional groups used to modify / replace / protect the N ⁴ position in the final product obtained can be optionally optionally removed (deprotection of the final product), depending on the chemical structure of each final product produced. However, in certain cases, for example, in the case of a capecitabine molecule, it is not necessary to deprotect the modified / substituted / protected cytosine N ⁴ group, since the modification is carried out within the final product (or API) Because it remains attached to the amino group. For capecitabine itself, the enzymatic method of the invention significantly shortens the time required when a traditional chemical production process is used.

Thus, the present invention provides novel nucleosides that are useful as anticancer and / or antiviral products by shortening conventional multistage synthesis, increasing overall yield, reducing side reactions and byproduct content, and therefore improving product purity and quality. Lt; RTI ID = 0.0 > analogous < / RTI >

For the purpose of detailed description, the following terms are further defined as follows.

The term "nucleoside" refers to any compound wherein a heterocyclic base is covalently linked to a sugar, and a particularly preferred coupling of a nucleoside to a sugar is a carbon- or heteroatom (typically nitrogen ) Of the carbon atoms in the sugar. For this reason, the term "nucleoside" in this context means a glycoside of a heterocyclic base. Similarly, the term "nucleotide" refers to a nucleoside in which the phosphate group is linked to the sugar.

The term "nucleoside" can be used broadly to include non-naturally occurring nucleosides, naturally occurring nucleosides as well as other nucleoside analogs. An illustrative example of a nucleoside is a deoxyribonucleoside comprising a deoxyribose moiety as well as a ribonucleoside comprising a ribose moiety. For bases of this nucleoside, it is meant to include any one of the naturally occurring bases, such as adenine, guanine, cytosine, thymine, and uracil, as well as any modified variants thereof or any possible non- It should be understood that it is possible.

The terms "nucleoside analog "," nucleoside analog ", "NA ", or" NAs ", as used herein, means that the sugar is preferably all ribofuranose or arabinofuranose Refers to all nucleosides that are not naturally occurring bases (e.g., A, G, C, T, I, etc.) where the nucleosides and / or the heterocyclic bases occur naturally.

As further used herein, the term "sugar" refers to all carbohydrates and derivatives thereof, wherein the derivatives specifically contemplated include deletions, substitutions or additions of chemical groups or atoms within the sugar. By way of example, particularly anticipated deletions include 2'-deoxy, 3'-deoxy, 5'-deoxy and / or 2 ', 3'-dideoxy-sugars. Particularly contemplated substitutions include the replacement of ring-oxygen with sulfur or methylene, or the replacement of hydroxyl groups with halogen, azido, amino-, cyano, sulfhydryl-, or methyl groups, Phosphonate group. More anticipated sugars also include sugar analogs (i.e., sugars that do not occur naturally), and in particular carbon ring system. The term "carbon cyclic ring system" as used herein refers to any molecule in which a plurality of carbon atoms form a ring, and in the particularly-contemplated carbon cyclic ring system, the ring may contain 3, 4, 5, or 6 Carbon atoms.

The term " chemoenzymatic "refers to a method for the synthesis of chemical compounds through a combination of chemical and biocatalytic steps. For a specific purpose of the present invention, the order of the reaction step is, in one of the preferred embodiments of the present invention, 1) a chemical modification of the cytosine base; 2) biocatalytic glycosyl transfer using NP enzymes; 3) Optionally, it should be an additional deprotection of cytosine-N ⁴ in the final product.

The term "enzymatic synthesis" refers to a process for the synthesis of chemical compounds by processes involving only biocatalytic steps, carried out by appropriate enzymes (NPs). Thus, other preferred embodiments of the synthetic process described herein include cytosine derivatives, such as those already described, represented by the general formula II, which are commercially available as already prepared or such cytosine derivatives, Thus demonstrating R ₂ and / or R ₃ at the N ⁴ position of the cytosine heterocyclic ring and, for this reason, a complete biocatalytic process starting from those which omit step 1 of the chemical modification of the cytosine backbone described above .

The term "heterocyclic ring" or "heterocyclic base" or "base" or "nucleobase" is used interchangeably herein and refers to any compound wherein a plurality of atoms form a ring through a plurality of covalent bonds , Wherein the ring contains at least one atom other than a carbon atom. Particularly contemplated heterocyclic bases are 5- and 6-membered rings containing at least one to four heteroatoms each independently selected from nitrogen, oxygen and sulfur as non-carbon atoms (such as imidazole, pyrrole, triazole, Dihydropyrimidine). More contemplated heterocycles may be fused (i.e., covalently bonded) to other rings or heterocycles and are thus termed "fused heterocycle" or "fused heterocyclic base ", as used herein. Particularly contemplated fused heterocycles include a 5-membered ring fused to a 6-membered ring (such as purine, pyrrolo [2,3- d ] pyrimidines), and 6- (E.g., pyrido [4,5- d ] pyrimidine, benzodiazepines). Examples of these and more preferred heterocyclic bases are provided below. Other more-anticipated heterocyclic bases may be aromatic, or they may include one or more double or triple bonds. In addition, the anticipated heterocyclic base and the fused heterocycle may be further substituted at one or more positions. And, any one having the Among these rings, halogen, hydroxy, nitro, cyano, carboxyl, C _1-6 alkyl, C _1-6 alkoxy, C _1-6 alkoxy C ₁ _-6 alkyl, C _1-6 alkyl carbonyl, amino, mono-or di-C ₁ _-6 alkylamino, azido, mercapto, C ₁ _-6 alkyl, C ₁ _-6 to be poly to poly-alkoxy, and the group consisting of C _3-7 cycloalkyl, 2, or 3 substituents each independently selected from lower alkyl, lower alkoxy, and lower alkoxy.

The term "nucleobase" covers naturally occurring nucleobases as well as non-naturally occurring nucleobases. It will be apparent to those skilled in the art that various nucleobases previously considered to be "naturally occurring" are subsequently discovered in nature. Thus, "nucleobases" include not only known purines and pyrimidine heterocycles, but also heterocyclic analogs (e.g., N -substituted heterocycles) and their tautomeric forms. Illustrative examples of nucleobases include adenine, guanine, thymine, cytosine, uracil, purine, xanthine, 2-chloroadenine, 2-fluoroadenine, pentyl (5-fluoro- 4-yl) carbamate, cytosine N -alkylcarbamate, cytosine N -alkyl ester, 5-azacytosine, 5-bromovinyluracil, 5- fluorouracil, 5-trifluoromethyluracil, 6- methoxy -9 H - purine-2-amine and (R) -3,6,7,8- tetrahydro an imidazo [4,5- d] [1,3] diazepin-8-ol.

The term "nucleobase" is intended to cover all of these examples as well as analogs, tautomers, and positional isomers thereof.

For the purposes of this specification, the term "nucleobase" refers primarily to the cytosine bases represented by formula II, in order to identify these "nucleobases" from other heterocyclic bases also present herein. However, the term "base" refers, for the purposes of this specification, to bases present predominantly in the nucleosides represented by formula III.

The term " tautomer "or" tautomeric form "refers to structural isomers of different energies compatible through low energy barriers. By way of example, proton variants (also known as protophilic fauids) include interconversions through the migration of protons, such as keto-enol and imine-enamine isomerization. The valence taut variants include interconversions by recombination of some of the coupled electrons.

The term " positional isomer "refers to a structural isomer or a constitutive isomer, in the sense that the atom is intended to refer to a molecule having the same molecular formula attached at a different degree of coupling.

The term "conversion" refers to the percentage of the starting material that is converted to the product, eg, the expected end product, by-product, or even the degradation product.

The term "yield" is the number of synthesized molecules of the product relative to the number of starting molecules. In multistage synthesis, the yield can be calculated by multiplying the yield of every single step.

The term "anomeric purity" refers to the amount of a particular anomer of a compound that is divided by the total amount of all anomers of the compound present in the mixture multiplied by 100%.

The term "cytosine modification / substitution / protection" means that at least one position (except nitrogen at position 1) on the original backbone of the cytosine is replaced by a functional group, such as a radical R ¹ , R ² , R ³ , R ⁴ , R ⁵ _, Refers to any nucleoside analog substituted by those described as X and / or Y, wherein one of each of these groups is independent of the others.

The term " cytosine modified / substituted / protected at position N ⁴ "means that at least one protons of the amino group at position 4 are substituted by functional groups, such as those described as radicals R ₂ and / or R ₃ Refers to a base having a cytosine backbone in which one of these substituents is independent from the others, and at least one of the substituents described above as a clue is different from hydrogen.

The term " intermediate "or" intermediate "refers to any nucleoside analog type compound that can be converted into the active pharmaceutical ingredient (API) of the nucleoside structure by a suitable additional chemical reaction. For this reason, intermediates are molecules that can be considered as API precursors. Purposes, the term "precursors" herein is N ⁴ - a modified compound of cytosine or a cytosine derivative, or when applied to the intermediate, a chemical group (II) in addition to the moiety bonded to the N ⁴ as those represented by R ² or R ³ Lt; / RTI >

The term "prodrug" as used throughout the text means pharmacologically acceptable derivatives, such as esters, amides, carbamates and phosphates, and thus the in vivo conversion product of the derivatives as a result of in vivo Is an active drug as defined in the compound of formula (I). (The Pharmacological Basis of Therapeutics, 8th Ed, McGraw-Hill, Int. Ed., 1992, "Biotransformation of Drugs ", p 13-15), which generally describes prodrugs, is incorporated herein by reference . Prodrugs preferably have good aqueous solubility, increased bioavailability, and are readily metabolized in vivo to an activity inhibitor. Prodrugs of the compounds of the present invention can be prepared by modifying the functional groups present in the compound in such a way that the modifications are modified by routine manipulation or in vivo in the parent compound.

Preferred prodrugs are pharmaceutically acceptable esters, amides and carbamates derived from compounds of formula I that are hydrolysable in vivo and have a hydroxy or amino group. In vivo hydrolysable esters, amides and carbamates are ester, amide or carbamate groups that hydrolyze in the human or animal body to produce the parent acid or alcohol. Suitable pharmaceutically acceptable esters, amides and carbamates for the amino group are C _1-6 alkoxymethyl esters, such as methoxymethyl, C _1-6 alkanoyloxymethyl esters, such as pivaloyl Oxymethyl, phthalidyl esters, C _3-8 cycloalkoxycarbonyloxy C ₁ _-6 alkyl esters such as ₁ _- cyclohexylcarbonyloxyethyl; 1,3-dioxolen-2-onyl methyl ester, for example, 5-methyl-l, 3-dioxolen-2-onylmethyl; And C _1-6 alkoxycarbonyloxyethyl esters which may be formed in any of the carboxy groups within the compounds of the present invention, for example, 1-methoxycarbonyl-oxyethyl.

In vivo hydrolysable esters of compounds of formula (I) containing hydroxy groups include compounds related to inorganic esters, such as phosphate esters and [alpha] -acyloxyalkyl ethers, which result in the in vivo hydrolysis of the ester And provides the parent hydroxy group. Examples of? -acyloxyalkyl ethers include acetoxymethoxy and 2,2-dimethylpropionyloxy-methoxy. Selection of the in vivo hydrolysable ester forming groups for hydroxy is accomplished by reacting alkanoyl, benzoyl, phenylacetyl and substituted benzoyl with phenylacetyl, alkoxycarbonyl (providing alkyl carbonate ester), dialkyl carbamoyl and N- (di Alkylaminoethyl) -N-alkylcarbamoyl (providing a carbamate), dialkylaminoacetyl and carboxyacetyl. Examples of substituents on benzoyl include morpholino and piperazino linked from the ring nitrogen atom via a methylene group to the 3- or 4-position of the benzoyl ring.

For therapeutic use, the salts of the compounds of formula (I) are those for which the counterion is pharmaceutically acceptable. However, salts of non-pharmaceutically acceptable acids and bases also find use, for example, in the preparation or purification of pharmaceutically acceptable compounds. All salts, whether pharmaceutically acceptable or not, are included within the scope of the present invention.

Pharmaceutically acceptable acid and base addition salts as mentioned above are meant to include the therapeutically active non-toxic acid and base addition salt forms which the compounds of formula (I) can form. Pharmaceutically acceptable acid addition salts may conveniently be obtained by treating the base form with such suitable acid. Suitable acids include, for example, inorganic acids such as hydrohalic acids, such as hydrochloric acid or hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and other acids; Or organic acids such as, for example, acetic acid, propanoic acid, hydroxyacetic acid, lactic acid, pyruvic acid, oxalic acid (i.e., ethanedioic acid), malonic acid, succinic acid (i.e. butanedionic acid), maleic acid, fumaric acid , Malic acid (i.e., hydroxybutanedionic acid), tartaric acid, citric acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclamic acid, salicylic acid, p-aminosalicylic acid, fumaric acid and other acids.

Conversely, the salt form can be converted into the free base form by treatment with an appropriate base.

Compounds of formula (I) containing acidic protons can also be converted into their non-toxic metal or amine addition salt form by treatment with appropriate organic and inorganic bases. Suitable base salt forms include, for example, ammonium salts, salts with alkaline and alkaline earth metal salts such as lithium, sodium, potassium, magnesium, calcium salts and the like, such as benzathine, N-methyl -D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine, and the like.

The term addition salt, as used hereinabove, also encompasses the solvates which the compounds of formula (I) as well as their salts can form. Such solvates are, for example, hydrates, alcoholates, and the like.

The term "quaternary amine" as used herein refers to a compound wherein the compound of formula (I) is reacted with a basic nitrogen of the compound of formula (I) and a suitable quaternarizing agent, A quaternary ammonium salt which may be formed by reaction between an aryl halide or an arylalkyl halide, e.g., methyl iodide or benzyl iodide. Other reactive materials with good leaving groups, such as alkyl trifluoromethanesulfonate, alkyl methanesulfonate salts, and alkyl p-toluenesulfonate salts, may also be used. The quaternary amine has positively charged nitrogen. Pharmaceutically acceptable counterions include chloro, bromo, iodo, trifluoroacetate and acetic acid salts. The selected counterion may be introduced using an ion exchange resin.

The N-oxide form of the compounds of the present invention is meant to include compounds of formula (I) wherein one or several nitrogen atoms are oxidized to the so-called N-oxide.

It will be appreciated that the compounds of formula (I) may have metal binding, chelating, complexing properties, and for that reason may exist as metal complexes or metal chelates. Such metallated derivatives of the compounds of formula (I) are intended to be included within the scope of the present invention.

Some of the compounds of formula (I) may also exist in their tautomeric form. This form is intended to be included within the scope of the present invention, although not explicitly indicated in the above formula.

The term "alkyl" as used herein refers to any linear, branched, or cyclic hydrocarbon in which all carbon-carbon bonds are single bonds.

The terms "alkenyl" and "unsubstituted alkenyl" are used interchangeably herein and refer to any linear, branched, or cyclic alkyl having at least one carbon-carbon double bond.

In addition, the term "alkynyl " as used herein refers to any linear, branched, or cyclic alkyl or alkenyl having at least one carbon-carbon triple bond.

The term "aryl" is a phenyl or a naphthalenyl group, or is a part of a group, each optionally substituted by halo, hydroxy, nitro, cyano, carboxyl, C _1-6 alkyl, C _1- As used herein ₆ alkoxy, C _1-6 alkoxy C ₁ _-6 alkyl, C _1-6 alkylcarbonyl, amino, mono-or di-C ₁ _-6 alkylamino, azido, mercapto, C ₁ _-6 alkyl in to poly, and it refers to any aromatic cyclic alkenyl, or alkynyl is optionally substituted with any by C ₁ _-6 _1, 2 or 3 substituents selected from alkoxy poly be. The term "alkaryl" is used when aryl is covalently bonded to alkyl, alkenyl, or alkynyl.

Refers to the replacement of an atom or chemical group (e.g., H, NH ₂ , or OH) with a functional group, as used herein, and the functional groups specifically contemplated are nucleophilic groups (e.g., -NH _2, -OH, -SH, -NC, etc.), an electrophilic group (e.g., C (O) OR, C (X) OH, etc.), polar groups (e.g., -OH), a non-polar group (e.g., aryl, Alkyl, alkenyl, alkynyl, etc.), ionic groups (e.g., -NH ₃ ⁺ ), and halogens (e.g., -F, -Cl), and combinations of all chemically reasonable ones. Accordingly, the term "functional group" and the term "substituent" is used herein to be replaced, and a nucleophilic group _{(e.g., -NH 2, -OH, -SH,} -NC, -CN , etc.), an electrophilic group (E.g., C (O) OR, C (X) OH, C (halogen) OR etc.), a polar group (e.g., -OH), a nonpolar group (e.g., aryl, alkyl, alkenyl, (E.g., -NH ₃ ⁺ ), and halogen.

The term "nucleoside phosphorylase" or "NP" or "NPs" refers to a pyrimidine nucleoside phosphorylase enzyme or PyNP (PyNPs) enzyme or PyNPs and PNPs (purine nucleoside phosphorylase enzyme) &Lt; / RTI > These enzymes can be obtained from naturally occurring microorganisms such as mesophiles, thermophilic bacteria, ultrahydrochemists or extreme organisms. For the purposes of detailed description, mesophilic or mesophilic organisms or NPs enzymes are those that can operate or perform NP activity at temperatures varying from 18 to 60 占 폚, at an optimal temperature range of 40 to 55 占 폚. Thermophilic or thermophilic organisms or NPs enzymes are those that can operate or operate at NP temperatures above 60 < 0 > C and up to 80 < 0 > C. The hyperthermophilic or hypertonic organisms or NPs enzymes are those that can operate or operate at NP temperatures above 80 ° C and up to 100 ° C, and at an optimal temperature range of 80-95 ° C. Additionally, these enzymes used in the present invention may be cloned enzymes obtained by genetic recombination techniques and expressed in host cells transformed with the corresponding vectors carrying the individual encoding nucleic acid sequences.

The nucleic acid molecule encoding the NP enzyme according to the present invention is preferably selected from the group consisting of SEQ ID NO: 1, 2, 5, 7, 9 or 11; or

a) a complementary nucleotide sequence of SEQ ID NO: 1, 2, 5, 7, 9 or 11; or

b) a nucleotide sequence which is flanked by SEQ ID NO: 1, 2, 5, 7, 9 or 11; or

c) under conditions of high stringency, in SEQ ID NO: 1, 2, 5, 7, 9 or 11; A complement of SEQ ID NO: 1, 2, 5, 7, 9 or 11; Or a nucleotide sequence which hybridizes to a hybridization probe derived from SEQ ID NO: 1, 2, 5, 7, 9 or 11; Or its complement; or

d) a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 1, 2, 5, 7, 9 or 11; or

e) a nucleotide sequence having at least 59% sequence identity with SEQ ID NO: 1, 2, 5, 7, 9 or 11; or

f) a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NO: 3, 4, 6, 8, 10 or 12.

Conditions of stringent hybridization in the sense of the present invention are defined as those described by Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), 1.1011.104. According to this, under stringent conditions, hybridization is carried out with 1 x SSC buffer and 0.1% SDS for 1 hour, in particular 55 ° C, preferably 62 ° C, at 55 ° C, preferably 62 ° C, most preferably 68 ° C, , Most preferably at 68 [deg.] C, for 1 hour with 0.2 x SSC buffer and 0.1% SDS.

Furthermore, in the sense of the detailed description, the present invention also encompasses a nucleotide or amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 (nucleotide) or SEQ ID NO: 3 or SEQ ID NO: 4 (amino acid) But covers at least 59%, particularly preferably at least 80%, most preferably at least 90% identity to the amino acid sequence. Percent identity is determined according to the following equation:

I = (n / L) x 100

Where I is percent identity, L is the length of the base sequence, and n is the number of nucleotides or amino acid differences in the sequence relative to the base sequence.

On November 15, 2013 Date of PNP protein and purine nucleoside kinases similar in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) (deoD) [alcohol Polo Booth Solpa Tari Syracuse (Sulfolobus solfataricus )] identity of the protein sequence Access number organism % Identity NP_342951.1 Sulfolobus solfataricus P2 (SEQ ID NO: 3) 100% NP_378450.1 Sulfurous booth Tokodai ( Sulfolobus tokodaii) str . 7 83% YP_005648757.1 Sulfurous booth Isradicus ( Sulfolobus islandicus) LAL14 /One 59% WP_016730864.1 Sulfurous booth Isradicus ( Sulfolobus islandicus) 64% WP_016730167.1 Sulfurous booth Isradicus ( Sulfolobus islandicus) 61%

UDP is similar to proteins in the GenBank (http://www.ncbi.nlm.nih.gov/genbank/) on 15 November 2013 and the date of uridine kinase [Erotic pirum Fernand Knicks (Aeropyrum pernix )] K1 protein sequence identity Access number organism % Identity NP_148386.2 Aeropyrum pernix ) K1 (SEQ ID NO: 4) 100% YP_008604720.1 Erofumi Carmini ( Aeropyrum glass ) SY1 93%

Another important feature of the present invention is a recombinant vector comprising at least one copy of a nucleic acid molecule as defined above operably linked to an expression control sequence. The vector may be any prokaryotic or eukaryotic vector. Examples of prokaryotic vectors are chromosome vectors, such as bacteriophage (e.g., bacteriophage lambda) and extrachromosomal vectors, such as plasmids (see, for example, Sambrook et al., Supra, Chapter 1-4). The vector may also be a eukaryotic vector, e. G., A yeast vector or a vector suitable for high cells, for example, a plasmid vector, a viral vector or a plant vector. Suitable eukaryotic vectors include, for example, Sambrook et < RTI ID = 0.0 > al ., described in Chapter 16 above. The invention further relates to a recombinant cell transformed with a nucleic acid or recombinant vector as described above. The cell can be any cell, e. G. Prokaryotic or eukaryotic. Prokaryotic cells, particularly E. coli cells, are particularly preferred.

For the purpose of detailed description, the present invention also covers mutants of SEQ ID NO: 1 to SEQ ID NO: 12, or orthologs.

The term "variant" is understood to mean the nucleotide sequence of the nucleic acid or the amino acid sequence of the protein or polypeptide, respectively, which is modified by one or more nucleotides or amino acids, as used throughout the specification. Variants can have a "conservative" change wherein the substituted nucleotide or amino acid has structural or chemical properties similar to the substituted nucleotide or amino acid. Variants may also have "non-conservative" changes or deletions and / or insertions of one or more nucleotides or amino acids. The term also includes within the scope any insertions / deletions of nucleotides or amino acids for a particular nucleic acid or protein or polypeptide. "Functional variant" shall be understood to mean a reference nucleotide sequence or a variant that retains the functional ability of the protein or polypeptide.

The term " complement "or" complementarity ", as used herein, means that 20 double-stranded nucleic acids, for example, each strand of DNA and RNA, are linked by two or three hydrogen bonds between them, May be meant to be complementary to the other in that it is connected to the other. In the case of DNA, the adenine (A) base supplements the thymine (T) base and vice versa; The guanine (G) base complements the cytosine (C) base and vice versa. In the case of RNA, the adenine (A) base is the same, except that it replaces the uracil (U) base instead of the thymine (T) base. Since there is only one 25 complementary base for each base found in DNA and RNA, complementary strands for any single strand can be reconstructed.

The term " orthologs "is understood to be sequences of homologous genes or miRNAs found in different species, as used throughout the specification.

"Recombinant DNA molecule" is understood herein as a DNA molecule that underwent molecular biological manipulation. The term "recombinant DNA" is, therefore, a form of DNA that does not exist naturally, which is created by joining DNA sequences that do not normally occur together.

Once introduced into a host cell, the "recombinant DNA molecule" is replicated by the host cell. The term "recombinant protein or enzyme" is used herein to mean a protein or enzyme produced by a method using a "recombinant DNA molecule ", and therefore also for the purposes of detailed description, the term recombinant enzyme or recombinant type enzyme refers to recombinant DNA Lt; / RTI > protein or enzyme.

Mutant enzymes are intended for the purposes of this specification by any enzyme that occurs naturally or that exhibits at least one mutation induced by human manipulation, including genetic engineering.

Conversely, enzymes that occur naturally, naturally or naturally occur for the purposes of this specification, are all considered synonymous and are meant by enzymes that preserve previous amino acid sequences that are found largely in nature, largely without mutations.

The functional portion of the enzyme of the present invention is meant to be the original enzyme or any sequence fragment of the DNA segment encoding it that maintains the ability of the complete enzyme sequence to perform all of the biocatalytic reaction properties.

The term cytosine nucleoside analog means, for the purposes of this specification, a nucleoside analog of a cytosine or a nucleoside analog of a cytosine derivative. Equally, the term cytosine base means a base cytosine or derivative thereof, as defined in the present patent specification. Similarly, the term modified cytosine base as used herein covers a compound represented by formula II wherein the backbone of the cytosine base is substituted at position 4, 5 or 6.

The detailed description discloses, in particular, an enzymatic process for producing cytosine nucleoside analogs useful as active pharmaceutical ingredients (APIs), intermediates or prodrugs thereof, which are active pharmaceutical ingredients (APIs) of formula I or their An intermediate, a nucleoside analogue (NAs):

(I)

here,

Z ₁ is O, CH ₂ , S, NH;

Z ₂ is independently from Z ₁ O, C (R ^S2 R ^S5 ), S (R ^S2 R ^S5 ), S (R ^S2 ), S (R ^S5 ), preferably SO or SO ₂ groups; ^{N (R S2 R S5),} N (R S2), N (R S5) , and;

Wherein R < ¹ ^> is hydrogen, OH, an ether or ester thereof,

,

n is 0 or 1, A is oxygen or nitrogen, and each M is independently selected from hydrogen, optionally substituted alkyl chains, optionally optionally substituted alkenyl chains, optionally optionally substituted alkynyl chains, Optionally substituted by any optionally substituted aryl, optionally optionally substituted alkyl, alkenyl, or alkynyl chain optionally optionally connected to P by an alkyl, alkenyl, or alkynyl chain substituted with A substituted heterocycle, or a pharmaceutically acceptable counterion, such as, without limitation, sodium, potassium, ammonium, or alkylammonium;

R ^S2 is hydrogen, OH or an ether or ester residue thereof, halogen, preferably F, CN, NH ₂ , SH, C≡CH, N ₃ ;

R ^S3 is hydrogen in the case of NA derived from a 2'-deoxyribonucleoside or arabinonucleoside, or OH, NH ₂ , halogen, preferably F when the NA is derived from a ribonucleoside , it is selected from OCH _3;

R ^S4 is hydrogen, OH or an ether or ester residue thereof, NH ₂ , halogen, preferably F, CN;

As a clue, R ^S1 and R ^S4 are different when both are ether or ester of the OH moiety;

R ^S5 is when Z ₂ be different from the oxygen, hydrogen, OH or an ether or ester residue thereof, NH ₂ or halogen, preferably F;

R ¹ is O, CH ₂ , S, NH;

R ² is selected from hydrogen, optionally substituted alkyl chains, preferably C _4-40 alkyl chains, optionally optionally substituted alkenyl chains, optionally optionally substituted alkynyl chains, optionally optionally substituted alkyl, alkenyl Or any optional substituted heteroaryl connected to N by an optionally substituted alkyl, alkenyl or alkynyl chain optionally joined to N by an alkynyl chain, COR ⁶ , CONR ⁶ R ⁷ , CO ₂ R ⁶ , C (S) OR ⁷ , CN, SR ⁶ , _{^{_{SO 2 R 6, SO 2 R}}} 6 R 7, CN, P (O) aryl, P (O) heterocycle, P (S) aryl, P (S) heterocycle, P (O) O ₂ R ^8, and;

R ³ is selected from the group consisting of hydrogen, optionally substituted alkyl chains, preferably C _4-40 alkyl chains, optionally optionally substituted alkenyl chains, optionally optionally substituted alkynyl chains, optionally optionally substituted alkyl, Or any optional substituted heteroaryl connected to N by an optionally substituted alkyl, alkenyl or alkynyl chain optionally joined to N by an alkynyl chain, COR ⁶ , CONR ⁶ R ⁷ , CO ₂ R ⁶ , C (S) OR ⁷ , CN, SR ⁶ , SO ₂ R ⁶ ; _{^{SO 2 R 6 R 7, CN}} , P (O) aryl, P (O) heterocycle, P (S) aryl, P (S) heterocycle, P (O) O _2, and R ^8; R ³ and R ² are independent of each other; And as a clue at least one of R ₂ or R ₃ is different from hydrogen;

here

And independently any optional optionally substituted heterocycle of R ¹ , R ² , R ³ , R ⁴ or R ⁵ , or optionally substituted aryl,

here

Y is C, N, S;

Wherein the process for producing a cytosine nucleoside analog comprises the following steps when a chemo-enzymic preferred embodiment of the process of the present invention is used:

(i) introducing a cytosine base into the cytosine derivative in order to incorporate the appropriate substitutions described as substituents R < ² > and R < ³ > into an intermediate compound of formula (II) optionally refined optionally at the end of step (i) Chemically reacting with an appropriate reagent for modifying the amino group at the ^4- position;

Alternatively, the process of the invention may start directly from the starting product (modified cytosine base) represented by formula II, which is commercially available or previously synthesized,

&Lt; RTI ID = 0.0 &

(ii) reacting the above-described modified cytosine base with a suitable nucleoside analog starting material, wherein the reaction is carried out in a suitable reaction aqueous medium and under suitable reaction conditions, with a cytosine derivative of formula (II) and a nucleoside analog of formula (NPs), preferably a pyrimidine nucleoside phosphorylase enzyme (PyNP), to a mixture of starting materials comprising the nucleoside phosphorylase enzyme,

(III)

here,

Z ₁ is O, CH ₂ , S, NH;

Wherein R < ¹ ^> is hydrogen, OH, an ether or ester thereof,

,

R ^S3 is hydrogen in the case of NA derived from a 2'-deoxyribonucleoside or arabinonucleoside, or OH when it is derived from a ribonucleoside, NH ₂ , preferably halogen (preferably F ), it is selected from OCH _3;

R ^S5 is the case where Z ₂ be different from the oxygen, hydrogen, OH or an ether or ester residue thereof, NH ₂ or halogen, preferably F;

R ¹ is O, CH ₂ , S, NH;

R ⁴ is hydrogen, OH, NH ₂ , SH, halogen (preferably F or I); An optionally substituted alkyl chain; An optionally substituted alkenyl chain; Optionally substituted alkynyl, trihaloalkyl, OR ⁶ , NR ⁶ R ⁷ , CN, COR ⁶ , CONR ⁶ R ⁷ , CO ₂ R ⁶ , C (S) OR ⁶ , OCONR ⁶ R ⁷ , OCO by _{^{2 R 6, OC (S)}} oR 6, NHCONR 6 R 7, NHCO 2 R 6, NHC (S) oR 6, SO 2 NR 6 R 7, any optionally substituted alkyl, alkenyl or alkynyl chain Any optional substituted heteroaryl optionally attached to Y by any optionally substituted alkyl, alkenyl or alkynyl chain, and independently selected from R ² , R ^3, , R < ⁴ > or R < ^{5 &} gt ;, or any optionally substituted aryl;

here

here

Y is C, N, S;

(iii) optionally deprotecting the amino group at the N ⁴ position in the cytosine nucleoside analog to recover the free primary amino N ⁴ in the cytosine nucleoside analog of formula I which is further purified by a conventional purification method .

Preferably, the heterocyclic ring constituting the base in formula (III) of the starting material is selected from: uracil, adenine, cytosine, guanine, thymine, hypoxanthine, xanthine, thiouracil, thioguanine, 9-H-purine 5-methyluronate, 5-methylcytosine, 5-hydroxymethylcytosine, and any of these. Substituted derivatives thereof.

In addition, free cytosine or cytosine derivatives that are modified to obtain the corresponding nucleobases of formula (II) are preferably selected from:

In addition, the cytosine nucleobase in the nucleoside analog of formula I is preferably selected from:

APIs, intermediates, or prodrugs thereof produced by the processes described herein are selected from the group consisting of: capecitabine, decitabine (aza-dCyd or DAC), 5-azacytidine (aza-Cyd), cytarabine ara-C), enocitabine (BH-AC), gemcitabine (dFdC), zalcitabine (ddC), ibatisitabine, sapacitabine, 2'- 2'-deoxy-4'-thiocytidine, tiarabine (T-araC), 2'-deoxy-4'-thiocytidine, -Deoxy-4 ' -thio-5-azacytidine or < / RTI >

More preferably, the APIs, intermediates or prodrugs thereof produced are selected from the group consisting of: capecitabine, decitabine (aza-dCyd or DAC), 5-azacytidine (aza-Cyd), cytarabine -C), and, more preferably, the described process is specifically intended for industrial production of capecitabine or cytarabine.

In a more preferred embodiment of carrying out the process described herein, the API, intermediate or prodrug thereof produced is capecitabine (Scheme 2).

[Reaction Scheme 2]

Synthesis of capecitabine by NPs or PyNPs

The biocatalytic step (enzymatic step) of capecitabine in enzymatic or chemoenzymatic synthesis was preferably carried out at a temperature in the range of 20-120 [deg.] C.

In a more preferred embodiment of carrying out the process described herein, the API, intermediate or prodrug thereof produced is cytarabine (Scheme 3).

[Reaction Scheme 3]

Synthesis of cytarabine by NPs or PyNPs

For the purpose of carrying out the biocatalytic process of the detailed description, the Applicant has thus selected an isolated NPs enzyme or an NPs enzyme, preferably an organism containing a PyNP enzyme or a PyNP / PNP enzyme mixture in both cases These enzymes or their containing microorganisms were mesophilic or mesophilic in the sense that they were able to operate and operate at temperatures ranging from 18 to 60 ° C, the optimal temperature range of 40-55 ° C, and the nucleoside transporter activity. Thermophilic or thermophilic organisms or NPs enzymes, in particular PyNP enzymes, are those which can operate or carry out nucleoside transmucosal activity at temperatures in the range of> 60 ° C and up to 80 ° C. The hyperthermophilic or hypertonic organisms or NPs enzymes, in particular the PyNP enzymes, are capable of operating or carrying out the nucleoside transferase activity at temperatures in excess of 80 ° C and up to 100 ° C, and in the optimum temperature range of 80-95 ° C.

The NPs (PyNP) enzymes used in the process of the invention can be obtained from microorganisms selected from mesophilic organisms, such as bacteria, in particular from E. coli , or from mesophilic, thermophilic or hyperthermophilic archaea, The Thermoprotein class, more particularly the classes selected from the genera and species disclosed in WO2011 / 076894. According to the present invention, the more preferred nucleoside phosphorylase is Sulfolobus < RTI ID = 0.0 > It is derived from solfataricus) and Aeropyrum pernix (Aeropyrum pernix).

Another embodiment of the present invention relates to the use of the recombinant NP enzyme in the disclosed process, comprising an amino acid sequence encoded by a nucleic acid sequence selected from SEQ ID NO: 1 or SEQ ID NO: 2, or a fragment thereof. Both DNA sequence is isolated from the bacteria and (Archaea) having the homologous sequence to that from bacteria (Archaea), more specifically, a nucleoside kinase enzyme.

As set forth in the detailed description, preferred embodiments for carrying out the process of the invention are mesophilic, hyperthermal or hypertonic NPs enzymes, such as Purine NPs (PNPs), or pyrimidine NPs (PyNPs), or mixtures thereof. Preferably, the thermophilic or hyperthermal NPs enzymes are incubated at a temperature in the range of greater than 60 < 0 > C and up to 100 < 0 > C, more preferably in the NP A gene sequence encoding an enzymatic activity, or a fragment thereof.

For the purpose of detailed description, preferred enzymes used in the biocatalytic processes of the invention, methods of cloning them, vectors carrying the nucleic acid sequences encoding them, and host cells transformed with the above-mentioned vectors are all disclosed in WO2011 / 076894 Which includes the names of some of the inventors of the present invention and the entirety of which is incorporated herein by reference in its entirety.

Suitable conditions for carrying out the different embodiments of the inventive process include the following:

a) varying temperatures in the range of 20-120 < 0 > C

b) Reaction times varying from 1-1000 h

c) Concentration of starting material varying from 1-1000 mM

d) stoichiometry The nucleoside starting material: nucleobase varies in the range of 1: 5 to 5: 1.

e) Amount of NP enzyme varying from 0.001-100 mg / ml

f) optionally, a nucleobase added to the reaction medium, dissolved in an organic solvent,

g) optionally an aqueous reaction medium also containing up to 40%, preferably up to 20%, more preferably up to 5% of a suitable organic solvent.

Optionally, organic solvents can be added to the reaction medium or used to pre-dissolve the nucleobases. Preferably, polar aprotic solvents selected from tetrahydrofuran, acetonitrile, acetone, dimethylformamide (DMF) or dimethylsulfoxide (DMSO) are preferred.

The process according to the invention also comprises the step of isolating and / or purifying the NA produced by standard working means selected from precipitation, filtration, concentration or crystallization.

Thermophilic NP enzymes can operate at higher temperatures, which makes the nucleobase transfer reaction more efficient in terms of time and overall yield.

As an embodiment of the present invention, the process of the present invention specifically discloses the production of capecitabine (Reaction Scheme 2), wherein the ribonucleosides used as starting materials are 5'-deoxyuridine, 5'-de 5-deoxyribofuranosyl nucleoside selected from oxy-5-methyluridine or 5'-chloro-5'-deoxyuridine; The nucleobase used as the starting material transferred by the PyNP enzyme is pentyl (5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl) carbamate, and PyNP is a non- It is a naturally occurring mesophilic, hyperthermal or hypertonic enzyme.

One alternative embodiment of the inventive process is where the API produced is capecitabine (Scheme 2) and the ribonucleosides used as starting material are 5'-deoxyuridine, 5'-deoxy-5- Methyluridine or 5'-chloro-5'-deoxyuridine, and the nucleobase also used as the starting material transferred by the PyNP enzyme is pentyl (5-fluoro-2-oxo-1,2-dihydropyridine 4-yl) carbamate, and PyNP is a recombinant mesophilic, hyperthermal or hyperthermophilic enzyme, and the cloned DNA of the enzyme has been isolated from bacteria.

A further embodiment of the process of the invention is characterized in that the API produced is capecitabine (Scheme 2) and the ribonucleosides used as starting materials are 5'-deoxyuridine, 5'-deoxy-5-methyluridine Or 5'-chloro-5'-deoxyuridine, and the nucleobase also used as the starting material which is transferred by the PyNP enzyme is pentyl (5-fluoro-2-oxo-1,2-dihydropyrimidin- -Yl) carbamate, and PyNP is a naturally occurring mesophilic, hyperthermal or hypertonic enzyme isolated from the archaea .

Another embodiment of the inventive process is where the API produced is capecitabine (Scheme 2) and the ribonucleosides used as starting materials are 5'-deoxyuridine, 5'-deoxy-5-methyl (5-fluoro-2-oxo-1,2-dihydropyrimidine-5-carboxamide) is used as the starting material which is transferred by PyNP enzyme, 4-yl) carbamate, and PyNP is a recombinant mesophilic, hyperthermal or hypertonic enzyme isolated from the archaea .

The process of the present invention is also characterized in that the API produced is cytarabine (Scheme 3), the ribonucleoside used as starting material is arabinofuranosyluracil or arabinonucleoside, the starting material transferred by PyNP enzyme (2-oxo-1,2-dihydropyrimidin-4-yl) pentanamide, and PyNP is a naturally occurring mesophilic, thermophilic or ultra-high temperature It is represented by being a sex sterile enzyme.

Another embodiment of the process of the invention is that the produced API is cytarabine (Reaction Scheme 3), the ribonucleoside used as the starting material is arabinofuranosyluracil or arabinonucleoside and is transferred by PyNP enzyme (2-oxo-1,2-dihydropyrimidin-4-yl) pentanamide and PyNP is a recombinant mesophilic, hyperthermal or hypertonic enzyme, The cloned DNA of the enzyme was isolated from bacteria.

A further embodiment of the process of the invention is characterized in that the produced API is cytarabine (Scheme 3), the ribonucleoside used as the starting material is arabinofuranosyluracil or arabinonucleoside, the starting (2-oxo-1,2-dihydropyrimidin-4-yl) pentanamide, and PyNP is a naturally occurring mesophilic, hyperthermal or hypertonic enzyme , The cloned DNA of the enzyme was isolated from the archaea .

Another embodiment of the process of the invention is characterized in that the API produced is cytarabine (Scheme 3), the ribonucleoside used as the starting material is arabinofuranosyluracil or arabinonucleoside, the starting (2-oxo-1,2-dihydropyrimidin-4-yl) pentanamide, and PyNP is a recombinant mesophilic, hyperthermal or hypertonic enzyme, and the enzyme Were isolated from the archaea .

The detailed description also discloses recombinant nucleoside phosphorylase enzymes (PNPs or PyNPs) for use in one of the processes of the invention as described above, forming part of the same inventive concept. Wherein said native or recombinant NP enzyme is from a mesophilic organism, more preferably from bacteria and Archaea ; Or from thermophilic organisms, more preferably from bacteria and archaea ; Or an ultra-high temperature organism, more preferably from bacteria and Archaea , and more particularly from Sulfolobus &lt; RTI ID = 0.0 > It can be isolated from solfataricus) and Aeropyrum pernix (Aeropyrum pernix).

Also on the same line incorporating the single inventive concept, the detailed description also discloses the use of mesophilic, hyperthermal or hypertonic nucleoside phosphorylase (NP or PyNP) in the production of APIs, intermediates or their prodrugs And these APIs, their intermediates or their prodrugs, cytosine nucleoside analogues (NAs) are useful as anticancer or antiviral agents. More preferably, the above-mentioned uses are achieved by the above-described production processes of NAs and variants thereof as APIs, intermediates or prodrugs thereof. In particular, with respect to the abovementioned uses, recombinant thermophilic nucleoside phosphorylases (PNPs, PyNPs or mixtures thereof) are preferred in the production of APIs, and these APIs or their intermediates, nucleoside analogues (NAs) Are useful as anticancer or antiviral agents.

Among the APIs, intermediates or prodrugs thereof produced by this use, the following may be found:

(Aza-dCyd or DAC), 5-azacytidine (aza-Cyd), cytarabine (ara-C), enocitabine (BH-AC), gemcitabine (dFdC), zalcitabine (ddC), ibatisitabine, sapacitabine, 2'-C-cyano-2'-deoxy-1-β-D-arabino- pentofuranosylcytosine (CNDAC), galactanabine, valproic (ATC), 2'-deoxy-4'-thio-5-azacytidine or amphotericin (ATC) .

More preferably, the APIs, intermediates or prodrugs thereof produced by the use of the enzymes disclosed herein are selected from: capecitabine, decitabine (aza-dCyd or DAC), 5-azacytidine -Cyd), cytarabine (ara-C), and, more preferably, the described processes are particularly intended for the industrial production of capecitabine or cytarabine and, respectively, intermediates or prodrugs thereof.

Also provided is a recombinant expression vector comprising a sequence encoding nucleoside phosphorylase enzymatic activity (NPs) operably linked to one or more control sequences driving expression or overexpression of a nucleoside phosphorylase in a suitable host Are included in the present invention. Preferred recombinant expression vectors according to the present invention are those which are present in high temperature and in archaea , preferably Crenarchaeote , and more preferably from the Thermoprotei to NP enzymatic Any transporting and expressing or overexpressing gene encoding the activity, for example: Thermofilum < RTI ID = 0.0 > A1RW90 (A1RW90THEPD) for hypothetical protein from pendens (strain Hrk 5); Sulfolobus Q97Y30 (Q97Y30SULSO) for a hypothetical protein from solfataricus); A3DME1 (A3DME1 STAMF) for hypothetical proteins from Staphylothermus marinus (strain ATCC 43588 / DSM 3639 / Fl); Aeropyrum Q9YA34 (Q9YA34AERPE) for a hypothetical protein from pernix; A2BJ06 (A2BJ06HYPBU) for hypothetical protein from Hyperthermus butylicus (strain DSM 5456 / JCM 9403); And D9PZN7 (D9PZN7ACIS3) for a hypothetical protein from Acidilobus saccharovorans (strain DSM 16705 / VKM B-2471/345).

Encoded NP enzyme Access number Uniprot
Genbank (NCBI) entry designation Protein name organism Gene name SEQ ID NO. (DNA / protein) A1RW90 YP_919473.1. A1RW90_THEPD Purine-nucleoside phosphorylase Thermofilm pendens (strain Hrk 5) Tpen_0060 5/6 Q97Y30
NP_342951.1 Q97Y30_SULSO Purine nucleoside phosphorylase (DeoD) Sulfolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) deoD SSO1519 1/3 A3DME1
YP _001040709.1. A3DME1_STAMF Purine-nucleoside phosphorylase Staphylothermus marinus (strain ATCC 43588 / DSM 3639 / F1) Smar_0697 7/8 Q9YA34
NP_148386.2 Q9YA34_AERPE Our Dean Phosphorylase Aeropyrum pernix (strain ATCC 700893 / DSM 11879 / JCM 9820 / NBRC 100138 / K1) udp APE_2105.1 2/4 A2BJ06
FC _001012312.1 A2BJ06_HYPBU Our Dean Phosphorylase Hyperthermus butylicus (strain DSM 5456 / JCM 9403) Hbut_0092 9/10 D9PZN7
FC _003815556.1 D9PZN7_ACIS3 Our Dean Phosphorylase Acidilobus saccharovorans (strain DSM 16705 / VKM B-2471 / 345-15) ASAC_0117 11/12

Preferred recombinant expression vectors according to the present invention comprise a nucleic acid sequence selected from SEQ ID NO: 1, 2, 5, 7, 9 or 11; or

a) a complementary nucleotide sequence of SEQ ID NO: 1, 2, 5, 7, 9 or 11; or

b) a nucleotide sequence which is flanked by SEQ ID NO: 1, 2, 5, 7, 9 or 11; or

f) carries and expresses or overexpresses a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NO: 3, 4, 6, 8, 10 or 12.

The present invention also covers the use of the recombinant expression vectors described above for the production of recombinant NPs or for the production of active pharmaceutical ingredients (APIs), intermediates or prodrugs thereof, and these APIs or their intermediates, nucleoside analogs (NAs) are particularly useful as anticancer or antiviral agents. In particular, the APIs, intermediates, or prodrugs thereof produced by the above-mentioned uses are selected from the following: capecitabine, decitabine (aza-dCyd or DAC), 5-azacytidine (aza-Cyd), cytarabine (B-AC), gemcitabine (dFdC), zalcitabine (ddC), ibatisitabine, sapacitabine, 2'-C-cyano-2'-deoxy- 2-deoxy-4'-thiocidin, tiarabine (T-araC), 2'-deoxy-4'-thiocytidine &Apos; -deoxy-4 ' -thio-5-azacytidine and aflativarine (ATC).

More preferably, the APIs, intermediates or prodrugs thereof produced are selected from the group consisting of: capecitabine, decitabine (aza-dCyd or DAC), 5-azacytidine (aza-Cyd), cytarabine -C); More preferably, the expression vectors described herein are particularly suitable for the industrial production of capecitabine or cytarabine, intermediates or prodrugs thereof.

More preferably, the above-mentioned uses for the production of APIs, intermediates or prodrugs thereof are achieved by the production processes described above and variants thereof.

The present invention also covers a host cell comprising the recombinant expression vector as described above, wherein said host cell is an Escherichia coli lt; / RTI > The use of a host cell comprising a recombinant expression vector as described above for the production of recombinant NPs (or PyNP), included as part of the same invention single-linked concept, is also contemplated. Similarly, the use of a host cell comprising a recombinant expression vector as described above for the production of active pharmaceutical ingredients (APIs), intermediates or prodrugs thereof is also part of the present invention, and these APIs or their intermediates , Nucleoside analogs (NAs) are useful as anticancer or antiviral agents. These host cells, particularly those containing recombinant expression vectors as described above, are used to produce APIs, intermediates or prodrugs thereof selected from: capecitabine, decitabine (aza-dCyd or DAC), 5- (Aza-Cyd), cytarabine (ara-C), enocitabine (BH-AC), gemcitabine (dFdC), zalcitabine (ddC), ibacitavin, (CNDAC), galactaparin, valproicitabine (NM283), 2'-deoxy-4'-thio-cysteine Dean, tiarabine (T-araC), 2'-deoxy-4'-thio-5-azacytidine and aflativarine (ATC).

More preferably, the APIs, intermediates, or prodrugs thereof produced using the host cells of the invention that have been transformed with the recombinant expression vectors described above are selected from: capecitabine, decitabine (aza-dCyd or DAC), 5-azacytidine (aza-Cyd), cytarabine (ara-C); More preferably, the host transformed cells described herein are particularly suitable for the industrial production of capecitabine or cytarabine, intermediates or prodrugs thereof.

More preferably, the above-mentioned uses are achieved by the production processes previously described above of NAs as APIs, intermediates or their prodrugs and variants thereof.

compare Example 1: unmodified 5- Fluorocytosine And From deoxyuridine Synthesis of starting 5-fluoro-5'-deoxycytidine

A solution of 2.5 mM 5-fluorocytosine and 8.0 mM 5'-deoxyuridine in 30 mM aqueous phosphate buffer and 10% DMSO at pH 7 was heated at 60 DEG C for 30 minutes. Then, PyNP (5.4 U / μmole _base ) was added and the reaction was stirred at 60 ° C for 4 hours under the same conditions. The raw reactant was then filtered through a 10 KDa membrane, and a portion was diluted and analyzed by HPLC. The expected product 5-fluoro-5'-deoxycytidine was not detected by UV-DAD (ultraviolet diode array detection).

compare Example 2: unmodified 5- Fluorocytosine And Chloro -5'- From deoxyuridine Synthesis of starting 5-fluoro-5'-deoxycytidine

A solution of 2.5 mM 5-fluorocytosine and 8.6 mM 5-chloro-5'-deoxyuridine in 30 mM aqueous phosphate buffer and 10% DMSO at pH 7 was heated at 60 ° C for 30 minutes. Then, PyNP (5.4 U / μmole _base ) was added and the reaction was stirred at 60 ° C for 4 hours under the same conditions. The raw reactant was then filtered through a 10 KDa membrane, and a portion was diluted and analyzed by HPLC. The expected product 5-fluoro-5'-deoxycytidine was not detected by UV-DAD.

compare Example 3: unmodified cytosine and From arabinofuranosyl uracil Synthetic synthesis of sitarabine

A solution of 3.0 mM cytosine and 10 mM 9- (bD-arabinofuranosyl) uracil in 30 mM aqueous phosphate buffer and 10% DMSO at pH 7 was heated at 60 ° C for 30 minutes. Thereafter, PyNP (4.4 U / μmole _base ) was added, and the reaction was stirred at 60 ° C. for 4 hours under the same conditions. The raw reactant was then filtered through a 10 KDa membrane, and a portion was diluted and analyzed by HPLC. The expected product 1- (bD-arabinofuranosyl) cytosine (cytarabine) was not detected by UV-DAD, but uracil was obtained from cytosine degradation through deamidation.

compare Example 4: unmodified cytosine and From arabinofuranosyl cytosine Synthetic synthesis of sitarabine

A solution of 3.0 mM cytosine and 7.6 mM 9- (bD-arabinofuranosyl) adenine in 30 mM aqueous phosphate buffer and 10% DMSO at pH 7 was heated at 60 DEG C for 30 minutes. Thereafter, PyNP (4.4 U / μmole _base ) was added, and the reaction was stirred at 60 ° C. for 4 hours under the same conditions. The raw reactant was then filtered through a 10 KDa membrane, and a portion was diluted and analyzed by HPLC. The anticipated product 1- (bD-arabinofuranosyl) cytosine (cytarabine) was not detected by UV-DAD but was not detected from adenine deamination by hypoxanthine and substrate deamidation from 9- (bD-arabinofuranosyl ) Hypoxanthin was obtained.

compare Example 5: unmodified cytosine and From arabinofuranosyl hypoxanthine Synthetic synthesis of sitarabine

A solution of 3.0 mM cytosine and 7.5 mM 9- (bD-arabinofuranosyl) hypoxanthin in 30 mM aqueous phosphate buffer and 10% DMSO at pH 7 was heated at 60 DEG C for 30 minutes. Thereafter, PyNP (4.4 U / μmole _base ) was added, and the reaction was stirred at 60 ° C. for 4 hours under the same conditions. The raw reactant was then filtered through a 10 KDa membrane, and a portion was diluted and analyzed by HPLC. The expected product 1- (bD-arabinofuranosyl) cytosine (cytarabine) was not detected by UV-DAD.

compare Example 6: unmodified cytosine and From arabinofuranosylguanine Synthetic synthesis of sitarabine

A solution of 3.0 mM cytosine and 8.6 mM 9- (bD-arabinofuranosyl) guanine in 30 mM aqueous phosphate buffer and 10% DMSO at pH 7 was heated at 60 ° C for 30 minutes. Thereafter, PyNP (4.4 U / μmole _base ) was added, and the reaction was stirred at 60 ° C. for 4 hours under the same conditions. The raw reactant was then filtered through a 10 KDa membrane, and a portion was diluted and analyzed by HPLC. The expected product 1- (bD-arabinofuranosyl) cytosine (cytarabine) was not detected.

Example 7: Pentyl (5-Fluoro-2-oxo-1,2- Dihydropyrimidine Yl) Carbamate synthesis

Under an inert atmosphere pentyl chloroformate (5 g, 1.0 eq.) Was added to a stirred solution of 5-fluorocytosine (1.0 eq.) In anhydrous pyridine and the reaction was heated to 60 < 0 > C. After 2 hours, no further conversion was observed, so the reaction was quenched by cooling at room temperature. The crude reactant was then extracted with AcOEt and HCl 1N, and the organic phase was dried over Na ₂ SO ₄ and the solvent evaporated. The obtained white solid is purified chromatographically using an SiO ₂ and AcOEt as mobile phase, and calculated the desired product (88%).

Example 8: N - (2-oxo-1,2-dihydropyrimidin-4-yl) acetamide

Under an inert atmosphere, triethylamine (18.1 μL, 0.13 mmol) and acetyl chloride (9.2 μL, 0.13 mmol) were added to a stirred solution of cytosine (10.0 mg, 0.09 mmol) in anhydrous DMF at room temperature for 25 h. The solvent was then evaporated and the resulting yellowish solid was washed with water, filtered and dried in vacuo to give the expected product in 55% yield.

Example 9: Synthesis of pentyl (2-oxo-1,2-dihydropyrimidin-4-yl) carbamate

Under an inert atmosphere, pentyl chloroformate (0.8 mmol, 1.0 eq.) Was added dropwise to a stirred solution of cytosine (0.8 mmol, 1.0 eq.) In anhydrous pyridine and the reaction was heated to 60 < 0 > C. After 2 hours, no further conversion was observed, so the reaction was quenched by cooling at room temperature. The crude reactant was then extracted with AcOEt and HCl 1N, and the organic phase was dried over Na ₂ SO ₄ and the solvent evaporated. The obtained white solid is purified chromatographically using an SiO ₂ and AcOEt as mobile phase, and calculated the desired product.

Example 10: From our Dean Starting, N ⁴ - Pentyloxycarbonyl - 5'- Deoxy -5- Fluorocytidine (Capecitabine) Synthesis of

(5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl) carbamate and 8.0 mM 5'-deoxycholate in 30 mM aqueous phosphate buffer and 10% DMSO at pH 7 0.0 > 60 C < / RTI > for 30 minutes. Thereafter, PyNP (5.0 U / μmole _base ) was added and the reaction was stirred at 60 ° C. for 4 hours under the same conditions. The raw reactant was then filtered through a 10 KDa membrane, and a portion was diluted and analyzed by HPLC. The expected product N ⁴ -pentyloxycarbonyl-5'-deoxy-5-fluorocytidine (capecitabine) was detected in a yield of 68% compared to Reference Example 1, where the final product was formed Or not detected.

Example 11: From deoxyuridine Starting, N ⁴ - Pentyloxycarbonyl - 5'- Deoxy Synthesis of 5-fluorocytidine (capecitabine).

2.5 mM pentyl (5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl) carbamate and 8.6 mM 5'-deoxycholate in 30 mM aqueous phosphate buffer and 10% DMSO at pH 7 0.0 > 60 C < / RTI > for 30 minutes. Then, PyNP (5.4 U / μmole _base ) was added and the reaction was stirred at 60 ° C for 4 hours under the same conditions. The raw reactant was then filtered through a 10 KDa membrane, and a portion was diluted and analyzed by HPLC. The expected product N ⁴ -pentyloxycarbonyl-5'-deoxy-5-fluorocytidine (capecitabine) was detected in a yield of 77% compared to Reference Example 2, where the final product was formed Or not detected.

Example 12: From arabinofuranosyl uracil Starting from 9- (b-D- Arabinofuranosyl ) Synthesis of uracil (cytarabine)

2.5 mM pentyl (2-oxo-1,2-dihydropyrimidin-4-yl) carbamate and 8.6 mM 9- (bD-arabinofuranosyl) carbamate in 30 mM aqueous phosphate buffer and 10% The solution of uracil was heated at 60 占 폚 for 30 minutes. Then, PyNP (5.4 U / μmole _base ) was added and the reaction was stirred at 60 ° C for 4 hours under the same conditions. The raw reactant was then filtered through a 10 KDa membrane, and a portion was diluted and analyzed by HPLC. The expected product N ⁴ -pentyloxycarbonyl cytidine was detected.

The N ⁴ -position can optionally be deprotected to provide 9- (bD-arabinofuranosyl) uracil (cytarabine).

Example 13: From arabinofuranosyladenine Starting from 9- (b-D- Arabinofuranosyl ) Synthesis of uracil (cytarabine)

2.5 mM pentyl (2-oxo-1,2-dihydropyrimidin-4-yl) carbamate and 8.6 mM 9- (bD-arabinofuranosyl) carbamate in 30 mM aqueous phosphate buffer and 10% The solution of adenine was heated at 60 DEG C for 30 minutes. Then, PyNP (5.4 U / μmole _base ) was added and the reaction was stirred at 60 ° C for 4 hours under the same conditions. The raw reactant was then filtered through a 10 KDa membrane, and a portion was diluted and analyzed by HPLC. The expected product N ⁴ -pentyloxycarbonyl cytidine was detected.

SEQUENCE LISTING <110> Plasmia Biotech <120> Enzymatic production of cytosinic nucleoside analogues <130> <160> 12 <170> BiSSAP 1.2 <210> 1 <211> 825 <212> DNA <213> Sulfolobus solfataricus <220> <221> source <222> 1..825 <223> / mol_type = "unassigned DNA" / note = "P2 Purine nucleoside phosporylase (deoD)" / organism = "Sulfolobus solfataricus" <400> 1 gtgccatttt tagaaaatgg ttccatggta tatggtgatt tcattagaaa tcaagaggta 60 agaaaaagaa ttacaaagga agaacttggg atagaagaag acgaaatccc ggaaagggta 120 gttgtaacac ctatgccatt taatactcaa tttcctaaaa actttgaaga tactttaact 180 aacttaggaa ttaaagtaaa taggttaaaa gtggaagacc aaatacttag acaattcgga 240 ggaaatttat tgcttgaaaa agacggtaat agaggattta ttgcgttcat aggcagaggt 300 ctgatagatt tcactgagag gataaggatt ttagctacag tttcgcgcat taaagatata 360 ttatttattg gtactgcagg atcgttatct aatgaaatat taataggaga tctaaatata 420 ccaaaatacg ccatcccatt cgaaaacgta agtgattttt acgctgatcc taccatagca 480 attccacaag ctgatgaaaa gttgctgaac gaagtttatg agtacgctga ggaaactgga 540 gttaaaaccc actcaacctt acatgcaaca ctacttttcc cttattccga aactactgag 600 ttcctaaact acttattaaa tatcggcgtt tctacgatag atatggaagt cagtgctttt 660 tataagatgt ctagatttta cggtaaaaga gctgttgcag tattacgaat ttcagatatg 720 cctttaatag aactgcataa gcaagaggaa ttgattaagg caagaaggga aattgcagtt 780 aatgctgttt tcagaattac cttaagattc ttaaaactga tttaa 825 <210> 2 <211> 849 <212> DNA <213> Aeropyrum pernix K1 <220> <221> source <222> 1..849 <223> / mol_type = "unassigned DNA" / note = "udp uridine phosphorylase" / organism = "Aeropyrum pernix K1" <400> 2 ttgggagacg agagtctaag gagcgccgcc cgtcccgagg gggagggagg gctgcagtac 60 catctgaggg tcaggagggg ggatgtggcc cgctacgttc tcctcccggg agaccccgag 120 aggacagacc ttatagcccg cctctgggat gaagcgaggc ttgtagcgca ccaccgggag 180 tacaggacgt ggaccggctt ctacaagggg acatcgataa gtgtaacaag caccgggata 240 ggctctccca gcacggcgat agccgttgag gagctgctga gggttggagc cgagactttc 300 ataagagtag gcactatggg cggtataagg gaggatctgc ggcccggcac cctggttata 360 gggagtgcgg cggttaggat ggaggggacg agcggccagt acgctccccg ggggttccca 420 gcggccgcca gctatgacgt tgtggcggcg ctggtggagg ctgctgaggc gctcggggtt 480 aggtatgagg ttggcgttgt tgccagcacg gacagcttct acctgggcca ggggaggccg 540 gggtacgggg ggtatatgac gccggaggct tcggaagtca tacccctcct caggtcagcc 600 ggcgtcctcg gcttcgagat ggaggcctcc gccctcttca ccctatccca gctctacggc 660 gccagggcag ggtgcgtgtg cgcggtagtg gcaaacaggg ttagcgggga gtttgtggta 720 aacgcggggg ttgaagacgc tgctagggtt gcctccgagg cggtagccat actagcaggc 780 tgggacaggg agagggagaa gaggggtaag aaatggtttt acccgagcct ggcgtgcaga 840 cgcacatag 849 <210> 3 <211> 274 <212> PRT <213> Sulfolobus solfataricus P2 <220> Purine nucleoside phosporylase (DeoD) <400> 3 Met Pro Phe Leu Glu Asn Gly Ser Met Val Tyr Gly Asp Phe Ile Arg 1 5 10 15 Asn Gln Glu Val Arg Lys Arg Ile Thr Lys Glu Glu Leu Gly Ile Glu 20 25 30 Glu Asp Glu Ile Pro Glu Arg Val Val Val Thr Pro Met Pro Phe Asn 35 40 45 Thr Gln Phe Pro Lys Asn Phe Glu Asp Thr Leu Thr Asn Leu Gly Ile 50 55 60 Lys Val Asn Arg Leu Lys Val Glu Asp Gln Ile Leu Arg Gln Phe Gly 65 70 75 80 Gly Asn Leu Leu Leu Glu Lys Asp Gly Asn Arg Gly Phe Ile Ala Phe 85 90 95 Ile Gly Arg Gle Leu Ile Asp Phe Thr Glu Arg Ile Arg Ile Leu Ala 100 105 110 Thr Val Ser Arg Ile Lys Asp Ile Leu Phe Ile Gly Thr Ala Gly Ser 115 120 125 Leu Ser Asn Glu Ile Leu Ile Gly Asp Leu Asn Ile Pro Lys Tyr Ala 130 135 140 Ile Pro Phe Glu Asn Val Ser Asp Phe Tyr Ala Asp Pro Thr Ile Ala 145 150 155 160 Ile Pro Gln Ala Asp Glu Lys Leu Leu Asn Glu Val Tyr Glu Tyr Ala 165 170 175 Glu Glu Thr Gly Val Lys Thr His Ser Thr Leu His Ala Thr Leu Leu 180 185 190 Phe Pro Tyr Ser Glu Thr Thr Glu Phe Leu Asn Tyr Leu Leu Asn Ile 195 200 205 Gly Val Ser Thr Ile Asp Met Glu Val Ser Ala Phe Tyr Lys Met Ser 210 215 220 Arg Phe Tyr Gly Lys Arg Ala Val Ala Val Leu Arg Ile Ser Asp Met 225 230 235 240 Pro Leu Ile Glu Leu His Lys Gln Glu Glu Leu Ile Lys Ala Arg Arg 245 250 255 Glu Ile Ala Val Asn Ala Val Phe Arg Ile Thr Leu Arg Phe Leu Lys 260 265 270 Leu Ile <210> 4 <211> 282 <212> PRT <213> Aeropyrum pernix K1 <220> <223> Uridine phosphorylase <400> 4 Met Gly Asp Glu Ser Leu Arg Ser Ala Ala Arg Pro Glu Gly Glu Gly 1 5 10 15 Gly Leu Gln Tyr His Leu Arg Val Arg Arg Gly Asp Val Ala Arg Tyr 20 25 30 Val Leu Leu Pro Gly Asp Pro Glu Arg Thr Asp Leu Ile Ala Arg Leu 35 40 45 Trp Asp Glu Ala Arg Leu Val Ala His His Arg Glu Tyr Arg Thr Trp 50 55 60 Thr Gly Phe Tyr Lys Gly Thr Ser Ile Ser Val Thr Ser Thr Gly Ile 65 70 75 80 Gly Ser Pro Ser Thr Ala Ile Ala Val Glu Glu Leu Leu Arg Val Gly 85 90 95 Ala Glu Thr Phe Ile Arg Val Gly Thr Met Gly Gly Ile Arg Glu Asp 100 105 110 Leu Arg Pro Gly Thr Leu Val Ile Gly Ser Ala Val Val Arg Met Glu 115 120 125 Gly Thr Ser Gly Gln Tyr Ala Pro Arg Gly Phe Pro Ala Ala Ala Ser 130 135 140 Tyr Asp Val Ala Leu Val Glu Ala Gla Ala Leu Gly Val 145 150 155 160 Arg Tyr Glu Val Gly Val Val Ala Ser Thr Asp Ser Phe Tyr Leu Gly 165 170 175 Gln Gly Arg Pro Gly Tyr Gly Gly Tyr Met Thr Pro Glu Ala Ser Glu 180 185 190 Val Ile Pro Leu Leu Arg Ser Ala Gly Val Leu Gly Phe Glu Met Glu 195 200 205 Ala Ser Ala Leu Phe Thr Leu Ser Gln Leu Tyr Gly Ala Arg Ala Gly 210 215 220 Cys Val Cys Ala Val Val Ala Asn Arg Val Ser Gly Glu Phe Val Val 225 230 235 240 Asn Ala Gly Val Glu Asp Ala Ala Arg Val Ala Ser Glu Ala Val Ala 245 250 255 Ile Leu Ala Gly Trp Asp Arg Glu Arg Glu Lys Arg Gly Lys Lys Trp 260 265 270 Phe Tyr Pro Ser Leu Ala Cys Arg Arg Thr 275 280 <210> 5 <211> 720 <212> DNA <213> Thermofilm pendens Hrk 5 <220> <221> source <222> 1..720 <223> / mol_type = "unassigned DNA" / note = "purine-nucleoside phosphorylase" / organism = "Thermophilum pendens Hrk 5" <400> 5 gtggctaagc cgttacacat actcgcaaag ccggaggaca tagcccccag ggttatcgcc 60 tcgggggacc ccgccagagt gaagcaactc tcaagctacc tcgataaccc caggctggtg 120 aacgagaaca ggggcttcct ggtatacacc ggcacgtaca agggggtacc cgtgactgtt 180 gctacccaca tgataggtgc tccctccgcc gcgatagtct tcgaggagct cataatgctg 240 ggtgcgaagc tgatagtcag gttcggcacc tgcggcggct tcctgccgga gatgcgcgta 300 ggggacttcg tgatagcgac gggcgcctcg tacagcggtg ggggaacaat gaatacctac 360 agccccggcg agtgcatggc cgccgtgccg gactacgacg tgctcagcgc actcgtcgag 420 agcgcttcga ggcacgggtt gaagtacttc ctggggcccg tggttagcag cgataacttc 480 tactcaggta tagagtacct gaacaggtgg ataaacaggg gcatgatagc cgtcgacatg 540 gaggctgcca cgctgttcgt cgtcggcagg cttaggcgcg tgaagaccgg tgcctccttc 600 gtcgtgagcg acgtgatcgg tgaggcttac aagaagatgg cgacggccga ggagctacgc 660 gaggctgtcg acaaggcttc gagagccgtg ctagacgcgg ttataagcgt gaaagtttga 720 <210> 6 <211> 239 <212> PRT <213> Thermofilm pendens Hrk 5 <220> <223> Purine-nucleoside phosphorylase <400> 6 Met Ala Lys Pro Leu His Ile Leu Ala Lys Pro Glu Asp Ile Ala Pro 1 5 10 15 Arg Val Ile Ala Ser Gly Asp Pro Ala Arg Val Lys Gln Leu Ser Ser 20 25 30 Tyr Leu Asp Asn Pro Arg Leu Val Asn Glu Asn Arg Gly Phe Leu Val 35 40 45 Tyr Thr Gly Thr Tyr Lys Gly Val Val Thr Val Ala Thr His Met 50 55 60 Ile Gly Ala Pro Ser Ala Ile Val Phe Glu Glu Leu Ile Met Leu 65 70 75 80 Gly Ala Lys Leu Ile Val Arg Phe Gly Thr Cys Gly Gly Phe Leu Pro 85 90 95 Glu Met Arg Val Gly Asp Phe Val Ile Ala Thr Gly Ala Ser Tyr Ser 100 105 110 Gly Gly Gly Thr Met Asn Thr Tyr Ser Pro Gly Glu Cys Met Ala Ala 115 120 125 Val Pro Asp Tyr Asp Val Leu Ser Ala Leu Val Glu Ser Ala Ser Arg 130 135 140 His Gly Leu Lys Tyr Phe Leu Gly Pro Val Val Ser Ser Asp Asn Phe 145 150 155 160 Tyr Ser Gly Ile Glu Tyr Leu Asn Arg Trp Ile Asn Arg Gly Met Ile 165 170 175 Ala Val Asp Met Glu Ala Ala Thr Leu Phe Val Val Gly Arg Leu Arg 180 185 190 Arg Val Lys Thr Gly Ala Ser Phe Val Val Ser Asp Val Ile Gly Glu 195 200 205 Ala Tyr Lys Lys Met Ala Thr Ala Glu Glu Leu Arg Glu Ala Val Asp 210 215 220 Lys Ala Ser Arg Ala Val Leu Asp Ala Val Ile Ser Val Lys Val 225 230 235 <210> 7 <211> 717 <212> DNA <213> Staphylothermus marinus F1 <220> <221> source <222> 1..717 <223> / mol_type = "unassigned DNA" / note = "Purine-nucleoside phosphorylase" / organism = "Staphylothermus marinus F1" <400> 7 atgaagcctg ctcttctaaa agatgttgca ggggttagtg atctagttat tgtaatgggt 60 gatcctgata gagtatactt attatcaaca ttgctggaaa accccaagat catatatgat 120 aggagaggaa tagttgttgt taatggagaa tataagggta gaaaaataac acttgccagc 180 cacggtattg gttgtccaat ggcatcaatt atattggagg aactgggtat gcttggtgct 240 aaaacaatta ttagaatagg cacagctggc tcacttgttg aaaacattgg tttaggcgat 300 attgtgttag tagcgggggc tgggtacatg cttaatgggt gtggaaacaa tatgtattct 360 cccgaaataa atggaggaac aagcccagat cctctactgc tcagcgaaat ctatcattat 420 ttatcacatt ataatattaa gccacatatt ggactagtat ttacaagcga tgcattctat 480 gcggaagaaa atataattga taaactaacc aataaaggat tcatcgctgt tgatatggag 540 acagcaattc tatacatgct tggatggatg aggaagtgga ggacactatc aatactggta 600 gtaagcaaca gcctcgtgaa gaaaacaccg ttgcttacaa catatgagct cgcagagaag 660 tttgttgaac tagcaaaact agtattagaa tatcttgcaa gaaacactca gcactaa 717 <210> 8 <211> 238 <212> PRT <213> Staphylothermus marinus F1 <220> <223> Purine-nucleoside phosphorylase <400> 8 Met Lys Pro Ala Leu Leu Lys Asp Val Ala Gly Val Ser Asp Leu Val 1 5 10 15 Ile Val Met Gly Asp Pro Asp Arg Val Tyr Leu Leu Ser Thr Leu Leu 20 25 30 Glu Asn Pro Lys Ile Ile Tyr Asp Arg Arg Gly Ile Val Val Val Asn 35 40 45 Gly Glu Tyr Lys Gly Arg Lys Ile Thr Leu Ala Ser His Gly Ile Gly 50 55 60 Cys Pro Met Ala Ser Ile Leu Glu Glu Leu Gly Met Leu Gly Ala 65 70 75 80 Lys Thr Ile Ile Arg Ile Gly Thr Ala Gly Ser Leu Val Glu Asn Ile 85 90 95 Gly Leu Gly Asp Ile Val Leu Val Ala Gly Aly Gly Tyr Met Leu Asn 100 105 110 Gly Cys Gly Asn Asn Met Tyr Ser Pro Glu Ile Asn Gly Gly Thr Ser 115 120 125 Pro Asp Pro Leu Leu Leu Ser Glu Ile Tyr His Tyr Leu Ser His Tyr 130 135 140 Asn Ile Lys Pro His Ile Gly Leu Val Phe Thr Ser Asp Ala Phe Tyr 145 150 155 160 Ala Glu Glu Asn Ile Ile Asp Lys Leu Thr Asn Lys Gly Phe Ile Ala 165 170 175 Val Asp Met Glu Thr Ala Ile Leu Tyr Met Leu Gly Trp Met Arg Lys 180 185 190 Trp Arg Thr Leu Ser Ile Leu Val Val Ser Asn Ser Leu Val Lys Lys 195 200 205 Thr Pro Leu Leu Thr Thr Tyr Glu Leu Ala Glu Lys Phe Val Glu Leu 210 215 220 Ala Lys Leu Val Leu Glu Tyr Leu Ala Arg Asn Thr Gln His 225 230 235 <210> 9 <211> 843 <212> DNA <213> Hyperthermus butylicus DSM 5456 <220> <221> source <222> 1..843 <223> / mol_type = "unassigned DNA" / note = "Uridine phosphorylase" / organism = "Hyperthermus butylicus DSM 5456" <400> 9 gtggagaggc cctcggcgaa agcgccaacc gttgagggta aaatgtacca tatcatgctc 60 ggcccaggcg aaattccgcc ctacgtcctc ctgccaggcg atccaggtag gatagacgat 120 atagtcgcga cgtgggatga atggcgggag ctagcatttc accgcgagta tcgtagtgtc 180 aagggcaggt ataagggtgt ggaaataggc gctgtcagca ctggcatagg cggaccgtca 240 actgcaattg ccgtcgagga gctggctagg ataggggtac atacgttcat ccgcgtgggc 300 accactggcg ctatacaacc ggatatagaa cttggaacag tcatcatagg ctatgcagct 360 gtaagatatg atggtgctag tggtgagtat gctccgccag agtaccccgc agctgcgaca 420 cccgaggtgg ttctagctct tgttgaggct gctgagaggc tcggggttacc ataccgtgtc 480 ggcgtggtgg cctcaacagc aacattccac ttggggcaga gccgcccagg gttccgtggg 540 tacgagtgga gccgtagcag ggagaggcta gcagacctac agcgcatggg tgtcctcagc 600 ttcgagatgg aggcggcgac gatattcact ctcgcgagtc tctatgggct gcgggcaggc 660 tgtgtctgcg ccgcaatagc caaccgcgtg accgacgagt ttaaaccggg ggtcggggtt 720 agggaggcga tactagtggc taatgaggct gtgaggatac tagccgaggc cgacagggaa 780 aagggcagga agccagctag tataacaacg ctctacaacg ccgtcagaaa gctgtacggc 840 tag 843 <210> 10 <211> 280 <212> PRT <213> Hyperthermus butylicus DSM 5456 <220> <223> Uridine phosphorylase <400> 10 Met Glu Arg Pro Ser Ala Lys Ala Pro Thr Val Glu Gly Lys Met Tyr 1 5 10 15 His Ile Met Leu Gly Pro Gly Glu Ile Pro Pro Tyr Val Leu Leu Pro 20 25 30 Gly Asp Pro Gly Arg Ile Asp Asp Ile Val Ala Thr Trp Asp Glu Trp 35 40 45 Arg Glu Leu Ala Phe His Arg Glu Tyr Arg Ser Val Lys Gly Arg Tyr 50 55 60 Lys Gly Val Glu Ile Gly Ala Val Ser Thr Gly Ile Gly Gly Pro Ser 65 70 75 80 Thr Ala Ile Ala Val Glu Glu Leu Ala Arg Ile Gly Val His Thr Phe 85 90 95 Ile Arg Val Gly Thr Thr Gly Ala Ile Gln Pro Asp Ile Glu Leu Gly 100 105 110 Thr Val Ile Ile Gly Tyr Ala Val Val Tyr Asp Gly Ala Ser Gly 115 120 125 Glu Tyr Ala Pro Pro Glu Tyr Pro Ala Ala Ala Thr Pro Glu Val Val 130 135 140 Leu Ala Leu Val Glu Ala Ala Glu Arg Leu Gly Val Tyr Arg Val 145 150 155 160 Gly Val Val Ala Ser Thr Ala Thr Phe His Leu Gly Gln Ser Arg Pro 165 170 175 Gly Phe Arg Gly Tyr Glu Trp Ser Arg Ser Ser Glu Arg Leu Ala Asp 180 185 190 Leu Gln Arg Met Gly Val Leu Ser Phe Glu Met Glu Ala Ala Thr Ile 195 200 205 Phe Thr Leu Ala Ser Leu Tyr Gly Leu Arg Ala Gly Cys Val Cys Ala 210 215 220 Ala Ile Ala Asn Arg Val Thr Asp Glu Phe Lys Pro Gly Val Gly Val 225 230 235 240 Arg Glu Ala Leu Val Ala Asn Glu Ala Val Arg Ile Leu Ala Glu 245 250 255 Ala Asp Arg Glu Lys Gly Arg Lys Pro Ala Ser Ile Thr Thr Leu Tyr 260 265 270 Asn Ala Val Arg Lys Leu Tyr Gly 275 280 <210> 11 <211> 846 <212> DNA <213> Acidilobus saccharovorans 345-15 <220> <221> source <222> 1..846 <223> / mol_type = "unassigned DNA" / note = "Uridine phosphorylase" / organism = "Acidilobus saccharovorans 345-15" <400> 11 gtggaggagc gcatgtcttc agcttcaagg cccttcgact cggagggcag ggcctaccac 60 ctgggcctca ggaagggcga cgtgccaaag tacgtgctta tgccaggcga ggttgagagg 120 gcctcaagga tagcctcctc ctgggacagg agctggaggc tggcccagag gagggagtac 180 tcaagcttca ggggcgttta caggggcgtt gacgtggcgg tggtctccac gggcataggg 240 ggacctgcca ccgccatagc agttgaggag ctgctggagc tcggcgctga cacgctgata 300 agggttggaa gcaccggcgc catacaggat gacatagaag ttggggatat aataataacc 360 accgccgcgg tcaggatgga cggcacgagc taccagtacg ccccggccgg ctacccggcc 420 tcggccagct acgaggtaat catggccctg gtcgaggccg ccgagagcct cggggtgagg 480 taccaccttg gcatcacggc ctctactgac agcttctacg tgggccaggg gaggccgggc 540 tacgggggtt acatgccgag ctggtcaagg aacctcgtgc ctgacctgag gcagatgagg 600 gtgctgaact ttgagatgga gagcgccacc ctccttacct tagcaaatat atacggcttc 660 agggcggggg cagtgcacgc cgtctacgcc cagagggtta aggatgagtt cgttgcccac 720 gctggcgagg agaacctgat aaaggtggct gacgaggccg tgaggatact tcacgagtgg 780 gacgaggtta aggggagggc cgggaagagg tacttctacc cctcgctcct gaggcctggc 840 ccctga 846 <210> 12 <211> 281 <212> PRT <213> Acidilobus saccharovorans 345-15 <220> <223> Uridine phosphorylase <400> 12 Met Glu Glu Arg Met Ser Ser Ala Ser Arg Pro Phe Asp Ser Glu Gly 1 5 10 15 Arg Ala Tyr His Leu Gly Leu Arg Lys Gly Asp Val Pro Lys Tyr Val 20 25 30 Leu Met Pro Gly Glu Val Glu Arg Ala Ser Arg Ile Ala Ser Ser Trp 35 40 45 Asp Arg Ser Trp Arg Leu Ala Gln Arg Arg Glu Tyr Ser Ser Phe Arg 50 55 60 Gly Val Tyr Arg Gly Val Asp Val Ala Val Val Ser Thr Gly Ile Gly 65 70 75 80 Gly Pro Ala Thr Ala Ile Ala Val Glu Glu Leu Leu Glu Leu Gly Ala 85 90 95 Asp Thr Leu Ile Arg Val Gly Ser Thr Gly Ala Ile Gln Asp Asp Ile 100 105 110 Glu Val Gly Asp Ile Ile Ile Thr Thr Ala Val Val Arg Asp Gly 115 120 125 Thr Ser Tyr Gln Tyr Ala Pro Ala Gly Tyr Pro Ala Ser Ala Ser Tyr 130 135 140 Glu Val Ile Met Ala Leu Val Glu Ala Ala Glu Ser Leu Gly Val Arg 145 150 155 160 Tyr His Leu Gly Ile Thr Ala Ser Thr Asp Ser Phe Tyr Val Gly Gln 165 170 175 Gly Arg Pro Gly Tyr Gly Gly Tyr Met Pro Ser Trp Ser Arg Asn Leu 180 185 190 Val Pro Asp Leu Arg Gln Met Arg Val Leu Asn Phe Glu Met Glu Ser 195 200 205 Ala Thr Leu Leu Thr Leu Ala Asn Ile Tyr Gly Phe Arg Ala Gly Ala 210 215 220 Val His Ala Val Tyr Ala Gln Arg Val Lys Asp Glu Phe Val Ala His 225 230 235 240 Ala Gly Glu Glu Aslan Leu Ile Lys Val Ala Asp Glu Ala Val Arg Ile 245 250 255 Leu His Glu Trp Asp Glu Val Lys Gly Arg Ala Gly Lys Arg Tyr Phe 260 265 270 Tyr Pro Ser Leu Leu Arg Pro Gly Pro 275 280

Claims

A method for producing cytosine nucleoside analogs, intermediates or prodrugs thereof of formula (I) by a method of chemical enzymatic or enzymatic synthesis,
(I)

here,
Z ₁ is O, CH ₂ , S, NH;
Z ₂ is independently from Z ₁ O, C (R ^S2 R ^S5 ), S (R ^S2 R ^S5 ), S (R ^S2 ), S (R ^S5 ), preferably SO or SO ₂ groups; ^{N (R S2 R S5),} N (R S2), N (R S5) , and;
Wherein R < ¹ ^> is hydrogen, OH, an ether or ester thereof,

,
n is 0 or 1, A is oxygen or nitrogen, and each M is independently selected from hydrogen, optionally substituted alkyl chains, optionally optionally substituted alkenyl chains, optionally optionally substituted alkynyl chains, Optionally substituted by any optionally substituted aryl, optionally optionally substituted alkyl, alkenyl, or alkynyl chain optionally optionally connected to P by an alkyl, alkenyl, or alkynyl chain substituted with A substituted heterocycle, or a pharmaceutically acceptable counterion, such as, without limitation, sodium, potassium, ammonium, or alkylammonium;
R ^S2 is hydrogen, OH or an ether or ester residue thereof, halogen, CN, NH ₂ , SH, C≡CH, N ₃ ;
R ^S3 is hydrogen in the case of NA derived from a 2'-deoxyribonucleoside or arabinonucleoside, or is selected from OH, NH ₂ , halogen, OCH ₃ when NA is derived from a ribonucleoside Being;
R ^S4 is hydrogen, OH or an ether or ester residue thereof, NH ₂ , halogen, CN;
As a clue, R ^S1 and R ^S4 are different when both are ether or ester of the OH moiety;
R ^S5 is Z ₂ is different from that when the oxygen, hydrogen, OH or an ether or ester residue thereof, NH ₂ or halogen;

R ¹ is O, CH ₂ , S, NH;
R ² is selected from hydrogen, optionally substituted C _4-40 alkyl chain, optionally optionally substituted alkenyl chain, optionally optionally substituted alkynyl chain, optionally optionally substituted alkyl, alkenyl or alkynyl chain Optionally substituted aryl, optionally substituted aryl, optionally substituted alkyl, alkenyl or alkynyl chain connected to N, COR ⁶ , CONR ⁶ R ⁷ , CO ₂ R ⁶ , C (S) OR ⁷ , CN, SR ⁶ , _{^{_{SO 2 R 6, SO 2 R}}} 6 R 7, CN, P (O) aryl, P (O) heterocycle, P (S) aryl, P (S) heterocycle, P (O) O ₂ R ^8, and;
R ³ is selected from hydrogen, optionally substituted C _4-40 alkyl chains, optionally optionally substituted alkenyl chains, optionally optionally substituted alkynyl chains, optionally optionally substituted alkyl, alkenyl or alkynyl chains Optionally substituted aryl, optionally substituted aryl, optionally substituted alkyl, alkenyl or alkynyl chain connected to N, COR ⁶ , CONR ⁶ R ⁷ , CO ₂ R ⁶ , C (S) OR ⁷ , CN, SR ⁶ , SO ₂ R ⁶ ; _{^{SO 2 R 6 R 7, CN}} , P (O) aryl, P (O) heterocycle, P (S) aryl, P (S) heterocycle, P (O) O _2, and R ^8; R ³ and R ² are independent of each other; And as a clue at least one of R ₂ or R ₃ is different from hydrogen;
R ⁴ is hydrogen, OH, NH ₂ , SH, halogen; An optionally substituted alkyl chain; An optionally substituted alkenyl chain; Optionally substituted alkynyl, trihaloalkyl, OR ⁶ , NR ⁶ R ⁷ , CN, COR ⁶ , CONR ⁶ R ⁷ , CO ₂ R ⁶ , C (S) OR ⁶ , OCONR ⁶ R ⁷ , OCO by _{^{2 R 6, OC (S)}} oR 6, NHCONR 6 R 7, NHCO 2 R 6, NHC (S) oR 6, SO 2 NR 6 R 7, any optionally substituted alkyl, alkenyl or alkynyl chain Any optional substituted heteroaryl optionally attached to Y by any optionally substituted alkyl, alkenyl or alkynyl chain, and independently selected from R ² , R ^3, , R < ⁴ > or R < ^{5 &} gt ;, or any optionally substituted aryl;

As a clue, Y is a carbon or sulfur atom and, alternatively, when R ⁴ is absent, Y is a nitrogen atom as a cue;
here
X is O, S, NR ^B2 , Se; R ^B1 is H, OH, NH ₂ , SH, straight or branched C _1-10 alkyl, F, Cl, Br, I, XR ^B2 , -C≡CR ^B2 , CO ₂ R ^B2 ; R ^B2 is H, OH, NH ₂ , straight or branched C _1-5 alkyl, phenyl;
R ⁵ is hydrogen, OH, NH _2, SH, halogen, and any optionally substituted alkyl chain, and any optional alkenyl of Al substituted by a chain, any optionally substituted alkynyl chain, alkyl, trihaloalkyl, OR ^6, NR ⁶ ^{R 7, CN, COR 6,} CONR 6 R 7, CO 2 R 6, C (S) OR 6, OCONR 6 R 7, OCO 2 R 6, OC (S) OR 6, NHCONR 6 R 7, NHCO 2 R ⁶ , NHC (S) OR ⁶ , SO ₂ NR ⁶ R ⁷ ; CH ₂ -heterocyclic ring, CN;
And independently any optional optionally substituted heterocycle of R ² , R ³ , R ⁴ or R ⁵ , or optionally substituted aryl,

here
X is O, S, NR ^B2 , Se; R ^B1 is H, OH, NH ₂ , SH, straight or branched C _1-10 alkyl, F, Cl, Br, I, XR ^B2 , -C≡CR ^B2 , CO ₂ R ^B2 ; R ^B2 is H, OH, NH ₂ , straight or branched C _1-5 alkyl, phenyl;
R ⁶ and R ⁷ are independently of each other hydrogen, optionally substituted alkyl chain, optionally optionally substituted alkenyl chain, optionally optionally substituted alkynyl chain, heterocyclic or optionally substituted aryl;
R < ⁸ > is hydrogen, optionally substituted alkyl chain, optionally optionally substituted alkenyl chain, optionally optionally substituted alkynyl chain, optionally optionally substituted aryl, or optionally substituted heterocycle;
Y is C, N, S;

Wherein the method for producing a cytosine nucleoside analog comprises the steps of:
(i) a cytosine nucleobase of formula (II) wherein Y, R ¹ , R ⁴ , R ⁵ , R ⁶ and R ⁷ are defined as above, to incorporate the appropriate substitutions described as substituents R ² and R ³ With a suitable reagent for modifying the amino group at the N < ⁴ > position, said modified cytosine nucleobases of formula II being formed are optionally selectively purified by conventional purification methods; Alternatively, the process starts directly from the starting product represented by such cytosine nucleobases of formula II:
&Lt; RTI ID = 0.0 &

(ii) biocatalytically reacting the above-described modified cytosine nucleobases of formula (II) with a suitable nucleoside analog substrate of formula (III)
(III)

Wherein Z _1, Z _2, R ^S1, R ^S2, R ^S3, R ^S4, R ^S5 is defined as above, and the base is selected from the following: uracil, adenine, cytosine, guanine, thymine, hypoxanthine, greater Thiuracil, thioguanine, 9- H -purine-2-amine, 7-methylguanine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, Cytosine and 5-hydroxymethyl cytosine, phthalidone, and any substituted derivatives thereof;

Wherein the foregoing reaction carried out in step ii) is carried out in a suitable reaction-aqueous medium and under suitable reaction conditions, with a mixture of the starting material comprising a cytosine nucleobase of formula (II) and a nucleoside analog of formula (III), with a nucleoside phosphorylase enzyme Comprising the addition of a pyrimidine nucleoside phosphorylase enzyme, a purine nucleoside phosphorylase enzyme, or a combination thereof,

(iii) optionally deprotecting the amino group at the N ⁴ position in the cytosine nucleoside analog to recover the free primary amino N ⁴ in the cytosine nucleoside analog of formula I which is further purified by a conventional purification method .

The method according to claim 1, wherein the cytosine nucleobase of formula (II), which is transferred by a pyrimidine nucleoside phosphorylase enzyme, is selected from:

The method according to any one of claims 1 or 2, wherein the produced nucleoside analog, intermediate or prodrug thereof is selected from the group consisting of capecitabine, decitabine, 5-azacytidine, cytarabine , Enocitabine, gemcitabine, zalcitabine, ibacitavin, sapacitabine, 2'-C-cyano-2'-deoxy-1-β-D-arabino-pentofuranosylcytosine, , Valproicitabine, 2'-deoxy-4'-thiocytidine, tiarabine, 2'-deoxy-4'-thio-5-azacytidine and aflativarbin.

4. The method according to any one of claims 1 to 3,
Characterized in that the source of the innate nucleoside phosphorylase enzyme and its mutants or variants, or of the recombinant nucleoside phosphorylase enzyme, is a mesophilic, pyrophoric or ultra-warm organism.

The use according to any one of claims 1 to 4, characterized in that the source of the innate nucleoside phosphorylase enzyme and its mutants or variants, or the recombinant nucleoside phosphorylase enzyme, is selected from Archaea or bacteria Way.

The method of claim 5, wherein the nucleoside phosphorylase enzyme is selected from the group consisting of Sulfolobus solfataricus ) or Aeropyrum ( Archaea ), which is selected from the group consisting of perennium , pernix , and the like.

3. The composition of claim 1, wherein the nucleoside phosphorylase enzyme, or functional portion thereof, is selected from the group consisting of SEQ ID NO: 1, 2, 5, 7, 9 or 11; or
a) a complementary nucleotide sequence of SEQ ID NO: 1, 2, 5, 7, 9 or 11; or
b) a nucleotide sequence which is flanked by SEQ ID NO: 1, 2, 5, 7, 9 or 11; or
c) under conditions of high stringency, in SEQ ID NO: 1, 2, 5, 7, 9 or 11; A complement of SEQ ID NO: 1, 2, 5, 7, 9 or 11; Or a nucleotide sequence which hybridizes to a hybridization probe derived from SEQ ID NO: 1, 2, 5, 7, 9 or 11; Or its complement; or
d) a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 1, 2, 5, 7, 9 or 11; or
e) a nucleotide sequence having at least 59% sequence identity with SEQ ID NO: 1, 2, 5, 7, 9 or 11; or
f) is encoded by a nucleotide sequence selected from a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NO: 3, 4, 6, 8, 10 or 12.

The method of claim 3, wherein the produced nucleoside analog is capecitabine and the ribonucleoside used as starting material is 5'-deoxyuridine, 5'-deoxy-5-methyluridine or 5'-de Oxy-5-chlorouridine; And the nucleobase also used as the starting material transferred by the pyrimidine nucleoside phosphorylase enzyme is pentyl (5-fluoro-2-oxo-1,2-dihydropyrimidin-4-yl) carbamate Lt; / RTI >

4. The method of claim 3, wherein the nucleoside analog produced is cytarabine, the ribonucleoside used as starting material is 9- (bD-arabinofuranosyl) uracil, and is transferred by pyrimidine nucleoside phosphorylase enzyme (2-oxo-1,2-dihydropyrimidin-4-yl) pentanamide. &Lt; / RTI >

In the production of cytosine nucleoside analogs, intermediates or prodrugs thereof useful as anticancer or antiviral agents, the native mesophilic, hyperthermal or hyperthermophilic nucleoside phosphorylase and its mutants or variants, or recombinant enzymes, Functional part; Or a naturally occurring or recombinant mesophilic, hyperthermal or hyperthermophilic nucleoside phosphorylase operably linked to one or more control sequences driving expression or overexpression of the nucleoside phosphorylase in a suitable host, A recombinant expression vector comprising a sequence encoding a portion; Or a microorganism or host cell containing them.

11. The method of claim 10, wherein the mesophilic, hyperthermal, or hyperthermal nucleoside phosphorylase enzyme, or functional portion thereof, is selected from the group consisting of SEQ ID NO: 1, 2, 5, 7, 9 or 11; or
a) a complementary nucleotide sequence of SEQ ID NO: 1, 2, 5, 7, 9 or 11; or
b) a nucleotide sequence which is flanked by SEQ ID NO: 1, 2, 5, 7, 9 or 11; or
c) under conditions of high stringency, in SEQ ID NO: 1, 2, 5, 7, 9 or 11; A complement of SEQ ID NO: 1, 2, 5, 7, 9 or 11; Or a nucleotide sequence which hybridizes to a hybridization probe derived from SEQ ID NO: 1, 2, 5, 7, 9 or 11; Or its complement; or
d) a nucleotide sequence having at least 80% sequence identity with SEQ ID NO: 1, 2, 5, 7, 9 or 11; or
e) a nucleotide sequence having at least 59% sequence identity with SEQ ID NO: 1, 2, 5, 7, 9 or 11; or
f) is encoded by a nucleotide sequence selected from a nucleotide sequence encoding an amino acid sequence selected from SEQ ID NO: 3, 4, 6, 8, 10 or 12.

Use according to any one of claims 10 or 11, wherein the produced cytosine nucleoside analogs, intermediates or prodrugs thereof are selected from the group consisting of capecitabine, decitabine, 5-azacytidine, Ribavirin, enocitabine, gemcitabine, zalcitabine, ibacitavin, sapacitabine, 2'-C-cyano-2'-deoxy-1-β-D- arabino- Bin, valproicitabine, 2'-deoxy-4'-thiocytidine, tiarabine, 2'-deoxy-4'-thio-5-azacytidine and apoptavarbin.

13. Use according to claim 12, characterized in that the produced cytosine nucleoside analog, intermediate or prodrug thereof is capecitabine.

The use according to claim 12, wherein the produced cytosine nucleoside analog, intermediate or prodrug thereof is cytarabine.