The N, N-deoxyribose transferase mutant and application thereof to synthesis of 2apos; -deoxy-2apos;-(2apos); fluorocytidine

Abstract

The invention provides an N-deoxyribose transferase mutant and application of the N-deoxyribose transferase mutant to synthesis of 2 '-deoxy-2'-fluorocytidine. Compared with the amino acid sequence shown in SEQ ID NO: 1, the amino acid sequence of the N-deoxyribose transferase is shown in SEQ ID NO: 1; the amino acid sequence has substitution mutation at any one position, any two positions, any three positions, any four positions, any five positions or any more than six positions of the 54th position, the 55th position, the 60th position, the 67th position, the 68th position, the 71th position, the 91th position, the 127th position, the 140th position and the 154th position. The invention also provides a corresponding polynucleotide molecule, a nucleic acid construct, a carrier, a host cell, an enzyme preparation and a synthetic method of 2 '-deoxy-2'-fluorocytidine.

Description

Technical Field

[0001] The invention belongs to the field of chemical engineering and enzyme engineering, and specifically relates to an N-deoxyribose transferase mutant and application thereof in the synthesis of 2'-deoxy-2'-fluorocytidine. Background Art

[0002] Nucleoside analogs are an important class of anticancer therapeutic agents and specific antiviral drugs, including various purine and pyrimidine nucleoside derivatives, mainly a class of antimetabolites, which are used to destroy viral transcription, interfere with or terminate the synthesis of viral nucleic acids, and are used to fight DNA and RNA viruses such as herpes virus, HIV, HBV, influenza and respiratory viruses. 2'-deoxy-2'-fluorocytidine analogs, as a type of nucleoside analog, have antiviral and anti-tumor activity, and can effectively embed into viral DNA and RNA chains, thereby inhibiting cell division and replication. Therefore, the demand for them in the domestic and foreign pharmaceutical markets and clinical treatments is also steadily increasing.

[0003] 2'-deoxy-2'-fluorocytidine (2'-F-dC) is generally synthesized by chemical synthesis and enzymatic method. Chemical synthesis is a multi-step process. In order to achieve regional and stereoselectivity, protection and deprotection processes are required. The operation steps are complicated, the cost is high, and toxic and harmful substances are easily produced, which pollutes the environment. Enzymatic synthesis is increasingly valued by people due to its high regional and stereoselectivity. Enzymatic synthesis can be carried out under mild reaction conditions, has high catalytic efficiency, is green and pollution-free, and can effectively reduce purification steps in the post-processing process.

[0004] N-deoxyribosyltransferase (EC.2.4.2.6) can catalyze the transfer of deoxyribose between different bases and can be divided into two categories according to the different substrate specificities: N-deoxyribosyltransferase I (PDT), which can only catalyze the transfer of deoxyribose between purines; and N-deoxyribosyltransferase II (NDT), which catalyzes the transfer of deoxyribose between purine and pyrimidine.

[0005] NDT has a wide base recognition range and can recognize a variety of natural and modified bases with a high conversion rate. Therefore, it is used in the synthesis of a variety of deoxynucleoside analogs, such as 5-fluoro-2'-deoxyuridine, 6-chloro-2'-deoxycytidine, etc. However, NDT has a high substrate specificity for the structure of the ribose donor. For nucleoside analogs with sugar modifications, such as cytarabine, stavudine, gemcitabine and zalcitabine, the efficiency of catalytic synthesis is extremely low, which limits the application of NDT. Therefore, it is necessary to expand the substrate recognition range of NDT and improve the activity of the enzyme through directed evolution of wild-type NDT. Summary of the invention

[0006] The purpose of the present invention is to provide an N-deoxyribosyltransferase mutant and its application in the synthesis of 2'-deoxy-2'-fluorocytidine.

[0007] In a first aspect of the present invention, there is provided an N-deoxyribosyltransferase, which has an amino acid sequence having a substitution mutation at any one position, any two positions, any three positions, any four positions, any five positions, or any six or more positions of the 54th, 55th, 60th, 67th, 68th, 71st, 91st, 127th, 140th and 154th positions compared to the amino acid sequence shown in SEQ ID NO: 1.

[0008] In one or more embodiments, the wild-type aspartic acid at position 54 is mutated to a polar uncharged amino acid, such as serine, threonine, cysteine, tyrosine, asparagine or glutamine;

[0009] The wild-type glutamic acid at position 55 is mutated into a polar positively charged amino acid, such as lysine, arginine or histidine;

[0010] The mutation at position 60 is that the wild-type leucine is mutated into a non-polar amino acid, such as alanine, valine, isoleucine, proline, phenylalanine, tryptophan, methionine or glycine;

[0011] The mutation at position 67 is that the wild-type serine is mutated into a non-polar amino acid, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine or glycine, or is mutated into a polar uncharged amino acid, such as threonine, cysteine, tyrosine, asparagine or glutamine;

[0012] The mutation at position 68 is that the wild-type alanine is mutated into a non-polar amino acid, such as valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine or glycine, or is mutated into a polar uncharged amino acid, such as serine, threonine, cysteine, tyrosine, asparagine or glutamine;

[0013] The mutation at position 71 is that the wild-type histidine is mutated into a polar positively charged amino acid, such as lysine or arginine, or is mutated into a non-polar amino acid, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine or glycine;

[0014] The wild-type glutamic acid at position 91 is mutated to a non-polar amino acid, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine or glycine;

[0015] The wild-type serine at position 127 is mutated to a polar uncharged amino acid, such as threonine, cysteine, tyrosine, asparagine or glutamine;

[0016] The wild-type leucine at position 140 is mutated to a polar uncharged leucine, such as serine, threonine, cysteine, tyrosine, asparagine or glutamine;

[0017] The wild-type aspartic acid at position 154 is mutated into a polar positively charged amino acid, such as lysine, arginine or histidine.

[0018] In one or more embodiments, the wild-type aspartic acid at position 54 is mutated to serine or threonine;

[0019] The wild-type glutamic acid at position 55 is mutated to histidine;

[0020] The wild-type leucine at position 60 is mutated to glycine, alanine, isoleucine or valine, preferably valine;

[0021] The wild-type serine at position 67 is mutated to glycine, alanine, leucine, isoleucine, valine or tryptophan, or is mutated to threonine;

[0022] The wild-type alanine at position 68 is mutated to phenylalanine or tyrosine;

[0023] The wild-type histidine at position 71 is mutated to arginine, or is mutated to methionine, phenylalanine, leucine, isoleucine or valine;

[0024] The wild-type glutamic acid at position 91 is mutated to phenylalanine;

[0025] The wild-type serine at position 127 is mutated to asparagine or glutamine;

[0026] The wild-type leucine at position 140 is mutated to cysteine;

[0027] The wild-type aspartic acid at position 154 was mutated to arginine.

[0028] In one or more embodiments, compared to SEQ ID NO: 1, the mutation of the N-deoxyribosyltransferase is selected from:

[0029] (1) D54S,

[0030] (2) E55H,

[0031] (3) L60V,

[0032] (4) S67L, S67T, S67I, S67W or S67A,

[0033] (5) A68F or A68Y,

[0034] (6) H71R, H71M, H71F or H71L,

[0035] (7) E91F,

[0036] (8)S127N,

[0037] (9) L140C, and

[0038] (10)D154R.

[0039] In one or more embodiments, compared to SEQ ID NO: 1, the mutation of the N-deoxyribosyltransferase is selected from the group consisting of: L60V, S67T or S67I or S67A, H71R or H71M or H71F or H71L, S127N, and L140C.

[0040] In a second aspect of the present invention, a polynucleotide molecule is provided, wherein the polynucleotide sequence is selected from:

[0041] (1) a polynucleotide sequence encoding the N-deoxyribosyltransferase according to the first aspect of the present invention; and

[0042] (2) A complementary sequence to the polynucleotide sequence described in (1).

[0043] The third aspect of the present invention provides a nucleic acid construct, which contains the polynucleotide molecule described in the second aspect of the present invention; preferably, the nucleic acid construct is an expression cassette.

[0044] The fourth aspect of the present invention provides a vector, which contains the polynucleotide molecule described in the second aspect of the present invention or the nucleic acid construct described in the third aspect of the present invention; preferably, the vector is a cloning vector or an expression vector.

[0045] The fifth aspect of the present invention provides a host cell, which: (1) expresses the N-deoxyribose transferase described in any one of claims 1 to 5, and / or (2) contains the polynucleotide molecule described in the second aspect of the present invention, the nucleic acid construct described in the third aspect of the present invention, or the vector described in the fourth aspect of the present invention; preferably, the host cell is Escherichia coli.

[0046] The sixth aspect of the present invention provides an enzyme preparation, which contains the N-deoxyribosyltransferase described in the first aspect of the present invention.

[0047] In one or more embodiments, preferably, the enzyme preparation further contains a buffer.

[0048] In one or more embodiments, the buffer is a phosphate buffer.

[0049] In one or more embodiments, the pH of the buffer is 5-10, preferably 6-9, and more preferably 7-8.

[0050] In one or more embodiments, the enzyme preparation is a powder, such as a lyophilized powder.

[0051] In one or more embodiments, the enzyme preparation is the supernatant obtained after the host cells expressing N-deoxyribosyltransferase are lysed, or is a concentrated solution of the supernatant.

[0052] The seventh aspect of the present invention provides a use of the N-deoxyribosyltransferase described in the first aspect of the present invention in catalyzing the transfer of a 2'-deoxy-2'-fluororibose group, or in preparing 2'-deoxy-2'-fluorocytidine, or in preparing an enzyme preparation for preparing 2'-deoxy-2'-fluorocytidine.

[0053] In an eighth aspect of the present invention, a method for preparing 2'-deoxy-2'-fluorocytidine is provided, the method comprising:

[0054]

[0055] The step of using the N-deoxyribosyltransferase described in the first aspect of the present invention to catalyze the reaction of cytosine C with a deoxyribose donor I to generate 2'-deoxy-2'-fluorocytidine II;

[0056] Wherein, in the deoxyribose donor I, R is a base, preferably selected from adenine, guanine, uracil, hypoxanthine and thymine; preferably, R is uracil, and the deoxyribose donor I is 2'-deoxy-2'-fluorouridine.

[0057] In one or more embodiments, the method has one or more of the following features:

[0058] The pH value of the reaction system of the reaction is 5 to 10, preferably 6 to 9, more preferably 7 to 8;

[0059] The reaction temperature of the reaction is 30 to 80°C, preferably 40 to 70°C;

[0060] The molar ratio of deoxyribose donor I to cytosine C in the reaction system is 1:(1-5), such as 1:(2-4);

[0061] The mass of the N-deoxyribosyl transferase in the reaction system is 10 to 80% of the mass of the deoxyribose donor, preferably 30 to 50%. DETAILED DESCRIPTION

[0062] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a preferred technical solution.

[0063] After extensive and in-depth research and a large number of screening tests, the inventors of the present application unexpectedly developed for the first time a biological preparation method for 2'-deoxy-2'-fluoro-cytidine with a high conversion rate, in which the deoxyribosyltransferase exhibits specificity and selectivity for the reaction substrate.

[0064] NDT mutant

[0065] As used herein, unless otherwise specified, the "N-deoxyribosyltransferase mutant", "NDT mutant", "mutant N-deoxyribosyltransferase" and "mutant NDT" are used interchangeably and refer to a polypeptide (protein) corresponding to the wild-type N-deoxyribosyltransferase (SEQ ID NO: 1, whose nucleotide sequence is shown in SEQ ID NO: 2) after mutation occurs at positions 54, 55, 60, 67, 68, 71, 91, 127, 140 and / or 154 of its amino acid sequence. The N-deoxyribosyltransferase mutant of the present invention may have a substitution mutation only at any one of positions 54, 55, 60, 67, 68, 71, 91, 127, 140 and 154 relative to SEQ ID NO: 1, or may have a substitution mutation at any two positions, any three positions, any four positions, any five positions or any six or more positions.

[0066] As used herein, "isolated" means that the modified object, such as the NDT mutant, is substantially free of other proteins, lipids, carbohydrates or other substances naturally associated therewith. Those skilled in the art can purify the NDT mutant using standard protein purification techniques. Substantially pure proteins can produce a single main band on a non-reducing polyacrylamide gel.

[0067] Herein, the mutation at position 54 may be that the wild-type D is mutated to a polar, uncharged amino acid (neutral amino acid), such as serine (S), threonine (T), cysteine ​​(C), tyrosine (Y), asparagine (N) or glutamine (Q), preferably mutated to S or T.

[0068] Herein, the mutation at position 55 may be that the wild-type E is mutated into a polar positively charged amino acid (basic amino acid), such as lysine (K), arginine (R) or histidine (H). In some embodiments, the mutation at position 55 is that the wild-type E is mutated into H.

[0069] Herein, the mutation at position 60 may be that the wild-type L is mutated into a non-polar amino acid (hydrophobic amino acid), such as alanine (A), valine (V), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M) or glycine (G), preferably mutated into G, A, I, L or V, more preferably V.

[0070] Herein, the mutation at position 67 may be that the wild-type S is mutated into a non-polar amino acid (hydrophobic amino acid), such as alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M) or glycine (G), or is mutated into a polar uncharged (neutral amino acid), such as threonine (T), cysteine ​​(C), tyrosine (Y), asparagine (N) or glutamine (Q). In some embodiments, the mutation at position 67 may be that the wild-type S is mutated into G, A, I, L, V or W, or is mutated into S or T. In some embodiments, the mutation at position 67 is that the wild-type S is mutated into L, T, I, W or A.

[0071] Herein, the mutation at position 68 may be that the wild-type A is mutated into a non-polar amino acid (hydrophobic amino acid), such as valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M) or glycine (G), or is mutated into a polar uncharged (neutral amino acid), such as serine (S), threonine (T), cysteine ​​(C), tyrosine (Y), asparagine (N) or glutamine (Q). Preferably, the mutation at position 68 is that the wild-type A is mutated into F or Y.

[0072] Herein, the mutation at position 71 may be that the wild-type H is mutated into a polar positively charged amino acid (basic amino acid), such as lysine (K) or arginine (R), or is mutated into a non-polar amino acid (hydrophobic amino acid), such as alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M) or glycine (G). In some embodiments, the mutation at position 71 may be that the wild-type H is mutated into R, M, F, I, L or V.

[0073] Herein, the mutation at position 91 may be that the wild-type E is mutated into a non-polar amino acid (hydrophobic amino acid), such as alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F), tryptophan (W), methionine (M) or glycine (G), preferably mutated into F.

[0074] Herein, the mutation at position 127 may be that the wild-type S is mutated to a polar, uncharged amino acid (neutral amino acid), such as threonine (T), cysteine ​​(C), tyrosine (Y), asparagine (N) or glutamine (Q), preferably mutated to N or Q.

[0075] Herein, the mutation at position 140 may be that the wild-type L is mutated into a polar, uncharged amino acid (neutral amino acid), such as serine (S), threonine (T), cysteine ​​(C), tyrosine (Y), asparagine (N) or glutamine (Q), preferably mutated into C.

[0076] Herein, the mutation at position 154 may be that the wild-type D is mutated into a polar positively charged amino acid (basic amino acid), such as lysine (K), arginine (R) or histidine (H). In some embodiments, the mutation at position 154 is that the wild-type D is mutated into R.

[0077] In some embodiments, compared with SEQ ID NO: 1, the mutation of the NDT mutant of the present application is selected from: (1) D54S, (2) E55H, (3) L60V, (4) S67L, S67T, S67I, S67W or S67A, (5) A68F or A68Y, (6) H71R, H71M, H71F or H71L, (7) E91F, (8) S127N, (9) L140C, and (10) D154R.

[0078] In some embodiments, compared with SEQ ID NO: 1, the mutation of the NDT mutant of the present application is selected from the group consisting of: L60V, S67T or S67I or S67A, H71R or H71M or H71F or H71L, S127N, and L140C.

[0079] In some embodiments, compared with SEQ ID NO: 1, the NDT mutant of the present application is a single mutant, and its mutation occurs at position 54, 55, 60, 67, 68, 71, 91, 127, 140 or 154, and the mutation that occurs is preferably as described in any of the embodiments above.

[0080] The present invention also includes fragments, derivatives and analogs of the NDT mutants. As used herein, the terms "fragment", "derivative" and "analog" refer to proteins that substantially retain the same biological function or activity as the natural NDT mutants of the present invention. The protein fragments, derivatives or analogs of the present invention may be (i) proteins in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) proteins having substitution groups in one or more amino acid residues, or (iii) proteins formed by the fusion of an additional amino acid sequence to this protein sequence (such as a leader sequence or secretory sequence or a sequence or proprotein sequence used to purify this protein, or a fusion protein). According to the definitions herein, these fragments, derivatives and analogs are within the scope known to those skilled in the art. However, the amino acid sequences of the NDT mutants and their fragments, derivatives and analogs must contain the mutations described above in the present invention; preferably, the mutations correspond to the 54th, 55th, 60th, 67th, 68th, 71st, 91st, 127th, 140th and / or 154th amino acids in SEQ ID NO:1 (that is, the mutation sites claimed in the present invention are conserved in the NDT fragments, derivatives and analogs).

[0081] In the present invention, the term "NDT mutant" also includes a conservative variant protein having several (usually 1-20, preferably 1-10, and even more preferably 1-8, 1-5, 1-3, or 1-2) amino acid deletions, insertions and / or substitutions compared to the mutant, and / or one or several (usually within 20, preferably within 10, and even more preferably within 5) amino acids added or deleted at the C-terminus and / or N-terminus of the mutant. For example, in the art, when amino acids with similar or similar properties are substituted, the function of the protein is usually not changed. For another example, adding or deleting one or several amino acids at the C-terminus and / or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of NDT mutants. However, among these variant forms, the mutations described above in the present invention are definitely present; preferably, the mutations are mutations corresponding to amino acids at positions 54, 55, 60, 67, 68, 71, 91, 127, 140 and / or 154 in SEQ ID NO:1.

[0082] In the present invention, the term "NDT mutant" also includes derivative conservative variant proteins that retain their protein activity but have an amino acid sequence identity of more than 80%, preferably more than 85%, more preferably more than 90%, further more preferably more than 95%, such as more than 98%, more than 99% with the NDT mutant. Similarly, these derivative conservative variant proteins must contain the mutations described above in the present invention, preferably, the mutations are mutations corresponding to amino acids at positions 54, 55, 60, 67, 68, 71, 91, 127, 140 and / or 154 in SEQ ID NO:1.

[0083] The NDT mutants of the present invention can be the product of chemical synthesis, or produced using recombinant technology from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher plants, insects and mammalian cells).

[0084] Polynucleotide molecules

[0085] The present invention also provides a polynucleotide molecule encoding the NDT mutant of the present invention or its conservative variant protein and its complementary sequence. The polynucleotide molecule of the present invention can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0086] The polynucleotide molecule encoding the mature protein of the mutant includes: a coding sequence encoding only the mature protein; a coding sequence of the mature protein and various additional coding sequences; a coding sequence of the mature protein (and optional additional coding sequences) and non-coding sequences.

[0087] A "polynucleotide molecule encoding a protein" may be a polynucleotide molecule that includes the protein, or may be a polynucleotide molecule that also includes additional coding and / or non-coding sequences.

[0088] The polynucleotide molecules of the present invention can usually be obtained by PCR amplification, recombination or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequences, and commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art are used as templates to amplify the relevant sequences. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then splice the fragments amplified in each time together in the correct order.

[0089] Nucleic acid constructs

[0090] The present invention also relates to nucleic acid constructs comprising one or more regulatory sequences operably linked to the polynucleotide molecules of the present invention.

[0091] As used herein, "operably linked" or similar descriptions refer to the arrangement of elements, wherein the components are arranged in a certain shape so as to perform their required functions. Thus, a given promoter operably linked to a coding sequence can effectively express the coding sequence when the correct transcription factors, etc., are present. The promoter does not need to be adjacent to the coding sequence, as long as it functions to direct the expression of the sequence. Thus, for example, a sequence that does not participate in translation but is transcribed can exist between the promoter sequence and the coding sequence, as can a transcribable intron; and the promoter sequence can still be considered to be "operably linked" to the coding sequence.

[0092] The polynucleotide encoding the present invention can be manipulated in a variety of ways to ensure expression of the NDT mutant or its conservative variant protein. Manipulation of the polynucleotide molecule prior to its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0093] The regulatory sequence can be a suitable promoter sequence, which is a nucleotide sequence recognized by the host cell for expressing the polynucleotide molecules of the present invention. The promoter sequence contains a transcriptional regulatory sequence for polypeptide expression. The promoter can be any nucleotide sequence that shows transcriptional activity in the selected host cell, including mutant, truncated and hybrid promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell.

[0094] In E. coli, suitable promoters include, but are not limited to, lac (lactose promoter), trp (tryptophan promoter), and tac (hybrid promoter of lactose and tryptophan).

[0095] The regulatory sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the selected host cell may be used in the present invention. In some embodiments, the transcription terminator sequence is a terminator that is functional in Escherichia coli.

[0096] In some embodiments, the nucleic acid construct is an expression cassette comprising a promoter and a transcription terminator operably linked to a polynucleotide molecule described herein.

[0097] Carrier

[0098] The present invention also relates to vectors comprising the polynucleotide molecules of the present invention, including but not limited to expression vectors and cloning vectors. For example, in certain embodiments, the nucleic acid constructs of the present invention are expression vectors or cloning vectors.

[0099] In expression vectors, various nucleic acid molecules and regulatory sequences can be linked together to produce a recombinant expression vector comprising one or more restriction sites that allow insertion of a polynucleotide molecule or nucleic acid construct described herein at such sites. When making an expression vector, the polynucleotide molecule described herein is located in the vector so that it can be operably linked to an appropriate regulatory sequence.

[0100] The recombinant expression vector may be any vector (such as a plasmid or virus) that can be easily subjected to recombinant DNA methods and can result in the expression of the nucleotide sequence of interest. The choice of vector generally depends on the compatibility of the vector with the host cell into which it is introduced. The vector may be a linear or closed circular plasmid.

[0101] The vector can be an autonomously replicating vector, i.e., exists as an extrachromosomal entity, and its replication is independent of the vector of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The vector can include any mode for ensuring self-replication. Alternatively, the vector can be a vector that is integrated into the genome and replicated together with the chromosome into which it has been integrated when being imported into the host cell. In addition, a single vector or plasmid or two or more vectors or plasmids, or a transposon, that together contain the total DNA that will be imported into the host cell genome can be used.

[0102] The vectors of the present invention may contain one or more selectable markers that allow easy selection of transformed, transfected, transduced, etc. cells. Selectable markers are genes whose products provide resistance to antibiotics or viruses, resistance to heavy metals, prototrophy to auxotrophy, and the like.

[0103] The vectors of the present invention may contain elements that permit integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0104] More than one copy of the polynucleotide of the present invention can be inserted into the host cell to increase the yield of the gene product. The increase in the number of polynucleotide copies can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selection marker gene and the polynucleotide, wherein the cells containing the amplified copy of the selection marker gene and thus the additional copy of the polynucleotide can be screened by culturing the cells in the presence of an appropriate selection agent.

[0105] The expression vector of the present invention is more preferably a vector that can be used for expression in Escherichia coli, such as pET-30a.

[0106] The cloning vector containing the polynucleotide sequence of the present invention can be used to replicate enough target plasmids. Therefore, the cloning vector of the present invention has strong self-replication elements, such as replication initiation sites, etc. Usually, the cloning vector of the present invention does not have expression elements.

[0107] Host cells

[0108] The host cell of the present invention can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell. Representative examples include: Escherichia coli, Bacillus subtilis, Streptomyces, Agrobacterium; eukaryotic cells such as yeast, plant cells, etc. In a specific embodiment of the present invention, Escherichia coli is used as a host cell.

[0109] The polynucleotide sequence of the present invention can be used to express or produce recombinant NDT mutants by recombinant DNA technology. Generally, the following steps are involved: (1) transforming or transducing a suitable host cell with a polynucleotide encoding an NDT mutant of the present invention, or with a recombinant expression vector containing the polynucleotide; (2) culturing the host cell in a suitable culture medium; (3) isolating and purifying the protein from the culture medium or cells.

[0110] In a specific embodiment of the present invention, the NDT mutant is expressed and purified in E. coli: the wild-type NDT gene is amplified from a cloning vector by PCR and subcloned into a prokaryotic expression vector. Using the NDT gene expression vector as a template, mutations are introduced by PCR primers, and a mutant protein expression vector is constructed after PCR amplification. The correctly sequenced mutant is heterologously expressed in E. coli. The obtained fermentation products can be mixed.

[0111] Enzyme preparations

[0112] In some embodiments, the present invention provides an enzyme preparation containing the NDT mutant according to any embodiment of the present invention. In some embodiments, the enzyme preparation is a buffer containing the NDT mutant. Preferably, the buffer is a phosphate buffer with a pH of 5 to 10, preferably 6 to 9, and more preferably 7 to 8. In some embodiments, the phosphate buffer is a dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer.

[0113] In some embodiments, the enzyme preparation is a powder, such as a lyophilized powder.

[0114] In some embodiments, the enzyme preparation is the supernatant obtained after lysis of the host cells expressing the NDT mutant described herein, such as Escherichia coli. Lysis can be performed by methods other than those known in the art. For example, the bacterial solution can be washed twice with 50 mmol / L Tris-HCl, and then the bacterial solution can be resuspended in 50 mmol / L Tris-HCl (pH 8.0) buffer, and the cells can be ultrasonically disrupted in an ice bath. The ultrasonically disrupted sample can be centrifuged to obtain a supernatant. In some embodiments, the enzyme preparation can be a concentrated solution of the supernatant, and the solvent in the supernatant can be removed by conventional methods.

[0115] application

[0116] Based on the novel findings of the present invention, the application of the NDT mutant is provided, which is used for carrying out 2'-deoxy-2'-fluororibose (group) transfer and adding 2'-deoxy-2'-fluororibose (group) to a substrate; or, for preparing a reagent (including a catalytic reagent) or a culture medium for catalyzing 2'-deoxy-2'-fluororibose (group) transfer.

[0117] In a preferred embodiment, the NDT mutant of the present invention is used to take cytosine as a substrate, and add a 2'-deoxy-2'-fluororibose (group) thereto to form 2'-deoxy-2'-fluorocytidine.

[0118] The mutant NDT of the present invention or the host cell expressing the mutant NDT can be formulated in a carrier acceptable for industrial synthesis to obtain a composition suitable for catalytic reaction or storage.

[0119] The mutant NDT of the present invention or the host cell containing the same can also be placed in a kit for industrial use or sale. In addition, the kit can also contain a deoxyribose acceptor and a deoxyribose donor involved in the reaction, as well as auxiliary reagents such as a reaction buffer and a reaction termination reagent. In the present invention, the deoxyribose acceptor is cytosine. Generally speaking, the kit also includes instructions for use.

[0120] It should be understood that in the present invention, the "deoxyribose donor" can be a variety of compounds, as long as they can be recognized and acted upon by the NDT enzyme of the present invention to obtain a 2'-deoxy-2'-fluororibonucleic acid group.

[0121] Synthesis of 2'-deoxy-2'-fluorocytidine catalyzed by NDT mutant

[0122]

[0123] The present invention provides a method for adding a 2'-deoxy-2'-fluororibose to cytosine. The method uses cytosine C as a substrate and uses the NDT mutant of the present invention for catalysis, so that the 2'-deoxy-2'-fluororibose in the ribose donor I is transferred to the cytosine to generate 2'-deoxy-2'-fluorocytidine.

[0124] In the present invention, 2'-deoxy-2'-fluororibose comes from a deoxyribose donor. The deoxyribose donor is a compound carrying 2'-deoxy-2'-fluororibose, as shown in Formula I. In Formula I, R is a base, which can be selected from adenine, guanine, uracil, hypoxanthine and thymine, etc. Exemplary compounds of Formula I include 2'-deoxy-2'-fluoroadenosine, 2'-deoxy-2'-fluoroguanosine, 2'-deoxy-2'-fluorothymidine, 2'-deoxy-2'-fluorouridine, and 2'-deoxy-2'-fluoroinosine. In some embodiments, the deoxyribose donor I is 2'-deoxy-2'-fluorouridine.

[0125] The pH value of the reaction system may be 5 to 10, preferably 6 to 9, more preferably 7 to 8. The reaction temperature may be 30 to 80°C, preferably 40 to 70°C.

[0126] The molar ratio of the deoxyribose donor to cytosine in the reaction system can be 1:(1-5), such as 1:(2-4).

[0127] The mass of the NDT mutant in the reaction system may be 10-80% of the mass of the deoxyribose donor, preferably 30-50%.

[0128] In some embodiments, the present invention provides a method for synthesizing 2'-deoxy-2'-fluorocytidine, comprising: using the N-deoxyribosyltransferase mutant described in any embodiment of the present invention to catalyze the reaction of cytosine with 2'-deoxy-2'-fluorouridine to generate 2'-deoxy-2'-fluorocytidine. In this method, a buffer solution can be used as a reaction solvent to carry out the reaction so that the reaction is carried out under certain pH conditions. In some embodiments, a phosphate buffer solution is used as a reaction solvent. The pH value of the reaction system can be 5 to 10, preferably 6 to 9, and more preferably 7 to 8. The reaction temperature can be 30 to 80°C; preferably, 40 to 70°C. The molar ratio of 2'-deoxy-2'-fluorouridine to cytosine can be 1:(2 to 4); preferably, the molar ratio is 1:(2.5 to 3.5). The mass of the N-deoxyribosyltransferase can be 10 to 80% of the mass of 2'-deoxy-2'-fluorouridine; preferably, 30 to 50%.

[0129] Amino acid sequence of wild-type N-deoxyribosyltransferase:

[0130] MNKKKTLYFGAGWFNEKQNKAYKEAMAALKENPTVDLENSYVPLENQYKGIRIDEHPEYLHNIEWASATYHNDLVGIKTSDVMLGVYLPEEEDVGLGMELGYALSQGKYILLVIPDEDYGKPINLMSWGVCDNAIKISELKDFDFNKPRYNFYDGAVY (SEQ ID NO: 1).

[0131] Advantages of the present invention include:

[0132] (1) The deoxyribosyltransferase mutant and preparation method of the present application are used to prepare 2'-deoxy-2'-fluorocytidine. Compared with the existing technology level, the design route is novel, green and environmentally friendly, and the reaction cost is reduced.

[0133] (2) The deoxyribosyltransferase mutant of the present application has substrate selectivity. With 2'-deoxy-2'-fluorouridine as the reaction substrate, the enzyme catalyzed reaction is carried out under the reaction conditions provided in the present application, and a high conversion rate is obtained.

[0134] (3) The deoxyribosyltransferase mutant of the present application can be prepared from 2'-deoxy-2'-fluoro-uridine and cytosine to obtain 2'-deoxy-2'-fluorocytidine through a one-step reaction. The reaction steps are short, the reaction conditions are mild, and the post-treatment only requires centrifugation and filtration. The operation is simple and easy to industrialize.

[0135] (4) The present invention discloses an evolutionary modified N-deoxyribosyltransferase and a method for synthesizing 2'-deoxy-2'-fluorocytidine, using 2'-fluoro-2'-deoxyribose donor and cytosine as raw materials, and adopting a one-pot method to obtain 2'-deoxy-2'-fluorocytidine. The synthetic process of the embodiment of the present invention is simple and safe to operate, low in cost, less polluting, and easy to realize industrialization.

[0136] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0137] Based on the attempts in the art, the above-mentioned preferred conditions can be combined arbitrarily to obtain the preferred embodiments of the present invention.

[0138] The reagents and raw materials used in the present invention are commercially available.

[0139] The experimental methods in the following examples without specifying specific conditions are generally carried out under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight. The experimental materials involved in the present invention are commercial reagents unless otherwise specified and can be obtained from commercial channels.

[0140] Example 1: Preparation of N-deoxyribosyltransferase NDT enzyme powder

[0141] 1. Construction and transformation of recombinant vector

[0142] Take the construction of the recombinant strain E. coli BL21 (DE3) pET-30a-LhNDT as an example.

[0143] Add 3-5 μL pET-30a-LhNDT (50 ng / μL) (commissioned by Jinweizhi Company to synthesize) to 50-100 μL E. coli BL21 (DE3) competent cells (purchased from Novezan Biotechnology Co., Ltd.), place on ice for 20 minutes, heat shock at 42°C for 90 seconds, quickly return to ice for 5 minutes, add 800 μL of resistance-free LB culture medium, incubate at 37°C, 200 rpm, and spread on LB agar plate medium containing kanamycin resistance. Incubate overnight at 37°C for 12 hours to obtain recombinant E. coli BL21 (DE3) containing pET-30a-LhNDT.

[0144] Among them, the Genbank sequence number of LhNDT is AY064167.1.

[0145] The formula of the non-resistant LB culture medium is as follows: yeast powder 5g / L, sodium chloride 10g / L, peptone 10g / L;

[0146] The formula of LB agar plate culture medium containing kanamycin resistance is as follows: yeast powder 5g / L, sodium chloride 10g / L, peptone 10g / L, agar powder 20g / L, kanamycin 50μg / mL.

[0147] 2. Construction of expression vector and induced expression

[0148] The recombinant E. coli BL21 (DE3) containing pET-30a-LhNDT obtained in 1 was directly spread on a solid LB plate containing 50 μg / mL kanamycin resistance, and cultured at 37°C for 12-14 hours to obtain a monoclonal colony. A single colony of E. coli BL21 (DE3) containing the LhNDT recombinant vector was picked from the kanamycin plate, inoculated into 1 mL of liquid LB medium containing 50 μg / mL kanamycin resistance, and cultured at 37°C for 12 hours. Then, the inoculation amount was 2% (v / v) and inoculated into a fresh 1L liquid LB medium containing 50 μg / mL kanamycin resistance, and cultured at 37°C until OD 600 When the pH value was about 0.6-0.8, IPTG (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.) was added to a final concentration of 1.0 mmol / L. Expression was induced at 200 rpm and 25°C for 20 h. The supernatant was removed by centrifugation (4°C, 4000 rpm, 30 min). The obtained bacterial sludge was washed with 30 ml of 0.9% NaCl solution and resuspended for later use.

[0149] 3. Preparation of freeze-dried enzyme powder

[0150] The bacterial solution obtained in 2 was washed twice with 50 mmol / L Tris-HCl, and then resuspended in 50 mmol / L Tris-HCl (pH 8.0) buffer, and the cells were ultrasonically disrupted in an ice bath (amplifier 6, power 500 W, on 2 s, off 5 s, 30 minutes). The ultrasonically disrupted sample was centrifuged at 12000 rpm and 4°C for 30 min, and the supernatant was freeze-dried in a -80°C freeze dryer for 24 h. The freeze-dried sample was ground to prepare freeze-dried enzyme powder.

[0151] Example 2: Reaction effects of different deoxyribosyltransferases

[0152] Add 15mg N-deoxyribose transferase (lyophilized enzyme powder) and 2mL phosphate buffer (pH8.0, 50mM) to a 4mL reaction bottle and incubate at 50℃ for 2h. After the incubation, add 0.2mmol (50mg) of 2'-deoxy-2'-fluorouridine and 0.6mmol (68mg) of cytosine to the reaction bottle, mix well, adjust the temperature to 50℃, and stir for 20 hours. After the reaction is completed, take 50μL of the reaction solution and add it to a 1.5mL centrifuge tube, then add 1ml of 50% methanol-water post-treatment solution to terminate the reaction, centrifuge the centrifuge tube at 10000rpm, and use HPLC to detect the supernatant after centrifugation. The detection conditions are: the chromatographic column is a WatersXbridge C18 chromatographic column; the mobile phase is: mobile phase A: 5% acetonitrile solution containing 10mM NH4OAc, mobile phase B: 95% acetonitrile solution; temperature 30℃; flow rate 1.3mL / min. The reaction results are shown in Table 1.

[0153] Table 1

[0154]

[0155] Comparison of experiments 1 to 5 shows that the reaction effects of different deoxyribosyltransferases are significantly different, among which NDT-X1 (its amino acid sequence is shown in SEQ ID NO: 1) has the best reaction effect.

[0156] Example 3: Reaction effects of different 2'-fluoro-deoxyribose donors

[0157] The same reaction operation as in Example 2 was performed, using different 2'-fluoro-deoxyribose donors (0.2 mmol of deoxyribose donor, 0.6 mmol of cytosine) and NDT freeze-dried enzyme powder (30% of the mass of the deoxyribose donor), NDT-X1 was selected. The reaction results are shown in Table 2.

[0158] Table 2

[0159] Test No. Ribose donor Conversion rate (%) 1 2'-Deoxy-2'-fluoroadenosine 10.9 2 2'-Deoxy-2'-fluoroguanosine 5.8 3 2'-Deoxy-2'-fluorothymidine 2.2 4 2'-Deoxy-2'-fluorouridine 14.3 5 2'-Deoxy-2'-fluoroinosine 9.7

[0160] Comparison of experiments 1-5 showed that NDT-X1 had a higher conversion rate using 2'-deoxy-2'-fluorouridine as the ribose donor.

[0161] Example 4: Reaction effects of different N-deoxyribosyltransferase mutants

[0162] The sequence of SEQ ID NO:1 (coding sequence as shown in SEQ ID NO:2) was used as the parent, and the directed evolution was transformed by roller PCR, iterative saturation mutation, and combination mutation strategies. The mutants were then transformed into E.coli BL21 (DE3) competent cells and evenly spread on LB agar plates containing 50 μg / mL kanamycin, and placed in a 37°C incubator for static culture for 18 hours. The mutants on the transformed plate were picked into a 96-well plate with a sterile toothpick and cultured overnight in a shaker at 37°C and 250 rpm. 50 μl of bacterial solution was drawn from the holes of the primary plate and inoculated into the corresponding holes of the secondary plate. After culturing at 37°C and 250 rpm for 2 to 3 hours, IPTG with a final concentration of 1 mM was added, and the mutants were cultured at 25°C for 20 hours to obtain the corresponding mutants for high-throughput screening. Combined with HPLC detection and rescreening, mutants with significantly improved activity and stability were identified for gene sequencing.

[0163] 2'-deoxy-2'-fluorouridine was used as the ribose donor (ribose donor 0.2 mmol, cytosine 0.6 mmol), and the reaction was carried out in the same manner as in Example 2. Different N-deoxyribosyltransferase mutants (the mass was 30% of the mass of the ribose donor) were used for the experiment, and the reaction results are shown in Table 3.

[0164] Table 3

[0165] Test No. Enzymes Mutation location Conversion rate (%) 1 NDT-X1 WT 14.3 2 NDT-E1 L60V 38.1 3 NDT-E2 A68F 23.9 4 NDT-E3 S67L 27.4 5 NDT-E4 A68Y 25.7 6 NDT-E5 S67T 38.7 7 NDT-E6 S67I 31.5 8 NDT-E7 S67W 20.0 9 NDT-E8 S67A 36.4 10 NDT-E9 A66E 11.8 11 NDT-E10 E55H 16.9 12 NDT-E11 K23S 5.9 13 NDT-E12 V82R 14.7 14 NDT-E13 H71R 45.2 15 NDT-E14 S127N 41.3 16 NDT-E15 D154R 28.9 17 NDT-E16 E91F 19.2 18 NDT-E17 D54S 21.3 19 NDT-E18 L140C 39.8 20 NDT-E19 H71M 44.7 21 NDT-E20 H71F 40.2 22 NDT-E21 H71L 43.1

[0166] Comparison of experiments 1-22 shows that the mutated N-deoxyribose transferase has a better reaction effect, among which NDT-E13 has the highest conversion rate of 45.2%.

[0167] In summary, the above embodiments are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. An N-deoxyribosyltransferase, which has a substitution mutation at any one, any two, any three, any four, any five or any six or more positions of the amino acid sequence of position 54, position 55, position 60, position 67, position 68, position 71, position 91, position 127, position 140 and position 154 compared to the amino acid sequence shown in SEQ ID NO:

1.

2. The N-deoxyribosyltransferase according to claim 1, characterized in that: The wild-type aspartic acid at position 54 is mutated to a polar uncharged amino acid, such as serine, threonine, cysteine, tyrosine, asparagine or glutamine; The wild-type glutamic acid at position 55 is mutated into a polar positively charged amino acid, such as lysine, arginine or histidine; The mutation at position 60 is that the wild-type leucine is mutated into a non-polar amino acid, such as alanine, valine, isoleucine, proline, phenylalanine, tryptophan, methionine or glycine; The mutation at position 67 is that the wild-type serine is mutated into a non-polar amino acid, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine or glycine, or is mutated into a polar uncharged amino acid, such as threonine, cysteine, tyrosine, asparagine or glutamine; The mutation at position 68 is that the wild-type alanine is mutated into a non-polar amino acid, such as valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine or glycine, or is mutated into a polar uncharged amino acid, such as serine, threonine, cysteine, tyrosine, asparagine or glutamine; The mutation at position 71 is that the wild-type histidine is mutated into a polar positively charged amino acid, such as lysine or arginine, or is mutated into a non-polar amino acid, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine or glycine; The wild-type glutamic acid at position 91 is mutated to a non-polar amino acid, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, methionine or glycine; The wild-type serine at position 127 is mutated to a polar uncharged amino acid, such as threonine, cysteine, tyrosine, asparagine or glutamine; The wild-type leucine at position 140 is mutated to a polar uncharged leucine, such as serine, threonine, cysteine, tyrosine, asparagine or glutamine; The wild-type aspartic acid at position 154 is mutated into a polar positively charged amino acid, such as lysine, arginine or histidine.

3. The N-deoxyribosyltransferase according to claim 2, characterized in that: The wild-type aspartic acid at position 54 is mutated to serine or threonine; The wild-type glutamic acid at position 55 is mutated to histidine; The wild-type leucine at position 60 is mutated to glycine, alanine, isoleucine or valine, preferably valine; The wild-type serine at position 67 is mutated to glycine, alanine, leucine, isoleucine, valine or tryptophan, or is mutated to threonine; The wild-type alanine at position 68 is mutated to phenylalanine or tyrosine; The wild-type histidine at position 71 is mutated to arginine, or is mutated to methionine, phenylalanine, leucine, isoleucine or valine; The wild-type glutamic acid at position 91 is mutated to phenylalanine; The wild-type serine at position 127 is mutated to asparagine or glutamine; The wild-type leucine at position 140 is mutated to cysteine; The wild-type aspartic acid at position 154 was mutated to arginine.

4. The N-deoxyribosyltransferase according to claim 1, characterized in that Compared to SEQ ID NO: 1, the mutation of the N-deoxyribosyltransferase is selected from: (1) D54S, (2) E55H, (3) L60V, (4) S67L, S67T, S67I, S67W or S67A, (5) A68F or A68Y, (6) H71R, H71M, H71F or H71L, (7) E91F, (8)S127N, (9) L140C, and (10)D154R.

5. The N-deoxyribosyltransferase according to claim 1, wherein Compared with SEQ ID NO: 1, the mutation of the N-deoxyribosyltransferase is selected from the group consisting of: L60V, S67T or S67I or S67A, H71R or H71M or H71F or H71L, S127N, and L140C.

6. A polynucleotide molecule, the polynucleotide sequence of which is selected from: (1) a polynucleotide sequence encoding the N-deoxyribosyltransferase according to any one of claims 1 to 5; and (2) A complementary sequence to the polynucleotide sequence described in (1).

7. A nucleic acid construct comprising the polynucleotide molecule according to claim 6; preferably, the nucleic acid construct is an expression cassette.

8. A vector comprising the polynucleotide molecule according to claim 6 or the nucleic acid construct according to claim 7; preferably, the vector is a cloning vector or an expression vector.

9. A host cell, which: (1) expressing the N-deoxyribosyltransferase according to any one of claims 1 to 5, and / or (2) containing the polynucleotide molecule according to claim 6, the nucleic acid construct according to claim 7 or the vector according to claim 8; preferably, the host cell is Escherichia coli.

10. An enzyme preparation comprising the N-deoxyribosyltransferase according to any one of claims 1 to 5; Preferably, the enzyme preparation further contains a buffer; preferably, the buffer is a phosphate buffer; preferably, the pH of the buffer is 5 to 10, preferably 6 to 9, more preferably 7 to 8; Preferably, the enzyme preparation is a powder, such as a lyophilized powder; Preferably, the enzyme preparation is the supernatant obtained by lysing the host cells expressing the N-deoxyribosyltransferase according to claim 9 or a concentrated solution of the supernatant.

11. Use of the N-deoxyribosyltransferase according to any one of claims 1 to 5 in catalyzing the transfer of 2'-deoxy-2'-fluororibose groups, or in preparing 2'-deoxy-2'-fluorocytidine, or in preparing an enzyme preparation for preparing 2'-deoxy-2'-fluorocytidine.

12. A method for preparing 2'-deoxy-2'-fluorocytidine, characterized in that: The method comprises: A step of using the N-deoxyribosyltransferase according to any one of claims 1 to 5 to catalyze the reaction of cytosine C with a deoxyribose donor I to generate 2'-deoxy-2'-fluorocytidine II; Wherein, in the deoxyribose donor I, R is a base, preferably selected from adenine, guanine, uracil, hypoxanthine and thymine; preferably, R is uracil, and the deoxyribose donor I is 2'-deoxy-2'-fluorouridine.

13. The preparation method according to claim 12, characterized in that: The method has one or more of the following features: The pH value of the reaction system of the reaction is 5 to 10, preferably 6 to 9, more preferably 7 to 8; The reaction temperature of the reaction is 30 to 80°C, preferably 40 to 70°C; The molar ratio of deoxyribose donor I to cytosine C in the reaction system is 1:(1-5), such as 1:(2-4); The mass of the N-deoxyribosyl transferase in the reaction system is 10 to 80% of the mass of the deoxyribose donor, preferably 30 to 50%.

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