HK1166645B - Enzymatic synthesis of carba-nad - Google Patents

Description

Enzymatic synthesis of CARBA-NAD

Technical Field

The present invention relates to the enzymatic synthesis of stable analogues of nicotinamide adenine dinucleotide NAD/NADH and nicotinamide adenine dinucleotide phosphate NADP/NADPH, so-called "carba-NAD", such as analogues of NAD/NADH or NADP/NADPH comprising, respectively, a carbocyclic sugar instead of ribose.

Background

Measurement systems for biochemical analysis are an important component of clinically relevant analytical methods. It relates firstly to the measurement of analytes, for example metabolites or substrates, which are determined directly or indirectly by means of enzymes. The target analyte is often converted by means of an enzyme-coenzyme complex and subsequently quantified via this enzymatic reaction. In this process, the analyte to be determined is brought into contact with a suitable enzyme and a coenzyme under suitable reaction conditions, whereby the coenzyme is changed, for example oxidized or reduced by an enzymatic reaction. The process can be determined electrochemically or photometrically, directly or with the aid of an intermediary. Typically, a standard curve provides a direct correlation between the measurement and the target analyte concentration from which the analyte concentration can be determined.

Coenzymes are organic molecules that bind covalently or non-covalently to enzymes and are altered by the conversion of an analyte. Prominent examples of coenzymes are Nicotinamide Adenine Dinucleotide (NAD) and Nicotinamide Adenine Dinucleotide Phosphate (NADP), which can form NADH and NADPH, respectively, by reduction.

As described in US 2008/0213809, the drawbacks of conventional measurement systems, such as limited shelf life, special requirements on storage conditions, such as cooling or dry storage, to achieve an improved shelf life, can be at least largely overcome by the stable nicotinamide adenine dinucleotide (NAD/NADH) and nicotinamide adenine dinucleotide phosphate (NADP/NADPH) derivatives disclosed herein. These stable nad (p) H analogues are suitable for eliminating false results due to inappropriate, inadvertent, poor storage, which is particularly important in tests performed by the end user himself, such as glucose self-monitoring, for example.

As described in US 2008/0213809, the chemical synthesis of carba-NAD is extremely challenging, requires at least 8 synthetic steps, has a rather low yield and is therefore rather expensive. FIG. 1 depicts the chemical route for the synthesis of carba-NAD. Alternative synthetic routes are urgently needed.

It is therefore an object of the present invention to provide carba-NAD in a less cumbersome manner, in high yields and at an attractive low cost.

It has now been unexpectedly found that carba-NAD can be provided in a cost effective and convenient manner using enzymes instead of traditional chemical means.

Disclosure of Invention

The present invention relates to a process for the enzymatic synthesis of carba-NAD or analogues thereof, comprising the following steps: a) phosphorylating 3-carbamoyl-1- (2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -pyridinium-methanesulphonate or an analogue thereof by means of NRK enzyme, b) adenylating the phosphorylated product of step (a) with adenosine or a structurally related compound by means of NMN-AT enzyme, thereby obtaining carba-NAD or an analogue thereof.

Detailed Description

In a preferred embodiment, the invention relates to a method for the synthesis of carba-NAD or analogues thereof, comprising the steps of:

a) phosphorylating a compound of formula I with the aid of Nicotinamide Ribokinase (NRK) enzyme,

wherein R1 is OH or NH₂O-methyl or N-dimethyl, methyl, Y-is a counter ion, X is O or S,

b) adenylating the phosphorylated product of step (a) with a compound of formula II by means of NMN-AT enzyme,

wherein R2 is NH₂An OH or NH alkyl group,

wherein R3 is H, OH or NH₂，

Whereby carba-NAD of formula III or an analogue thereof is obtained,

formula III

Wherein R1, R2, R3, Y-and X are as defined above.

The above process is also exemplified by the reaction scheme shown in figure 2.

The term "carba-" is used to indicate the presence of 2, 3-dihydroxycyclopentane in place of the ribosyl sugar residue. In other words, the carba-analogue of oxidized nicotinamide adenine dinucleotide (NAD '), for example, is the same compound as (NAD') except that the 2, 3-dihydroxycyclopentane ring replaces the D-ribonucleotide ring of the nicotinamide riboside moiety (Slama, j.t. and Simmons, a.m., Biochemistry 27(1988) 1831).

Enzymes are known to be highly specific catalysts that allow reactions to occur under more or less physiological conditions, requiring harsh conditions or sometimes even little completion when the enzyme is not present. To enable such specific reactions, enzymes tend to be very specific in terms of both substrate specificity and the reactions catalyzed as a result of evolution through generations under selective pressure. It has now surprisingly been found that nicotinamide ribokinase accepts pyridinium compounds of formula I containing a 2, 3-dihydroxycyclopentane ring instead of a ribose residue as substrate and can phosphorylate these compounds.

According to the international nomenclature for enzymes, Nicotinamide Ribokinase (NRK) belongs to the EC 2.7.1.22 class (ATP: N-ribosylnicotinamide 5' -phosphotransferase). For phosphorylating compounds of formula I, it is preferred to use an enzyme selected from the group of EC 2.7.1.22 in the method according to the invention. Preferred NRKs are those known from Saccharomyces cerevisiae (Saccharomyces cerevisiae), Pseudomonas aeruginosa (Pseudomonas aeruginosa), Streptococcus sanguinius (Streptococcus sanguinius) and human (Homo sapiens). It is also preferred that the NRK used in the method of the invention is one known from Streptococcus sanguis and human. In a preferred embodiment, NRK1 from human is known to be used for carrying out the first step in the method of the invention.

As shown in formula I, not only R1 is NH₂Also other compounds such as carba-nicotinamide analogues as defined and outlined by the replacement of R1 appear to be suitable substrates for certain NRK enzymes. One skilled in the art of understanding the present disclosure will readily investigate the ability of compounds of formula I and related compounds to be efficiently phosphorylated by NRK enzymes. Preferably, the pyridinium compound as defined in formula I is used for enzymatic phosphorylation in the process according to the invention. Nicotinamide analogs are compounds as defined in formula I wherein R1 is other than NH₂. Superior foodR1 of formula I is selected from the group consisting of OH, NH₂And O-methyl. In one preferred embodiment, R1 is OH, and in another preferred embodiment, R1 is NH₂. The alkyl group in R1 or R2 is preferably a C1 to C6 straight or branched chain alkyl group, preferably a straight chain alkyl group.

Residue X in formula I may be O or S. In a preferred embodiment, X in formula I is O.

Counterion Y^-Preferably selected from the group consisting of methylsulfonate (methylsulfonate), Cl^-、PF6^-、BF4^-And ClO4^-A group of which. It is also preferred that the counterion is BF4^-Or a methyl sulfonate.

Surprisingly, nicotinamide nucleotide adenyl transferase (NMN-AT) can use the phosphorylated carba-nicotinamide obtained as described above as receptor molecule and is able to adenylate these compounds. In the second step of the enzymatic synthesis of carba-NAD or an analogue thereof, nicotinamide mononucleotide adenyl transferase is thus used to transfer an adenosine residue or an analogue thereof to phosphorylated carba-nicotinamide or an analogue thereof, thereby forming carba-NAD or an analogue thereof.

According to the international nomenclature of enzymes, nicotinamide nucleotide adenyl transferase (NMN-AT) belongs to the EC 2.7.7.1 class (ATP: nicotinamide-nucleotide adenyl transferase). In order to use the compounds of formula II to adenylate the phosphorylated compounds of formula I, it is preferred to use an enzyme selected from the group consisting of EC 2.7.7.1 in the methods of the present invention. Preferred NMN-ATs are those known from Bacillus subtilis, Escherichia coli, Methanococcus jannaschii, Sulfolobus solfataricus, Saccharomyces cerevisiae and human. In a preferred embodiment, NMN-AT known from human origin, e.g.expressed in E.coli or Bacillus subtilis, is used for carrying out the second enzymatic reaction in the method according to the invention. Although adenine groups and analogs thereof can be used as substrates for NMN-AT in the disclosed methods, the terms adenylation, adenylation or adenylation are used for convenience for all of the above common substances (substances unissous).

It was also surprisingly observed that the two steps in the enzymatic synthesis of carba NAD or analogues thereof can be carried out in the form of a single reaction mixture. In a further preferred embodiment, the present invention relates to a process for the enzymatic synthesis of carba-NAD or analogues thereof, comprising the following steps: a) phosphorylating 3-carbamoyl-1- (2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -pyridinium-sulfonate or an analogue thereof by means of NRK enzyme, b) adenylating the phosphorylated product of step (a) with adenosine or a structurally related compound by means of NMN-AT enzyme, thereby obtaining carba-NAD or an analogue thereof, wherein both enzymatic reactions are performed as a single reaction mixture.

It has surprisingly been found that, based on the process disclosed in the present invention, the biologically relevant enantiomer of cdna based on the 1R, 2S, 3R, 4R enantiomer of carbamoyl-1- (2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -pyridinium is obtained in pure form and in high yield. In a preferred embodiment, the disclosed method is used to synthesize a cdna comprising the 1R, 2S, 3R, 4R enantiomer of carbamoyl-1- (2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -pyridinium.

As shown in formula II, not only adenosine triphosphate, but also other structurally related compounds such as those characterized and summarized by the definitions of R2 and R3 in formula II, respectively, compounds of formula II having various possible combinations of each of R2 and R3 also represent suitable substrates for certain NMN-AT enzymes. The ability of compounds of formula II and structurally related compounds to be effectively adenylated by NMN-AT enzyme will be readily investigated by those skilled in the art who are knowledgeable of the present disclosure. Compounds structurally related to adenosine are as defined for formula II wherein R2 is not NH₂And R3 is not H. Purine compounds as defined via the respective groups R2 and R3 in formula II are preferably used for the enzymatic adenylation of phosphorylated carba-nicotinamide or analogues thereof.

In a further preferred embodiment, the present invention relates to the use of a compound, which is related to the compound of formula II and is selected from the group consisting of the following triphosphates: nebularine, synbiotics, arinomycin, 7-deazaadenosine, 7-deazaguanosine, 7-deazainosine, 7-deazaxanthosine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-azaadenosine, 7-deaza-8-azaguanosine, 7-deaza-8-azainosine, 7-deaza-8-azaxanthosine, 7-deaza-8-aza-2, 6-diaminopurine, 8-azaadenosine, 8-azaguanosine, 8-azainosine and 8-xanthosine, and 8-aza-2, 6-diaminopurine. These compounds can also be used to produce the corresponding dinucleotides comprising the carba analogues of nicotinamide in the process of the invention.

Preferably R2 of formula II is selected from the group consisting of NH₂Or OH. In one preferred embodiment R2 is OH, and in another preferred embodiment R2 is NH₂。

Preferably R3 of formula II is selected from the group consisting of H or OH. In a preferred embodiment R3 is H.

In a preferred embodiment, the process of the invention is carried out with compounds given in formulae I, II and III, wherein R1 is NH₂R2 is NH₂R3 is H and X is O.

It is obvious to the skilled person that carba-NAD or analogues thereof do not function exactly the same as the various different enzymes that require NAD as a coenzyme or cofactor. However, the skilled person is able to select the most suitable analogue from these presently unforeseeable options.

The following examples and figures are provided to aid the understanding of the present invention and the precise scope of protection set forth in the appended claims. It is to be understood that variations may be made in the procedures set forth without departing from the spirit of the invention.

Drawings

FIG. 1: FIG. 1 schematically illustrates a standard route for the chemical synthesis of carba-NAD (cNAD). As shown by the percentages given, the overall yield of the process is rather low.

FIG. 2: FIG. 2 schematically illustrates the two enzymatic reaction steps used in the synthesis of carba-NAD disclosed in the present invention.

Example 1:

synthesis of 5-dimethylamino-4-methoxycarbonyl-penta-2, 4-dienylidene-dimethyl-ammonium tetrafluoroborate

Example 1.1: synthesis of methyl- (2E) -3- (3-dimethylamino) prop-2-enoate

To 700ml of a solution of methyl propiolate (68.0ml, 0.764mol) in dry Tetrahydrofuran (THF) was added a solution of 2M N, N-dimethylamine in the same solvent (392ml, 0.783mol) over 1 hour at room temperature. After removal of the solvent, the residue was dried in an evaporator for 1 hour (37 ℃ C., 10-20 mbar) to give a pale yellow solid. The crushed solid was washed with n-hexane to yield 93.0g (94%) of methyl- (2E) -3- (3-dimethylamino) prop-2-enoate, which was purified by TLC and 1H NMR.

Example 1.2: synthesis of pyridinium tetrafluoroborate

Tetrafluoroboric acid (250ml, 2.00mol) was added to cold (0 ℃ C.) pyridine (157.7ml, 1.95mol) over 25 minutes to give a colorless precipitate. After the acid was completely added, the mixture was further stirred at the same temperature for 30 minutes. The reaction mixture was then filtered. The residue was washed twice with cold ethanol and dried under high vacuum for 12h, yielding 201.9g (60%) of pyridinium tetrafluoroborate in the form of colorless crystals.

Example 1.3: synthesis of 5-dimethylamino-4-methoxycarbonyl-penta-2, 4-dienylidene-dimethyl-ammonium tetrafluoroborate

Pyridinium tetrafluoroborate (283.7g, 1.70mol) was added to 442.5ml of acetic anhydride/acetic acid (2: 1) solution of methyl- (2E) -3- (3-dimethylamino) prop-2-enoate. The resulting suspension was cooled to 0 ℃ and 3-dimethylaminoacrolein (169.9ml, 1.70mol) was added slowly (3h) with vigorous stirring and ice-bath cooling to yield a yellow-brown precipitate. After further stirring at room temperature for 2h, the reaction mixture was filtered. The residual solid was washed several times with diethyl ether and dried under reduced pressure. Recrystallization from isopropanol/ethanol (2: 1) gave 326.7g (65%) of pentamethinium salt (pentamethinium salt) in the form of yellow crystals. .

Example 2:

synthesis of 2, 3-dihydroxy-4-hydroxymethyl-1-aminocyclopentane

A1M solution of KOH in EtOH (54.5ml, 54.5mmol) was added to a 540ml solution of hydrogen chloride (10.0g, 54.5mmol) in EtOH at 0 deg.C. After stirring at room temperature for 15min, the colorless precipitate formed was removed by filtration. The filtrate was concentrated under reduced pressure. The residual oil was dried in an evaporator (1h, 40 ℃) yielding 9.01g (112%) of amino carba ribose (carbaribose) as a pale yellow oil. The resulting product was used in the subsequent step without further purification.

This procedure was used to synthesize (1R, 2S, 3R, 4R) -2, 3-dihydroxy-4-hydroxymethyl-1-aminocyclopentane and its enantiomers.

Example 3:

synthesis of 1- (2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -3-methoxycarbonyl-pyridinium-methanesulfonate

Vinamidinium salt (298.1g, 1.00mol) was dissolved in 1500ml of DMF solution, and 1 equivalent of methanesulfonic acid (65.02ml, 1.00mol) was added. The mixture was added dropwise continuously and very slowly (over 5 h) to a refluxing solution (90 ℃ C.) of 3-amino-5-hydroxymethyl-cyclopentane-1, 2-diol (165.3g, 0.90mol) and 3-amino-5-hydroxymethyl-cyclopentane-1, 2-diol (25.8g, 0.15mol) in 1250ml MeOH. After the addition of the vinamidite base salt solution was complete, the reaction mixture was cooled to room temperature and 0.15 equivalents of methanesulfonic acid were added. The mixture was stirred at the same temperature for 12 h. After removal of the solvent under reduced pressure, a red-brown oil was obtained which was further dried for 3h (45 ℃, 4 mbar). And (3) output: 693.0g (191%, containing salt and bulk solvent).

This procedure was used to synthesize 3-methoxycarbonyl-1- ((1R, 2S, 3R, 4R) -2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -pyridinium salt and its enantiomer.

Example 4:

3-carbamoyl-1- (2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -pyridinium methanesulfonate

The crude 1- (2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -3-methoxycarbonyl-pyridinium-methanesulfonate starting material from example 3 was rapidly converted to the corresponding acid amide without further purification.

Crude 1- (2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -3-methoxycarbonyl-pyridinium methanesulfonate (118.3g, 173.7mmol) was dissolved in 100.0ml methanol. After addition of methanolic ammonia (7M, 350.0ml, 2.45mol), the reaction mixture was stirred for 2.5 h. After removal of the solvent under reduced pressure, a red-brown oil was obtained which was further dried for 3h (40 ℃, 10 mbar). This crude product is pre-purified with activated carbon and can be used directly, for example, in the chemical synthesis of cdna (WO2007/012494) or in the enzymatic synthesis of cdna as described herein below.

Other compounds suitable for use in the methods of the invention, for example, compounds defined with reference to formula I, may be synthesized in a manner analogous to the procedures described in examples 1 to 4 above herein.

This procedure was used to synthesize 3-carbamoyl-1- (1R, 2S, 3R, 4R) -2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -pyridinium salt and its enantiomer.

Example 5:

enzymatic phosphorylation of several compounds of formula I using nicotinamide ribokinase

(recombinant NRK1 from human (SwissProt ID: Q9NWW6) or NRK (nadR) from Streptococcus sanguis (S. sanguinis) (SwissProt ID: A3CQV5) heterologously expressed in E.coli).

The general process comprises the following steps:

creatine phosphate (14.5mg) and creatine kinase (0.1mg) were dissolved in TRIS buffer (pH 7.5, 15mM MgCl)₂960. mu.l) and ATP (100mM/1 aqueous solution, 40. mu.l). Then a nucleoside solution (compound 14 or an analog given above) (100mg/ml aqueous solution, 100. mu.l),ribose kinase (0.7U/ml, 230. mu.l) was then added. The reaction mixture was incubated at 37 ℃ for 16 h. After a brief heating at 80 ℃, the mixture was filtered and studied by High Performance Liquid Chromatography (HPLC).

In all three cases (compounds 14, 12 or 17), complete consumption of the nucleoside and formation of new compounds (corresponding phosphorylation products as given above for compounds 15, 16 or 18, respectively) can be detected by HPLC.

The correct mass of the desired phosphorylated product was found via LC/MS: (MS: ESI: M)^-330.75 (compound 15), 345.74 (compound 16), 358.79 (compound 18)).

Compound 15 was purified by chromatography on cation exchange resin Dowex and elution with water.

Example 6:

enzymatic transformation of Carba-nicotinamide and analogues thereof,

using NMN-AT separately

15：R＝NH₂ [331.24] [551.15] 20：R＝NH₂ [660.46]

16：R＝OMe [346.26] 19 21：R＝OMe [675.47]

18：R＝NMe₂[359.30] 22：R＝NMe₂[688.51]

From substances 15, 16 and 18 (from example 5), respectively

Crude material enzymatically phosphorylated) ca. 10.0.0 mg

Substance 19 (adenosine triphosphate, disodium salt) 22.6mg

NMN-AT：，(32U/ml) 4.8μl(0.153U)

(recombinant nicotinamide mononucleotide-adenyltransferase from human (NMN-AT) (SwissProt ID: Q9HAN 9.) alternatively, NMN-AT from E.coli (SwissProt ID: P0A752) or NMN-AT from Bacillus subtilis (SwissProt ID: P54455) can be used for heterologous expression in E.coli.)

Process flow

ATP disodium salt (22.6mg) and nicotinamide mononucleotide adenyl transferase (NMN-AT, 4.8. mu.l, 0.153U) were added to the filtrate containing mononucleotides (compound 15 or analogs, e.g. compounds 16 and 18) obtained from the enzymatic phosphorylation. The reaction mixture was incubated at 37 ℃ for 18 h. After a brief heating at 80 ℃, the mixture was filtered and studied by HPLC and LC/MS.

In all three experiments, complete consumption of the mononucleotide (compound 15, 16 or 18) and formation of new compound was detected by HPLC.

The correct mass of Compound 20 was found (MS: ESI: M)^-＝659.77)。

Example 7:

one-step process for the conversion of 3-carbamoyl-1- ((1R, 2S, 3R, 4R) -2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -pyridinium salt to carba-nicotinamide

1g (2.16mmol) of 3-carbamoyl-1- ((1R, 2S, 3R, 4R) -2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -pyridinium; chloride, 0.242g (0.4mmol) ATP disodium salt, 300mg MgCl₂×6H₂O (1.45mmol)16U ribokinase, 1.45g (4.43mmol) phosphocreatine and 4.27kU creatine kinase were dissolved in 25ml sterile water. The mixture was incubated overnight at 35 ℃. 2.42g (4mmol) of ATP disodium salt, 440mg of MgCl were then added₂×6H₂O (2.16mmol) and 32U NMNAT. Subjecting the mixture to a temperature of 35 deg.CIncubate overnight. Then it was heated to 90 ℃ for 5min, cooled and filtered. Purification was performed using ion exchange chromatography as described in WO 2007/012494.

Example 8:

conversion of 3-carbamoyl-1- ((1R, 2S, 3R, 4R) -2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -pyridinium salt to carba-nicotinamide in the presence of the enantiomer 3-carbamoyl-1- ((1S, 2R, 3S, 4S) -2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -pyridinium salt

1g (2.16mmol) of a mixture of 3-carbamoyl-1- ((1R, 2S, 3R, 4R) -2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -pyridinium; a 1: 1 mixture of chloride and 3-carbamoyl-1- ((1S, 2R, 3S, 4S) -2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -pyridinium salt; chloride 0.242g (0.4mmol), ATP disodium salt, 300mg MgCl₂×6H₂O (1.45mmol)16U ribokinase, 1.45g (4.43mmol) phosphocreatine and 4.27kU creatine kinase were dissolved in 25ml sterile water. The mixture was incubated overnight at 35 ℃. The reaction was monitored by reverse phase HPLC analysis (ODS Hypersil, 5 μ M, 250X 4, 6mm Thermo Scientific, Part-Nr.: 30105-. At 2.96 min, corresponding to 3-carbamoyl-1- ((1R, 2S, 3R, 4R) -2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -pyridinium; the peaks for the chloride and (1S, 2R, 3S, 4S) enantiomers disappeared, and at 3.45 min, a new peak corresponding to the phosphorylated product appeared.

2.42g (4mmol) of ATP disodium salt, 440mg of MgCl are subsequently added₂×6H₂O (2.16mmol) and 32U NMN-AT. The mixture was incubated overnight at 35 ℃. Thereafter, the mixture was heated to 90 ℃ for 5min, cooled and filtered. Reverse phase HPLC analysis showed the peak at 7.92 min to correspond to cdna. At 3.45 min, the peak corresponding to the phosphorylated (1S, 2R, 3S, 4S) enantiomer remained. Addition of alkaline phosphatase, 7 th.The 92 min peak was unaffected, while the peak for the 3.45 min phosphorylated (1S, 2R, 3S, 4S) enantiomer disappeared, and the peak corresponding to 3-carbamoyl-1- ((1S, 2R, 3S, 4S) -2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -pyridinium salt appeared at 2.96 min. Thus, cdna (based on the 1R, 2S, 3R, 4R enantiomer) is unaffected, while the remaining phosphorylated (1S, 2R, 3S, 4S) enantiomer is dephosphorylated by alkaline phosphatase.

As a control, the use of 3-carbamoyl-1- ((1S, 2R, 3S, 4S) -2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -pyridinium alone was performed; same test for chloride and monitoring by HPLC. Upon addition of ribokinase, a peak (corresponding to the phosphorylated enantiomer) was formed AT 3.45 min, but no peak with a retention time of 7.92 min was found in the HPLC chromatogram after NMN-AT addition.

Thus, synthesis of cdna using a mixture of enantiomers of 2, 3-dihydroxy-4-hydroxymethyl-1-aminocyclopentane consisting of the (1R, 2S, 3R, 4R and 1S, 2R, 3S, 4S) enantiomers can be initiated and the biologically relevant cdna obtained separately by the method disclosed herein.

Then 2.42g (4mmol) ATP disodium salt, 440mg MgCl were added₂×6H₂O (2.16mmol) and 32U NMN-AT. The mixture was incubated overnight at 35 ℃. Thereafter, the mixture was heated to 90 ℃ for 5min, cooled and filtered. HPLC analysis showed a peak corresponding to cdna at 7.92 min. At 3.45 min, the peak corresponding to the phosphorylated (1S, 2R, 3S, 4S) enantiomer remained. Upon addition of alkaline phosphatase, the peak at 7.92 min was unaffected, while the peak at 3.45 min for the phosphorylated (1S, 2R, 3S, 4S) enantiomer disappeared, and a peak at 2.96 min appeared, corresponding to the 3-carbamoyl-1- ((1S, 2R, 3S, 4S) -2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -pyridinium salt. Thus, cdna (based on the 1R, 2S, 3R, 4R enantiomer) was unaffected, while the remaining phosphorylated (1S, 2R, 3S, 4S) enantiomer was dephosphorylated by alkaline phosphatase.

As a control, the use of 3-carbamoyl-1- ((1S, 2R, 3S, 4S) -2, 3-dihydroxy-4-hydroxymethyl-cyclopentyl) -pyridinium alone was performed; same experiment for chloride and monitoring by HPLC. When ribokinase was added, a peak (corresponding to the phosphorylated enantiomer) was formed AT 3.45 minutes, but no peak with a retention time of 7.92 minutes was found in the HPLC chromatogram after NMN-AT addition.

Thus, the synthesis of cdna can be initiated using a mixture of enantiomers of 2, 3-dihydroxy-4-hydroxymethyl-1-aminocyclopentane consisting of the (1R, 2S, 3R, 4R and 1S, 2R, 3S, 4S) enantiomers and the cdna based only on the biologically relevant 1R, 2S, 3R, 4R enantiomer can be obtained by the method disclosed in the present invention.

Example 9:

enzymatic conversion of carba-nicotinamide mononucleotide (substance 15) using NMN-AT and N6 hexylamino ATP

The general process comprises the following steps:

N6-Hexylamino ATP disodium salt Jena Bioscience (0.33mg) and nicotinamide mononucleotide adenyl transferase (NMN-AT, 4.8. mu.l, 0.153U) were added to a solution of 1mg of Compound 15. The reaction mixture was incubated at 37 ℃ for 18 h. After a brief heating at 80 ℃, the mixture was filtered and studied by HPLC and LC/MS.

Carba-NMN (compound 15) was completely consumed and new compound (corresponding adenosine derivative) was detected by HPLC.

The correct mass was found (MS: ESI: M)^-＝759.77)。

Claims (8)

1. A method for the synthesis of carba-NAD, said method comprising the following steps

   a) Phosphorylation of compounds of formula I by nicotinamide ribokinase

   Wherein R1 is OH or NH₂O-methyl or N-dimethyl, methyl, Y^-Is a counterion and X is O or S,

   b) adenylating the phosphorylated product of step a) with a compound of formula II by means of nicotinamide mononucleotide adenyl transferase

   Wherein R2 is NH₂An OH or NH alkyl group,

   wherein R3 is H, OH or NH₂，

   Whereby carba-NAD of the formula III is obtained

   Wherein, R1, R2, R3 and Y^-And X is as defined above.
2. 2. The method of claim 1, wherein said nicotinamide ribokinase is selected from the group consisting of known nicotinamide ribokinases from Saccharomyces cerevisiae, Pseudomonas aeruginosa, Streptococcus sanguis, and human.
3. 3. The method of claim 1 or 2, wherein said nicotinamide mononucleotide adenyl transferase is selected from the group consisting of known nicotinamide mononucleotide adenyl transferases from bacillus subtilis, escherichia coli, methanococcus jannaschii, sulfolobus solfataricus, saccharomyces cerevisiae, and human.
4. 4. The method according to any one of claims 1 to 3, wherein R1 in the compound of formula I is selected from the group consisting of OH, NH₂And O-methyl.
5. 5. The method of any one of claims 1 to 3, wherein R2 in the compound of formula II is NH₂Or OH.
6. 6. The method of any one of claims 1 to 3, wherein R3 in the compound of formula II is H or OH.
7. 7. The method according to any one of claims 1 to 3, wherein X in the compound of formula I is O.
8. 8. The method of any of the above claims, wherein R1 is NH₂R2 is NH₂R3 is H, and X is O.