ANALYTICAL BIOCHEMISTRY 68, 128-137 (1975)

Convenient Method for Preparation and Purification of Nicotinamide Mononucleotide Analogs ~ WERNER HENSEL, DAGMAR RAKOW, AND W O L F R A M CHRIST lnstitut far Neuropsychopharmakologie der Freien Universiti~t Berlin, Ulmenallee 30, D 1000 Berlin 19, Germany (FRG) Received January 28, 1975; accepted April 10, 1975 1. Nucleotide pyrophosphatase from Crotalus adamanteus venom cleaves the pyrophosphate linkage of N A D P +, thio-NADP +, 3-AcPyr-ADP +, selenoN A D P +, thio-NAD +, 3-AcPyr-AD + and 3-OHCPyr-AD +. 2. The nicotinamide mononucleotide (NMN) analogs can be obtained from reaction mixtures in good yield and high purity by chromatography on Whatman DE 52 cellulose. 3. The uv spectra and specific extinction coefficients of the N M N analogs were almost identical with those of the corresponding 1-methylpyridinium derivatives. 4. Thio-NMN, 3-AcPyr-MN and 3-OHCPyr-MN can be condensed with AMP to the corresponding N A D + analogs using dicyclohexylcarbodiimide.

In our laboratory we have been investigating the in vivo biosynthesis of abnormally structured NAD(P) ÷ analogs. It is well established that 6aminonicotinamide, 3-acetylpyridine and thionicotinamide are converted in vivo into their corresponding NAD(P) ÷ analogs (1-4). The exchange reaction catalyzed by the NAD(P)-glycohydrolase (EC 3.2.2.6) is thought to be responsible for the in vivo incorporation of these nicotinamide analogs. It is not known whether the de novo biosynthesis of NAD(P) + analogs occurs via the "nicotinate" or the "nicotinamide" pathway. We first investigated the substrate specifity of N A D pyrophosphorylase (EC 2.7.7.1) with respect to nicotinamide mononucleotide requirement. We also designed experiments to prepare the N A D ÷ analogs selenonicoI Abbreviations used: NMN, nicotinamide mononucleotide; thio-NAD(P) +, thionicotinamide adenine dinucleotide (phosphate); 3-AcPyr-AD(P) +, 3-acetylpyridine adenine dinucleotide (phosphate); seleno-NADP +, selenonicotinamide adenine dinucleotide phosphate; 3-OHCPyr-AD +, pyridine-3-carbaldehyde adenine dinucleotide; thio-NMN, thionicotinamide mononucleotide; 3-AcPyr-MN, 3-acetylpyridine mononucleotide; 3-OHCPyrMN, Pyridine-3-carbaldehyde mononucleotide. Enzymes: 1) Nucleotide pyrophosphatase (dinucleotide nucleotidohydrolase, EC 3.6.1.9); 2) N A D pyrophosphorylase ( A T P : N M N adenylyltransferase, EC 2.7.7.1); 3) Alcohol dehydrogenase (alcohol: N A D oxidoreductase, EC 1.1.1.1). 128 Copyright© 1975by AcademicPress, Inc. All rightsof reproductionin any formreserved.

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tinamide adenine dinucleotide and 5-fluoronicotinamide adenine dinucleotide either enzymatically (using N A D pyrophosphorylase) or chemically by condensation of the N M N analogs with ATP and AMP, respectively. Many nicotinamide mononucleotide derivatives have been prepared chemically as intermediary products in different syntheses of N A D ÷ and N A D + analogs (for example, 5-8). N M N analogs can be prepared by convenient procedures when stable nicotinamide compounds, such as 4-methylnicotinamide (9), were used as precursors. However, the preparative methods for chemical synthesis of N M N molecules become complicated when labile pyridine derivatives such as thionicotinamide and selenonicotinamide are used as starting material. It has been shown that thionicotinamide cannot be Nl-alkylated directly (10) and that selenonicotinamide is stable in aqueous solutions for only a few hours. Therefore, we tried to develop a simple enzymatic method to obtain N M N analogs with alterations to the pyridine moiety of the molecule. In the present study we investigated the possible hydrolysis of N A D P ÷ and NAD(P)-- analogs by nucleotide pyrophosphatase as well as the purification and identification of the N M N analogs formed.

MATERIALS AND METHODS Enzymes The following enzymes were obtained from Boehringer Mannheim GmbH (Germany): NAD-pyrophosphorylase (hog liver) and alcohol dehydrogenase (yeast and horse liver). Crotalus adamanteus venom as source of nucleotide pyrophosphatase was supplied by Sigma.

Coenzymes NMN, N A D +, N A D P +, thio-NAD +, thio-NADP +, 3-AcPyr-AD +, ATP, AMP (disodium salt and free acid) were purchased from Boehringer Mannheim GmbH. 3-AcPyr-ADP ÷ was a product of Schwarz/Mann, Orangeburg, NY, and 3-OHCPyr-AD + was supplied by Sigma. Selenonicotinamide and seleno-NADP ÷ were prepared according to the methods described (10,11). N,N-dicyclohexylcarbodiimide was supplied by E. Merck AG, Germany.

Preparation of N M N and N M N Analogs from NADP + and NAD(P) + Analogs Previously we investigated the conversion of N A D P ÷ analogs into N A D + analogs by enzymic dephosphorylation (12). We found that triethanolamine hydrochloride buffer (0.05 M, pH 7.4) was a suitable medium. This buffer was also convenient for nucleotide pyrophospha-

tase ;in c]ea,cing ~he pyropho~l~hate ~ldnk~ngeii~n N A D P + and N A D P + analogues. ~For spRtfing ;the N A I ) + analogues, +however, ~the reaction was performed ixa~aN~atlCO3~/Na~I303 b~ffer (0.~05 r~., p H 9.2). To ~isotate ~NMN t'rom ,the ;reat~/io~n ,rr/i~xt,ure we used a :modification of ~the procedure described b2¢ .Apps ,(13~. a9 ~he :inc:uba.t~on cmixtt~re for :otoava,ge of pyrophosphate linkage in ,NA,D~ analogs. ~ i s corrgisted ~of 1130 m g of N A D P + :(NADP + analog), ;50 mg Of A M 9 ('sodi,n~m ,s~lf)and 59 ~xng ofCr~ot~ltts "adamanteus venom in 15 m] ~of :rrietbanolmmine 'hydroelCtoride !buffer (0.05 M, p H 7.4, 0.2 mM ~nNO4). I~ncubatJorr: ~I~l25 tl~] ErIenmeyer flasks at 30°C :in a Dubnoff me~tab01ic s / ~ k e r u~der air. Ia~cubafion thnes: ,F o r N A D P+, 90 min; :for ~tliio;NA:O9+ ;and 3,Ac~y,r~ADP*, 120 rain; for seleno-NADP +, 65

&9 g'he ~tiba~i, on :mi~tttres for c~tea~age o f pyrophosphate linkage in NA.O + ;an~iogs. T~liis cons:ist.ed ~of IO0 mg of N A D + analog, 50 mg of A M P (sodium ~salt) and 30 mg of Crotalus adamanteus venom in 10 mi ,o~f N:a~H'COJNa~COz 'buffer (0.05 M, p:I-I 9.2, 0.1 mM ZnSO4). Incuba6on amder ~tbe :seine eondifior~s as ~given,above. 'Incubation times: For 3OHCPyr-A:D*,/1:00 mien and :fo~"3-AcPyr~AD + a n d thio-NAD +, 90 min. 'Chromato, graphy ~on D~AE-Ce~lu~o~e After ~incubation, the 'crude reaction mixtures (with enzyme protein) were applied ~to a~40 '× 2.5-cm column of Whatman D E 52 (approx 130 g 'wet weight). T h e :ceIlu'lose was equilibrated with 10 mM ammonium bicarbonate (,adjusted Wi~h CO2 ,gas to p H .7.4) and with 80 mM ammo~ ~bicarbormte ,(pH 7A'). The p H o f the column material was finally adjus,teM to p H 7..4 with "0.5 N I~CI. Elufion was performed with a linear gr~dier~t ~of 10-80 m g N H ~ H C O , '(by running 80 mM NH4HCOa ;into a 250-ml reservoir of the dilute buffer). Fractions of 2.5 ml were obtained ~ n g an L~KB fraction .collector.. T h e percent transmittance at 2"60 ~m~ was 'recorded ~usi~r~g:an U'Cicord absorptiometer. To achieve a :flow rate o f about ,1~00-115 m~t~hT.,:~h~e,column was run under pressure. T ypica'l ¢61m:ion:diagrams ,for a load ,0f:an incubation mixture with 50 mg ,of3zOHCPyr~A~I) + a~d :thio-NA:D+, ~res~ectively, are shown in Figs. 2a ai~d ~b. T h e nucleotide fractions were combined 'and lyophilized. The ammonium :salts o f 3-AcPy,r-MN ::and 3-ObICPyr-MN proved to be very 'unstable. Therefore in :later runs 'these zfracfions were adjusted to pH 4 with HC;I before ~lyophilizafion. The material obtained after freezeclr~ing was (dissolved in 2 ml Of 0.,001 N HCL This solution was exTracted ~ i c e with 5 ml ~of efhyl acetate. The organic layer was dis~ d o d . ~be m o n o n u d e o f i d e s were precipitated by acetone from the ,re~g ~aqueous phase, ,collected b y ,centfifugation, dried under vac-

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uum (room temperature) and stored a t - 2 0 ° C . The ammonium salts of NMN and thio-NMN were stable and could be converted into potassium salts. The yield of N M N , thio-NMN, 3-AcPyr-MN and 3-OHC-Pyr-MN was 30-70%, based on N A D + and N A D P + derivatives. It depended on the rate of hydrolysis of the pyrophosphate linkage of the starting material and on the rate of dephosphorylation of the N M N analog. Approximately 8-17% of total N M N compound formed was converted into the corresponding riboside by 5'-nucleotidase. In most cases, however, the rate of formation of riboside was below 10%. Synthesis of N A D + and N A D + Analogs from the Corresponding Mononucleotides The chemical synthesis of N A D ÷, thio-NAD +, 3-AcPyr-AD + and 3OHC-Pyr-AD + from the corresponding N M N derivatives and 5'-AMP (free acid) was performed essentially according to the procedure described by Jarman and Searle for synthesis of 4-methylnicotinamideAD + (9). The yield of N A D + and its analogs, based on the N M N compounds used, was 15-29%, The existence of synthesized N A D ÷ and N A D ÷ analogs was demonstrated by enzymic reduction using alcohol dehydrogenase and by thin layer chromatography using reference substances. Assay of N A D + Pyrophosphorylase We used the commercially available N A D pyrophosphorylase from hog liver. The assay was similar to that described in Bergmeyer (14) except that the reaction was terminated by the addition of 0.1 ml of HCIO4 (60%) and not by heating. Assay of N A D + and N A D + Analogs The assay was performed essentially according to that described in Bergmeyer (15). The overall reaction was followed at 340 nm (NADH), 398 nm (thio-NADH), 365 nm (3-AcPyr-ADH), 358 nm (3-OHCPyrADH) in a Zeiss PMQ II spectrophotometer. Assay of Phosphate and Ribose of the N M N analogs Ribose was determined according to a modified orcinol method (16,17). For determination of phosphate a modified Berenblum-Chain (18,19) procedure was used. Isobutanol and SnC12 were added to the phosphomolybdate complex and the absorbance was measured spectrophotometrically at 725 nm. Commercially available N M N was used as reference substance. The orcinol method did not give reproducible results when 3-OHCPyr-MN was analyzed.

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HENSEL, RAKOW AND CHRIST

TABLE 1 Rs VALUESOF NMN, NAD AND NADP ANALOGS R s value a

Compound

System (a)

System (b)

NMN Thio-NMN 3-AcPyr-MN 3-OHCPyr-MN NAD Thio-NAD 3-AcPyr-AD 3-OHCPyr-AD NADP Thio-NADP 3-AcPyr-ADP

0.42 0.46 0.52 0.50 0.43 0.41 0.42 0.41 0.32 0.32 0.33

0.78 0.81 0.82 0.81 0.58 0.46 0.58 0.50 0.27 0,24 0,35

Adenosine

0.77

--

The values represent the mean of three chromatograms. The compounds were located under a uv lamp,

Thin Layer Chromatography

To identify NAD + analogs and to check for possible impurities of N M N analogs the following tlc systems were used: a) Pre-coated cellulose plates (Merck) and isobutyric acid/NH4OH (25%)/water (66[1/33 by volume) as solvent; b) DEAL-cellulose (Serva, Heidelberg, Germany) plates (layer thickness about 250 /xm) and 0.02 N HCI as solvent. D E A L plates were pre-developed once in 0.02 N HCI and dried at room temperature before loading. For Rf values, see Table 1.

RESULTS AND DISCUSSION It is known that the pyrophosphate linkage in the N A D + molecule can be cleaved by nucleotide pyrophosphatase from potatoes (20-22) or from Crotalus adamanteus venom (13,23,24). Siegel et al. (24) reported the enzymatic hydrolysis of the pyrophosphate linkage in different N A D + analogs with alterations to the pyridine moiety. Honjo et al. (25) showed the splitting of some N A D + analogs with modifications of the AMP moiety of the molecule using pyrophosphatase from potatoes and from snake venom. In our study we used the nucleotide pyrophosphatase from Crotalus venom. We found that the pyrophosphate linkages of N A D P + and of the NAD(P) + analogs so far investigated are hydrolyzed due to the low substrate specificity of this nucleotide pyrophosphatase (Fig. 1). The resulting N M N analogs could be obtained in good yield and high purity by column chromatography on Whatman DE 52 cellu-

NICOTINAMIDE

MONONUCLEOTIDE

133

ANALOGS

80

70

60

40

i '° 30

2O

10

J 10

I 20

I 30

I 40

I 50

I 60

J 70

I 80

I 90

I 100

I 110

I 120

I 130

I 140

I 150

I 160

Time [minutes]

FIG. 1. T i m e course of nucleotide p y r o p h o s p h a t a s e activity of Crotalus adamanteus v e n o m with N A D P + and N A D P + analogs as substrates. Incubation mixtures consisted of 10 mg of N A D P + or N A D P + analog, 5 mg of 5 ' - A M P , 5 mg of Crotalus adamanteus v e n o m in 5 ml of triethanolamine hydrochloride buffer (0.05 M, p H 7.4, 0.2 mM ZnSO4). A t intervals indicated, aliquots of 0.1 ml were taken from the mixtures and c o e n z y m e concentration was determined enzyrnatically using glucose-6-phosphate d e h y d r o g e n a s e or isocitrate d e h y d r o g e n a s e (for 3 - A c P y r - A D P + reduction). 0 - - 0 , N A D P + ; A - - A , thioN A D P + E I - . - [ ~ , 3 - A c P y r - A D P + ; x - - - - x , s e l e n o - N A D P +.

lose. The elution profiles of the incubation mixtures with N A D + and N A D P + analogs were nearly identical. Usually three main peaks were obtained; the first represented the pyridinium riboside, the second adenosine and the third N M N analog. When the amount of Crotalus venom added to the incubation mixture was not sufficient to cleave the N A D + molecule in the time given, noncleaved N A D + analog appeared as a fourth band (see Figs. 2a and b). This was not observed with N A D P + analogs, because the N A D P + molecule is not eluted from the column material by 10-80 mM NHaHCO3 buffer. This separation procedure seems to have advantages compared with those previously reported, e.g., adsorption on charcoal (21) or electrophoresis (26), for the following reasons: Adsorption on charcoal is not very specific and has to be followed by subsequent chromatography on a Dowex resin; electrophoresis enables separation of very small amounts only. 5'-AMP was completely dephosphorylated to adenosine by 5'-nucleotidase present in the crude Crotalus venom. However, under the reaction conditions selected (e.g., buffer medium, pH value, concentration of covalent cations, addition of 5'-AMP), the N M N analogs formed were

134

H E N S E L , R A K O W A N D CHRIST

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Convenient method for preparation and purification of nicotinamide mononucleotide analogs.

ANALYTICAL BIOCHEMISTRY 68, 128-137 (1975) Convenient Method for Preparation and Purification of Nicotinamide Mononucleotide Analogs ~ WERNER HENSEL,...
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