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Biocatalysir, 1987, Vol. 1, pp. 173-186 Photocopying permitted by license only
0 1987 Hanvood Academic Publishers GmbH Printed in the United Kingdom
A NEW SYNTHESIS OF COENZYMICALLY
NAD AND NADP DERIVATIVES ACTIVE WATER-SOLUBLE MACROMOLECULAR
ANDREAS F. BUCKMANN GBF, Gesellschaft fur Biotechnologische Forschung mbH., Mascheroder Weg 1,
0-3300 Braunschweig, FRG
(Received I 1 July 1987)
Based on an unexpected transformation of N(1)-(2-aminoethyl)-NAD(P) to N6-(2-aminoethy1)- NAD(P) under mild aqueous conditions (fH 6.0-6.5, 50°C) synthesis of uniform macromolecular derivatives of N6-alkylated NAD and N -alkylated NADP was possible, with, in most cases, acceptable overall yields (6-37%). The usual steps of (a) the chemical reduction with Na,S,O,, (b) the Dimroth rearrangement under harsh alkaline conditions and (c) the enzymatic or chemical oxidation were omitted. This represents a significant simplification of the procedure. A common procedure for the synthesis of macromolecular N6-(2-aminoethyl)-NAD(P) derivatives was pursued, coupling N6-(2-aminoethyl)-NAD(P) to several water-soluble copolymers containing maleic acid anhydride. PEG (M, = 20 OOO)-N6-(2-aminoeth I)-NAD, polyvinylpyrrolidone (M, = 160 OOO)-N6-(2- aminoethyl NAD and dextran (M, = 70 000)-N l -(2-aminoethyl)-NAD were synthesized by covalently binding N 2- -(2-aminoethyl)-NAD ’
N t? -(2-aminoethyl)-NAD(P) derivatives was satisfactory (90-95%).
to the corresponding carboxylated polymers by the carbodiimide method. PEG (M, = 4000 and 20 000)-p-(2-aminoethyl)-NADP was efficiently synthesized by covalent attachment of N6-(2-aminoethyl)-NADP to N-hydroxy-succinimide activated carboxylated PEG (M, = 4000 and 20 OOO), avoiding the carbodiimide method, which would lead simultaneously to 2’,3’-cyclic NADP derivatives. Except for the macromolecular cofactor derivatives based on co olymers containing maleic acid anhydride, the total enzymatic reducability of the macromolecular
KEY WORDS N(1)-(2-aminoethyl)-NAD(P) to N6-(2-aminoethyl)-NAD(P), fast Dimroth rearrange- ment, synthesis of macromolecular uniform NAD(P) derivatives.
INTRODUCTION
Water-soluble macromolecular NAD(H) and NADP(H) derivatives have been synthesized and applied in continuously operating NAD(H) and NADP(H) dependent enzyme systems with simultaneous coenzyme regeneration (Wichmann et al., 1981; Okuda, Urabe and Okada, 1985, Wandrey and Bossow, 1986) or in automated analysis (Sakaguchi et al., 1981).
A common strategy for the synthesis of well-defined macromolecular NAD(H) or NADP(H) derivatives has been as follows: alkylation of the N(1)-position of the adenine ring of NAD(P); chemical reduction with Na2S,0, to N(1)-alkylated NAD(P)H; Dimroth rearrangement under harsh conditions (e.g. pH 11, 70T, 2 h) to N6-alkylated NAD(P)H; optional enzymatic or chemical oxidation to N6-alkylated NAD(P); covalent attachment of N6-alkylated NAD(P)(H) to a water-soluble polymer (e.g. dextran, polyethyleneimine or polyethylene glycol) or copolymerization of vinyl derivatives of N6-alkylated NAD(P).
With respect to NAD(P) this reaction strategy has used alkylating reagents 173
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174 ANDREAS F. BUCKMANN
such as iodoacetic acid (Lindberg, Larsson and Mosbach, 1973; Lowe and Mosbach, 1974), propiolactone (Muramatsu et al., 1977; Okuda, Urabe and Okada, 1985), 3.4-epoxybutyric acid (Zapelli, Rossodivita and Re, 1975; Zapelli et al. , 1977) and ethyleneimine (Weibel et al. , 1974; Schmidt and Grenner, 1976; Buckmann, 1979; Buckmann et al. , 1981). Reactive carboxyl- or amino groups are thus introduced by which chemical coupling of the coenzyme derivatives to water-soluble polymers becomes possible, while coenzyme activity is maintained.
Based on this common strategy, more simple alternative methods have been developed by carrying out some of the successive reaction steps simultaneously (LeGoffic, Sicsic and Vincent, 1980; Fuller, Rubin and Bright, 1980). The main disadvantage of these simple methods is the occurrence of side reactions, which result in non-uniform macromolecular NAD(H) derivatives with restricted total enzymatic oxidizability or reducability .
A compromise between the simplified and laborious procedures, which still has the advantage of uniformity of the coupled NAD(H) analog, is described by Buckmann (1979; Buckmann et al., 1981). The essential steps of the procedure are synthesis and purification of N( 1)-(2-aminoetyl)-NAD, covalent attachment of N( 1)-(2-aminoethyl)-NAD to carboxylated water-soluble polymers and, while covalently bound, chemical reduction and subsequent rearrangement to N6-(2- aminoethy1)-NADH, followed by an optional enzymatic oxidation to N6-(2- aminoethy1)-NAD. Based on this strategy a method was developed for the synthesis of technical grade polyethylene glycol (M, = 20 000)-N6-(2-aminoethy1)- NADH with 33.5% overall yield; this can easily be adapted to large-scale synthesis (Buckmann, Morr and Kula, 1987).
The harsh alkaline conditions for the Dimroth rearrangement lead to con- siderable losses if low molecular weight derivatives of NAD(P)H are involved. Because of the instability of NAD(P) under alkaline conditions, the Dimroth rearrangement under usual conditions cannot be used.
This paper describes a new method for the synthesis of macromolecular NAD and NADP derivatives based on an unexpected and relatively fast transformation of N( 1)-(2-aminoethyI)-NAD(P) to N6-(2-aminoethyl)-NAD(P) under exception- ally mild aqueous conditions (e.g. pH 6.5, 50"C, 4-7h) with very acceptable yields. For the first time macromolecular N6-alkylated NAD(P) can be synthes- ized, omitting the three steps common to all the procedures described so far. An important simplification is obtained, maintaining uniformity with respect to the attached coenzyme derivative.
MATERIALS AND METHODS Materials P-NAD (free acid) was a product from Oriental Yeast Co., Tokyo (Japan); P-NADP (disodium salt), yeast alcohol dehydrogenase and yeast glucose-6- phosphate dehydrogenase were purchased from Boehringer , Mannheim (FRG); ethyleneimine from Serva, Heidelberg (FRG); PEG ( M , = 20 000) and polyv- inylpyrrolidone 160 ( M , = 160 OO0) from Fluka, Buchs (Switzerland); PEG (M, = 4000) from Riedel deHaen, Hannover (FRG); Dextran-T 70 (M, = 70 OOO), Sephadex G-50 and G-10 from Pharmacia, Uppsala (Sweden); Polyethylene/maleic acid anhydride (M, , unknown) from EGA, Steinheim (FRG); Polymethylvinylether/maleic acid anhydride (LM, M, = 20 000) from
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MACROMOLECULAR NAD(P) DERIVATIVES 175
Polysciences, Warrington (USA); Biorex 70 (50-100 mesh), AGSOW-X4 (100- 200 mesh), and AG1X4 (100-200 mesh) from Biorad, Munich (FRG); 1-(3- dimethylaminopropyl)-3-ethyl-carbodiimide-HC1 from Sigma, Deisenhofen (FRG); poly-divinylether/maleic acid anhydride (DIVEMA, M, = 18 000), was a gift from Hercules Inc., Wilmington (USA). Silica gel 6O-FZs4 (0.2 mm) aluminum thin-layer chromatography plates and all other chemicals of reagent grade were purchased from E. Merck, Darmstadt (FRG).
Analytical Procedures
Thin-layer chromatography (TLC) was carried out on silica gel 60-F254 in isobutyric acid [25% aqueous NH3/H20, pH 3.7, 66/1/33 (v/v/v)] to follow the progress of the conversion of NAD(P) by reaction with ethyleneimine. Spots were qualitatively visualized under UV-light (254 nm) and by spraying the TLC plates with ninhydrin.
Quantitative determinations were performed by scanning at 259 nm with a Shimadzu CS-920 high-speed TLC scanner. UV-spectra were obtained with a Perkin-Elmer 554 spectrophotometer using the standard medium (200 mM potassium phosphate, pH 7.0). The coenzyme concentration was determined spectrophotometrically using the following molar absorption coefficients: 18 000 M-' cm-' at 259 nm for NAD, NADP, N(1)-(2-aminoethyl)-NAD(P); 21 600 M-' cm-' at 267 nm for N6-(2-aminoethyl)-NAD(P) and their macro- molecular derivatives. Overall yields for all the coenzyme derivatives were calculated based on NAD(P) present in the solution of the alkylation reaction at time zero.
Using a Perkin-Elmer 554 UV-VIS spectrophotometer at 340 nm, the total reducability of all the N6-(2-aminoethyl)-NAD derivatives was determined using yeast alcohol dehydrogenase under the following assay conditions at room temperature (RT): TrislHCl (100 mM, pH 8.2), ethanol (100 mM), semicar- bazide/HCl (7 mM), N6-(2-aminoethyl)-NAD or -derivatives (0.1 mM) and yeast alcohol dehydrogenase (0.3 mg/ml).
The total reducability of all the N6-(2-aminoethyl)-NADP derivatives was determined at 340 nm using glucose-6-phosphate dehydrogenase under the following assay conditions at RT: triethanolamine/HCl (50 mM, pH 8.0), MgClz (5.5 mM), glucose-6-phosphate (4.5 mM), N6-(2-aminoethyl)-NADP or -derivatives (0.1 mM), glucose-6-phosphate dehydrogenase (0.02 mg/ml).
The total reducability of N6-(2-aminoethyl)-NAD(P), with a characteristic ratio of absorbance A267nm/A340nm = 3.2 in the reduced form, was set at 100%
Effluents from gel filtration and ion exchange columns were monitored with an ISCO absorption monitor UA5. Coupling yields were checked by gel filtration of samples (200-1000 pl) on a Sephadex G-50 (medium) column (0.5 x 60 cm) with 0.1% KCl as eluant at RT and by comparison of the fraction of the first peak with that of the original sample by absorbance measurement at 267 nm. In the case of the maleic acid copolymers, coupling yields were checked by absorbance measurement at 267 nm after exhaustive dialysis.
Synthesis of Macromolecular N6-(2-aminoethyl)-NA D N( 1)-(2-aminoethyl)-NA D Ethyleneimine (850 mmol) was slowly added to a solution of NAD (300 mmol) in distilled water (400ml), maintaining the pH at 3.2 by the simultaneous addition
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176 ANDREAS F. BUCKMANN
of 70% HC104 (total volume 650 mi). The reaction was gently stirred in the dark at 30°C for 50 h, and the pH was kept at 3.2 with 70% HC104. After this time an optimal transformation was obtained as judged by quantitative TLC analysis after the alkylation of NAD. The reaction mixture was diluted to 11 and the product precipitated with cold technical grade ethanol (4"C, 5 X 10 1). The coenzyme precipitate was recovered by subsequent centrifugation. In this way unused ethanol-soluble ethyleneimine was removed. The final precipitate was dried under vacuum at 25°C and stored over NaOH in a desiccator at 4°C. The precipitate contained NAD (69 rnmol, 23%), N(1)-(2-aminoethyl)-NAD (195 mmol, 65%) and unidentified by-products (36 rnmol, 12%), which are probably aminoethy- lated derivatives of N( 1)-(2-aminoethyl)-NAD.
Dry reaction mixture (20 g), with composition NAD (5.3 mmol), N(1)-(2- aminoethy1)-NAD (15 mmol) and N(1)-NAD by-products (2.6 mmol), were dissolved in distilled water (40ml). After pH adjustment to 5.0 with NaOH (10N) the solution was applied to a Biorex-70 cation exchange column (2.6 X 100cm), pre-equilibrated against LiCl (lOmM, pH 4.5). Elution with the equilibration solution produced two fractions: NAD (5.3mmol) in 21 and a mixture (2.5 mmol) of N6-(2-aminoethyl)-NAD and N6-ethanoadenine-NAD (easily formed from N(1)-(2-aminoethyl)-NAD at elution conditions on the column-see Results) in 21. After removal of the lower part of the Biorex-70 column, to leave a column of 50 cm length, pure N(1)-(2-aminoethyl)-NAD (11.3 mmol) was obtained in 1.5 1 by elution with LiCl (200 mM, pH 4.7).
N( 1)-NAD by-products (2.6 mrnol), formed during the alkylation reaction from N( 1)-(2-aminoethyl)-NAD, were not further isolated. The three fractions were concentrated by flash evaporation to 30 ml and precipitated with cold technical ethanol (5 x 100 ml) to remove the ethanol-soluble LiCl. The final precipitate was dissolved in a minimal amount of distilled water, lyophilized and stored in a desiccator over NaOH at 4°C.
N6-(2-aminoethyl)-NA D Purified N( 1)-(2-aminoethyl)-NAD (2.78 mmol) was dissolved in distilled water (200ml) and the pH adjusted to 6.5 with LiOH (1 N). This solution was incubated at 50°C in a water-bath, keeping the pH at 6.5 (1 N LiOH). The composition of the solution was determined by quantitative scanning of the TLC pattern. After 7 h N(1)-(Zaminoethyl)-NAD had disappeared and two new compounds were visualized and quantified: N6-(2-aminoethyl)-NAD [ ~ f = 0.13, 62.5% (1.74 mmol)] and 1,N6-ethanoadenine-NAD [rf = 0.068, 37.5% (1.04 mmol)] (see Results).
The lyophilized reaction mixture was dissolved in distilled water (16.5 ml), the pH adjusted to 5.5 with LiOH (1 N), and applied to a Biorex-70 cation exchange column (1.6 x 100cm), pre-equilibrated with LiCl (lOmM, pH 3.5) at 4°C. By elution with equilibration buffer, three fractions were obtained: 1,N6- ethanoadenine-NAD (0.98mmol) in 250rn1, a mixture of the two products (0.2 mmol) in 120 ml and N6-(2-aminoethyl)-NAD (1.56 mmol) in 660 ml. The fractions were concentrated by flash evaporation to approximately 3 ml and precipitated with cold ethanol (2 x 60 rnl) to remove the ethanol-soluble LiCl. The precipitates were dissolved in a minimal volume of distilled water, lyophil- ized and stored over NaOH in a desiccator at 4°C.
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MACROMOLECULAR NAD(P) DERIVATIVES 177
PEG (M, = 20 000)-N6-(2-aminoethyl)-NAD N6-(2-aminoethyl)-NAD (125 pmol) was added to poly(ethy1ene glycol) (PEG) (2 g, M, = 20 000, carboxylated according to Buckmann, Morr and Johansson (1981), 0.1 mmol with 0.14 mmol carboxyl groups), dissolved in distilled water (6 ml). After pH adjustment to 4.7 with 1 N HCl, 1-(3-dimethylaminopropyl)-3- ethylcarbodiimide-HC1 (1.04 mmol) was added in two equal portions within 10 min. The solution was gently stirred for 2 h at RT, with the pH maintained in the range 4.6-4.8 by the addition of HCl (1 N) or NaOH (1 N) and stored for 16 h at 4°C. The reaction mixture (7ml) was fractionated by gel filtration in two successive aliquots (3.5 ml) applied to a Sephadex G-50 column (2.6 x 100 cm), pre-equilibrated against distilled water at 4°C. The pooled fractions containing PEG (M, = 20 000)-N6-(Zaminoethyl)-NAD were reduced in volume by flash evaporation to 15 ml. PEG (M, = 20 000)-N6-(2-aminoethyl)-NAD (85 pmol) was isolated, with a coupling yield of 68%.
Dextran (M, = 70 000)-N6-(2-aminoethyf)-NAD N6-(2-aminoethyl)-NAD (65 pmol) was added to dextran-T70 (0.2 g, carboxy- lated according to Buckmann, Morr and Johansson (1981), 1 mmol anhydroglu- cose monomers with 0.3 mmol carboxyl groups), dissolved in distilled water (3 ml). After pH adjustment to 4.7 with HCl (1 N), 1-(3-dirnethylaminopropyl)-3- ethylcarbodiimide-HC1 (0.78 mmol) was added in two equal portions within 10min. The reaction was carried out as described above. The reaction mixture was fractionated by gel filtration on Sephadex G-50 (2.6 X 100 cm), pre- equilibrated against distilled water at 4°C. The fraction containing dextran ( M , = 70 000)-N6-(Zaminoethyl)-NAD was reduced by flash evaporation to 10 ml. Dextran-T7O (M, = 70 000)-N6-(2-aminoethyl)-NAD (14 pmol) was isolated with a coupling yield of 22%.
Polyvinylpyrrolidone (M, = 160 000)-N6-(2-aminoethyl)-NAD N6-(Zaminoethyl)-NAD (63 pmol) was added to polyvinylpyrrolidone-160 (0.3 g, carboxylated according to von Specht, Seinfeld and Brendel (1973), 2.7 mmol vinylpyrrolidone monomers with 0.135 mmol carboxyl groups), dissolved in distilled water (3 ml). After pH adjustment to 4.7 with HCl (1 N), 1-(3-dimethyl- aminopropyl)-3-ethylcarbodiimide-HC1 (0.78 mmol) was added in two equal portions within 10min. The reaction was carried out as described above. The reaction mixture (3.8 ml) was fractionated by gel filtration on Sephadex G-50 (2.6 x 100 cm), pre-equilibrated against distilled water at 4°C. The fraction con- taining polyvinylpyrrolidone (M, = 160 000)-N6-(2-aminoethyl)-NAD was reduced in volume by flash evaporation to 10 ml. Polyvinylpyrrolidone (M, = 160 000)- N6-(2-aminoethyl)-NAD (10 pmol) was isolated with a coupling yield of 16%.
Poly -(ethylene/maleic acid)-N6-(2-aminoe~hyf)-NAD (M,, unknown) N6-(2-aminoethyl)-NAD (20 pmol) was added to 50 mg poly-(ethylene/maleic anhydride) (50 mg, with 0.4 mmol ethylene maleic acid anhydride monomers) dissolved in distilled water (10 ml) at pH 7.5 (1 N NaOH). The solution was gently stirred for 5 h at RT at pH 7.5 (1 N NaOH) and subsequently dialysed for
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I 78 ANDREAS F. BUCKMANN
16 h against distilled water ( 5 1) at 4°C. Poly-(ethylene/maleic acid)-N6-(2- aminoethy1)-NAD (13 pmol) was obtained with a coupling yield of 65%.
Poly -(methylvinylether/maleic acid)-N6-(2-aminoethyl)-NA D (M, = 20 000)
Nh-(2-aminoethyl)-NAD (20 pmol) was added to poly-(methylvinylether/maleic acid anhydride) (50 mg, 0.32 mmol methylvinylether/maleic acid anhydride mon- omers), dissolved in distilled water (10 ml) at pH 7.5 (1 N NaOH). The solution was gently stirred and subsequently dialysed as described above. Poly- (methylvinylether/maleic acid)-N6-(2-aminoethyl)-NAD (M, = 20 000) (17 pmol) was isolated with a coupling yield of 85%.
Poly-(divinylerherlmaleic acid)-N6-(2-aminoethyl)-NAD (M, = 18 000)
N6-(2-aminoethyl)-NAD (20 pmol) was added to poly-(divinylether/maleic acid anhydride) (50 mg, with 0.19 mmol divinylether/maleic acid anhydride mono- mers), dissolved in distilled water (10ml) at pH 7.5 (1 N NaOH). The solution was gently stirred and dialysed as above to yield poly-(divinylether/maleic acid)-N6-(2-aminoethyl)-NAD (M, = 18 000) (20 pmol, coupling yield 100%).
Synthesis of N ( l)[N-(2-carboxy -propioamido)-ethyl]-NA D
Succinic anhydride (0.17 mmol) was dissolved in portions (10 mg, 3 mg, 2 mg and 2 mg) at R T in an aqueous solution of N(1)-(2-aminoethyl)-NAD (0.14 mmol in 3 ml) at pH 7.5 (1 N NaOH) over a period of 10 min. After 30 min this solution gave a negative reaction with ninhydrin and was diluted with distilled water (9.3 ml, final concentration 15 mM). The yva lue of the product was 0.165.
Synthesis of Macromolecular N6-(2-aminoethyl)-NA D P N( 1)-(2-aminoethyl)-NA D P
Ethyleneimine (28 mmol) was slowly added to a solution of NADP (9.51 mmol) in distilled water (10 ml) and the pH maintained at 3.3 by simultaneous addition of 70% HClO, (total volume 25 ml). The solution was gently stirred in the dark at 30°C for 120h, the pH maintained at 3.3 with 70% HClO,. The subsequent removal of ethyleneimine was carried out as described for the reaction mixture with N( 1)-(2-aminoethyl)-NAD, while the same volume ratio was maintained. A mixture of NADP (2.61 mmol, 27.5%), N( 1)-(2-aminoethyl)-NADP (6.47 mmol, 68%) and by-products of N( 1)-(2-aminoethyl)-NADP (0.43 mmol, 4.5%) was obtained. Lyophilized reaction mixture (0.75 g), with composition NADP (0.234 mmol), N( 1)-(2-aminoethyl)-NADP (0.575 mmol) and N(1)-NADP by- products (0.038 mmol), was dissolved in distilled water (22 ml). After pH adjustment to 3.5 with HCl (1N) the solution was applied to an AGSOW-X, cation-exchange column (1.6 X 60 cm), equilibrated against triethanolamine- bicarbonate (10 mM, pH 3.5) at 4°C. Elution with the equilibration buffer gave three fractions: NADP (0.235 mmol including 0.055 mmol by-products in 25 ml), by-product (0.115 mmol. spontaneously formed on the column from N(1)-(2- aminoettiy1)-NADP in 100 ml) and pure N( 1)-(2-aminoethyl)-NADP (0.39 mmol in 200ml). The fractions were concentrated by flash evaporation to 25m1, lyophilized to remove triethanolamine-bicarbonate and stored over NaOH in a desiccator at 4°C.
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MACROMOLECULAR NAD(P) DERIVATIVES 179
N6-(2-aminoethyl)-NA D P
N( 1)-(2-aminoethyl)-NADP (0.24 mmol) was dissolved in distilled water (500 ml), the pH adjusted to 6.0 with LiOH (1 N) and the solution incubated at 50°C in a water bath while the pH was maintained at 6.0 (1 N LiOH). The composition of the solution was followed by quantitative scanning of the TLC pattern. After 4 h N( 1)-(2-aminoethyl)-NADP had disappeared and two new compounds were visualized and quantified: 1 ,N6-ethanoadenine-NADP [rf = 0.031, 75% (0.18 mmol)] and N6-(2-aminoethy1)-NADP [rf = 0.048, 25% (0.06 mmol)] (see Results).
The solution was reduced in volume to 7ml by flash evaporation, the pH adjusted to 5.0 with HCl (1 N) and applied to a AG1X4 anion exchange column (1.6 X 100 cm), pre-equilibrated against triethanolamine-bicarbonate (10 mM, pH 3.5) at 4°C. Elution with the equilibration buffer yielded 1 ,N6-ethanoadenine- NADP (0.17 mmol in 1 1, and N6-(2-aminoethyl)-NADP (0.053 mmol in 250 ml). The two fractions were reduced in volume to 2ml by flash evaporation and applied to a Sephadex G-10 column (1.5 x 100 cm), pre-equilibrated against distilled water, to remove triethanolamine-bicarbonate. After lyophilization the dry fractions were stored over NaOH in a desiccator at 4°C.
PEG (M, = 4000)-N6-(2-aminoethyl)-NADP N-hydroxysuccinimide activated carboxylated PEG (60 mg, M, = 4000, synthesized according to Buckmann, Morr and Johannson (1981), 0.015 mmol with 0.03mmol carboxyl groups) was added to an aqueous solution of N6-(2- aminoethy1)-NADP (15 pmol, 1 ml, pH 7.2, 1 N NaOH). The reaction mixture was stirred at RT for 5 h at pH 7.2 (1 N NaOH) and subsequently fractionated on a Sephadex G-50 column (2.6 x 100 cm), pre-equilibrated against distilled water at 4°C. The fraction containing PEG (M, = 4000)-N6-(Zarninoethyl)-NADP was reduced in volume by flash evaporation to 10 ml. PEG (M, = 4000)-N6-(2- aminoethy1)-NADP (12 pmol) was obtained with a coupling yield of 80%.
PEG (M, = 20 000)-N6-(2-aminoethyl)-NADP N-hydroxysuccinimide activated carboxylated PEG (325 mg, M, = 20 000, also equivalent to 0.015 mmol), was reacted and processed exactly as described above. PEG (M, = 20 000)-N6-(2-aminoethyl)-NADP (15 pmol) was obtained with a coupling yield of 100%.
Poly-(ethylene/maleic acid)-N6-(2-aminoethyl)-NA D P (M,, unknown) N6-(2-aminoethyl)-NADP (5.5 pmol) was added to poly-(ethylene/maleic acid anhydride) (12 mg with 0.1 mmol ethylene/maleic acid anhydride monomers), dissolved in distilled water ( lml , pH7.5, 0.2M NaOH). This solution was processed as described for the corresponding NAD derivative. Poly- (ethylene/maleic acid)-N6-(2-aminoethyl)-NADP (0.8 pmol, M,, unknown) was isolated with a coupling yield of 15%.
Poly -(methylvinylether/maleic acid)-N6-(2-aminoethyl)-NA D P (M, = 20 000)
N6-(2-aminoethy1)-NADP (5.5 pmol) was added to poly-(methylvinylether/maleic acid anhydride) (12 mg, with 0.077 mmol methylvinylether/maleic acid anhydride
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180 ANDREAS F. BUCKMANN
monomers) and coupled as described above. Poly-(methylvinylether/maleic acid)-N6-(2-aminoethyl)-NADP (0.6 pmol, M, = 20 000) was obtained with a coupling yield of 11 % .
Poly-(divinyIether/maleic acid)-N6-(2-aminoethyl)-NADP (M, = 18 000) N6-(2-aminoethyl)-NADP (5.5 pmol) was added to poly-(divinylether/maleic acid anhydride) (12 mg, with 0.046 pmol divinylether/maleic acid anhydride mono- mers) and coupled as described above. Poly-(divinylether/maleic acid)-N6-(2- aminoethy1)-NADP (0.7 pmol, M , = 18 000) was obtained with a coupling yield of 13%.
RESULTS AND DISCUSSION
Reaction conditions have been established for the alkylation of NAD and NADP with ethyleneimine to N( 1)-(2-aminoethyl)-NAD and N( 1)-(2-aminoethyl)-NADP with transformation percentages of 65% and 68% (Figure l ) , restricting the formation of N(1)-NAD- and N(1)-NADP-by-products (12% and 4.5%). The reaction of NADP with ethylenimine is significantly slower compared to that of NAD under similar reaction conditions, possibly due to a higher percentage of NADP in a folded conformation, with the adenine stacked intramolecularly on the nicotinamide.
The products synthesized in this work, N(1)-(2-aminoethyl)-NAD, N6-(2- aminoethy1)-NAD, N(1)-(2-aminoethyl)-NADP and N6-(2-aminoethyl)-NADP, have been characterized by UV, NMR and MS. Under alkaline conditions the UV-spectra of N(1)-(2-aminoethyl)-NAD(P) (A,, = 259 nm) show shoulders in the range 300-310 nm, characteristic for N(1)-adenine alkylation, and give a positive reaction with ninhydrin. This shoulder is absent in the spectra of N6-(2-aminoethyl)-NAD(P) (Amm = 267 nm) under alkaline conditions.
Detailed data of NMR and MS studies to confirm the structure of these compounds and the 1 ,N6-ethano-adenine derivatives of NAD and NADP, formed by a parallel tricyclization reaction, will be reported in a separate publication dealing with the reaction mechanism of the unexpected transformations of N(1)-(2-aminoethyl)-NAD(P) under mild aqueous conditions (Buckmann, Wray and van der Plas, 1987).
The conversion of N( 1)-alkylated NAD(P)H to N6-alkylated NAD(P)H by Dimroth rearrangement is usually carried out under harsh alkaline conditions, achieving an acceptable conversion rate. A striking feature is the different reaction behaviour of N( 1)-(2-aminoethyl)-NAD(P) with respect to the Dimroth rearrangement to N6-(2-aminoethyl)-NAD(P). This rearrangement can still be carried out at an acceptable rate, but now under unusual mild conditions, with the consequence that no previous chemical reduction of the C-4-position of the nicotinamide is needed (Figure 2a). This particular reaction behaviour is an exception, applying only to N( 1)-(2-aminoethyl)-adenine derivatives. Normal Dimroth rearrangement character (slow reaction at mild aqueous conditions) is observed for N( l)-[N-(2-carboxy-propioamido)-ethyl]-NAD, obtained after chemical modification of the aminoethyl group of N( 1)-(2-arninoethyl)-NAD with succinic anhydride (Figure 2b).
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A
pH 3.2-33. 30%
I 0
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X:H - NAD
X=PO
,H, - NADP
100 90
80
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50
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60
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Figu
re 1
R
eact
ion
sche
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and
com
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of t
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(%)
as a
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or th
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tion
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AD
(A
) and
NA
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(B) w
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e (se
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ds). (0
) NA
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= 2
-am
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thyl
.
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182 ANDREAS F. BUCKMANN
o / 1'0 iGC
80
60
40
20
I I r 1
Figure 2 Comparison of the transformation (%) of N( 1)-(2-aminoethyl)-NAD (A) and N(l)-[N-(2- carboxy-propioamido)-ethyl]-NAD (B) under mild aqueous conditions: distilled water, pH 7.0, 50°C. initial concentration 15 mM. A: (0) N(1)-(2-aminoethyl)-NAD; (W) Nh-(2-aminoethyl)-NAD; (+) l.N'-ethanoadenine-NAD. B: (V) N(l)-[N-(2-carboxy-propioamido)-ethyl]-NAD; (A) N6-[N-(2- carboxy-propioamido)-ethyl]-NAD .
Another striking aspect of the transformations described in this paper is the lower conversion of N(1)-(2-aminoethyl)-NADP to N6-(2-aminoethyl)-NADP (max. 25-30%) obtained, compared to the corresponding transformation of the NAD derivative (max. 60-65%). The phosphate group at the 2-position of the ribose of N( 1)-(2-aminoethyl)-NADP has a similar catalytic effect as inorganic phosphate on the tricyclization reaction of N( 1)-(2-aminoethyl)-NAD, producing 1 ,N6-ethano-adenine-NADP as the major product and decreasing the yield of N6-(2-aminoethyl)-NADP (Buckmann and Wray, 1986).
The fractionation procedures for the separation of N6-(2-aminoethyl)-NAD(P) from 1,N6-ethano-adenine-NAD(P) are similar, in that an eluent with a low constant ion strength can be used.
Several examples of coupling N6-(2-aminoethyl)-NAD(P) to water-soluble polymers are given (Figure 3). For N6-(2-aminoethyl)-NADP the coupling yields are low in case of the maleic acid anhydride copolymers (11-15%), resulting in low overall yields (Table 2). Presumably, this may be due to the repulsive interaction between both the negatively charged carboxyl groups and N6-(2- aminoethy1)-NADP at pH 7.5.
In the case of N6-(2-aminoethyl)-NADP N-hydroxy-succinimide activated carboxyl-PEG (Mr = 4000 and 20 0o0) can be used to avoid the disadvantage of the carbodiimide coupling method. Besides synthesizing amide bonds, the carbodiimide reagent would induce 2'-3'-cyclization via the 2'-phosphate group on the ribose, giving inactive NADP derivatives (Sogin, 1976).
A much simpler method for the synthesis of PEG-NADP of high molecular weight (M, = 20000) with an overall yield up to 15% can now be pursued. The laborious procedure of Okuda, Urabe and Okada (1985), coupling carbodiimide activated and simultaneously 2' ,3'-cyclized N6-(2-carboxyethyl)-NADP to an excess of amino-PEG ( M , = 3000), can be avoided. This latter procedure required an enzymatic step with the expensive 2',3'-cyclic-nucleotide-3'-phosphodiesterase to prepare PEG ( M , = 3000)-N6-(2-~arboxyethyl)-NADP with 13% overall yield.
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MACROMOLECULAR NAD(P) DERIVATIVES 183
Nil)- I2A EI-NA DIP)
6 0-6 5
OH OH
xo on no on
/ N6- E THANOA DENINE-NA DIP)
POLYMER R T .pH 7 5
POLYMER ( - C O O H I
OR NHS ACTIVATED, pH7 2 R T
EDC ACTIVATED, pH1 6 - L 7
MACROMOLECULAR N6- (2AEI-NADIPI
OH I
I I 0
x = P-OH - NADP
Figure 3 Reaction pathway for the new synthesis of water-soluble macromolecular N6-(2- aminoethy1)-NAD and N6-(2-aminoethyl)-NADP. X = H+ NAD; X = PO,H,+ NADP; EDC = 1-(3- dimethylaminopropyl)-3-ethyl-carbodiimide-HCl; NHS = N-hydroxy-succinimide; 2-AE = 2-amino- ethyl; RT = room temperature.
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184 ANDREAS F. BUCKMANN
Table 1 Overall yield and total reducability of macromolecular N6-(2AE)-NAD compared to N6-(2AE)-NAD (see Methods).
Overall Total yield reducability (%)
N6-( 2-AE)-NAD 36.7 100 PEG ( M , = 20 OOO)-N6-(2AE)-NAD 25 90 Dextran (M, = 70000)-N6-(2-AE)-NAD 8.1 90 Polyvinylpyrrolidone ( M , = 160 OOO)-
Poly(ethylene/maleic acid)-Nb-
Poly(methylvinylether/maleic acid)-N6- (2-AE)-NAD ( M , = 20 OOO) 31.2 70 Poly(divinylether/maleic acid)-N6-(2-AE)-NAD
N6-(2-AE)-NAD 5.9 90
(2-AE)-NAD (M, , unknown) 23.8 60
(M, = 18 OOO) 36.7 60
As summarized in Tables 1 and 2 the macromolecular N6-(2-aminoethy1)- NAD(P) derivatives described here exhibit similar total reducabilities, depending on the type of water-soluble polymer chosen. In particular, the maleic acid copolymerisate-coenzyme derivatives may have a conformation, with partly buried NAD or NADP inaccessable to the active centers of the dehydrogenases tested. Studies on the coenzymatic properties of macromolecular N6-(2- aminoethy1)-NAD with respect to several NAD dependent dehydrogenases have demonstrated the superiority of PEG (M, = 20 000)-N6-(2-aminoethyl)-NAD (Buckmann et al., 1981; Riva et al., 1986). The coenzymatic properties of PEG ( M , = 20 000)-N6-(2-aminoethyl)-NADP will be the subject of a forthcoming paper (Biickmann et a/ . , unpublished work).
Based on the unexpected transformation of N(1)-(2-aminoethyl)-NAD(P) to N6-(2-aminoethyl)-NAD(P) under mild aqueous conditions (pH 6.0-6.5, 50"C, 4-7 h) it is now possible, for the first time, to synthesize macromolecular N6-alkylated NAD and -NADP derivatives as uniform products with acceptable overall yields (Tables 1 and 2) omitting (a) the chemical reduction reaction with
Table 2 Overall yield and total reducability of macromolecular N6-(2AE)- NADP compared to N6-(2AE)-NADP (see Methods).
Overall Total yield reducability (%I _________ ______-____
N6-(2-AE)-NADP 14.75 100 PEG ( M , = 4W)-N6 - (2-AE)-NADP 12.0 95 PEG ( M , = 20 000)-N6-(2-AE)-NADP 14.75 95 Poly(ethylene/rnaleic acid)-N6- (2-AE)-NADP (M, , unknown) 2.2 50 Poly(rnethylvinylether/rnaleic acid)-
Poly(divinylether/maleic acid)-N6 (2-AE)-NADP ( M r = 18 OOO) 2.0 63
N6-(2-AE)-NADP (M, = 20 OOO) 1.6 60
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MACROMOLECULAR NAD(P) DERIVATIVES 185
Na2S204, (b) the Dimroth rearrangement at harsh conditions (pH 11, 70°C, 2 h), and (c) the enzymatic or chemical oxidation reaction. This results in a significant simplification in comparison to the procedures described so far (Buckmann, 1986). Currently, the procedure is being optimized for larger scale synthesis, with emphasis on finding optimal conditions for the direct conversion of N( 1)-(2- aminoethy1)-NAD(P) to N6-(Zaminoethyl)-NAD(P) at higher concentrations as reported in this publication.
Ackno wledgernents The work described has been supported by a grant from the Biotechnology Program of the Bundesministerium fur Forschung und Technologie (BCT 310A). I thank DEGUSSA-AG. for the gift of NAD and gratefully acknowledge the expert technical assistance of Miss Britta Neumann and Miss Annette Stawski.
References Biickmann, A. F. (1979) Process for the production of adenine ring system containing coenzymes
bound to macromolecules. German Patent DP 28.41.414. Biickmann, A. F. (1986) Process for the production of N6-substituted NAD, NADP or FAD. German
Patent DP 36.17.535.8. Buckmann, A. F. and Wray, V. (1986) An unexpected transformation of N(l)-(Zaminoethyl)-
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Buckmann, A. F., Morr, M. and Kula, M.-R. (1987) Preparation of technical grade polyethylene glycol (PEG)-(M, = 20 000)-N6-(2-aminoethyI)-NADH by a procedure adaptable to large-scale synthesis. Biotechnol. Appl. Biochem., 9,258-268.
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Sogin, D. C. (1976) 2',3'-Cyclic NADP as a substrate for 2'.3'-cyclic nucleotide 3'-phosphohydrolase. J . Neurochem., 27, 1333-1337.
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