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Biosynthesis of Riboflavin. A Simple Synthesis of the Substrate and Product of the Pyrimidine Deaminase and of Structural Analogs

P. Nielsen3 and A . Bacher*

Lehrstuhl für Organische Chemie und Biochemie der Technischen Universität München. Lichtenbergstraße 4, D-8046 Garching. FRG Z. Naturforsch. 43b, 1358-1364 (1988); received March 11, 1988

Riboflavin, Biosynthesis, Pyrimidine, Deaminase, 5'-Phosphates of Ribitylaminopyrimidines

2-Amino-5-nitro-6-ribitylamino-4(3H)-pyrimidinone (7) was phosphorylated with chlorophos-phoric acid yielding an isomer mixture containing about 63% of the 5'-phosphate 7a together with other monophosphates and bisphosphates of 7. Preparative HPLC afforded pure 7a. Catalytic hydrogenation of 7a yields the labile substrate of pyrimidine deaminase, 2,5-diamino-6-ribityl-amino-4(3H)-pyrimidinone 5'-phosphate (2a). The product of the enzyme, 5-amino-6-ribityl-amino-2,4(l H.3H)-pyrimidinedione 5'-phosphate (4a), can be obtained from 5-nitro-6-ribityl-amino-2,4(lH,3H)-pyrimidinedione (8) by an analogous procedure. The 3'- and 4'-monophos-phates of 2,5-diamino-6-ribitylamino-2,4(lH,3H)-pyrimidinedione are not substrates for the deaminase. 5'-Phosphates of a variety of ribityl-substituted pyrimidines and pteridines could also be obtained by phosphorylation with chlorophosphoric acid without the use of protecting groups.

Introduction

The biosynthesis of riboflavin is summarized in Fig. 1 (for review see Refs . [1 — 3]). It is firmly estab-lished that the first biosynthetic step consists in the opening of the imidazole ring of G T P leading to the pyrimidine in te rmedia te l a [4—6].

In yeasts, the ribose side chain of the in termedia te l a is reduced yielding 2,5-diamino-6-ribitylamino-4(3H)-pyr imidinone 5 ' -phospha te (2a) [7—10], Sub-sequent ly, the position 2 amino g roup of the py-rimidine ring is hydrolyzed yielding 5-amino-6-ribityl-amino-2,4( l H ,3H) -pyr imid ined ione 5 ' -phospha te

GTP ^ Y ^ N H HN^N^NH

1a

OH OH

OH OH

3a

a Present address: Physiologisch-Chemisches Institut, Ab-teilung Medizinische Biochemie. Universitätskranken-haus Eppendorf, Martinistraße 52, D-2000 Hamburg 20. FRG.

* Reprint requests to Dr. A. Bacher.

Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen 0932-0776/88/1000-1358/$ 01.00/0 Fig. 1. Biosynthesis of riboflavin.

-C-0H CH20H

P. N i e l s e n - A . Bacher • Biosynthesis of Riboflavin 1359

(4a) [11 — 14], The sequence of deamination and side chain reduction appears to be reverse in Escherichia coli with 3a as intermediate [15].

Dephosphorylation of 4 a yields 4 which is subse-quently converted to 6,7-dimethyl-8-ribityllumazine (5) by condensation with 3,4-dihydroxy-2-butanone 4-phosphate [16-19], The dismutation of 5 yielding riboflavin (6) and 4 which is catalyzed by riboflavin synthase has been studied extensively by Plaut and his coworkers [20, 21].

The detailed investigation of the early biosynthetic steps has been hampered by the instability and poor availability of the respective intermediates. The phosphoric acid esters 2 a and 4a have been prepared by a sequence of tritylation and acetylation, followed by removal of the trityl protecting group and phos-phorylation with cyanoethylphosphate [22], Due to the insufficient regioselectivity of the tritylation reac-tion, the final product of this reaction contains only about 80% of the desired 5'-phosphate together with other phosphates.

This paper describes a simple procedure for the synthesis of the pyrimidines 2 a and 4 a without the use of protecting groups. The regioselectivity is com-parable to the more laborious protection group ap-proach [22], Pure 5'-isomer can be obtained by pre-parative HPLC. A wide variety of structural analogs was also obtained by this method. A preliminary re-port of part of this work has been published [23],

Experimental

General methods

Visible and U V spectra were obtained with a photometer PM 6 (Zeiss). The following extinction coefficients were used (pH 1.0): 7 a, £334 = 14200 M - 1 cm - 1 ; 8a , £323 = 14200 M"1 cm"1. High performance liquid chromatography (HPLC) was performed with instruments from Waters Inc. and Hupe und Busch Co. Columns of Nucleosil 100—10 C18 (Macherey and Nagel, Düren; 4x250 mm, with a minimum of 4000 theoretical plates as determined with phenylacetic acid as solute and 30% methanol, adjusted to pH 3.0 by the addi-tion of formic acid) were used for analytical work. Columns of Lichrosorb RPig (Merck, Darmstadt; 16x250 mm) were used for preparative work. Phos-phate was determined by published procedures [24]. Phosphoric acid esters were hydrolyzed by treatment with alkaline phosphatase (Boehringer, Mannheim)

prior to the phosphate determination. Pyrimidine deaminase was partially purified from cell extract of Candida guilliermondii as published earlier [14].

Chemicals

The following compounds were prepared by pub-lished procedures: 6-chloro-5-nitro-2,4(l H,3H)-py-rimidinedione [25], 6,7-dimethyl-8-ribityllumazine [26], 5-nitroso-6-ribitylamino-2,4(lH,3H)-pyrimi-dinedione [27], 2-amino-6,7-dimethyl-8-ribityl-4(3 H)-pteridinone [28].

5-Nitro-6-ribitylamino-2,4( 1 H,3 H)-pyrimidinedione (8) [25]

A solution of 5-nitro-6-chloro-2,4(l H,3H)-py-rimidinedione (18 g, 94 mmol) in 360 ml of ethanol was added to a solution of 94 mmol ribitylamine [27] in 360 ml of water. The pH was repeatedly adjusted to 8 by the addition of 1 M K 2 HP0 4 . The mixture was heated at 60 °C for 1 h and then kept at room temperature overnight. The pH was adjusted to 10.7 by the addition of concentrated ammonia, and the precipitate formed was removed by filtration. The solution was evaporated to dryness. The residue was dissolved in 100 ml of water and placed on a column of Dowex 1X8 (100-200 mesh, formate form, 2 .5x40 cm). The column was developed with (a) 1 1 of water, (b) 1 1 of 0.01 M formic acid, (c) 2 1 of 0.1 M formic acid. Fractions containing 8 were pooled and concentrated to a small volume. The pre-cipitate formed was collected. Yield, 5.37 g, 18,7%, m.p . 155 °C.

Chlorophosphoric acid [29]

Freshly distilled phosphorous oxitrichloride (7.3 ml, 80 mmol in a flask with stirrer) was im-mersed in an ice bath. Water (2.9 ml, 0.16 mol) was slowly added by a peristaltic pump over a period of 2—3 h. The product was used immediately.

2-Amino-5-nitro-6-ribitylamino-4(3 H)-pyrimidinone 5'-phosphate (7a)

A suspension of 7 (270 mg, 0.89 mmol) in 1.25 ml (16.5 mmol) of freshly prepared monochlorophos-phoric acid was stirred for 26 h at room temperature. A stream of dry nitrogen was bubbled through the mixture to remove HCl. The reaction mixture was poured into 50 ml of dry ether. The solid was col-lected on a sintered glass filter by suction. The hygro-scopic material was dissolved in 27 ml of water and brought to pH 4 by the addition of aqueous NH 4 OH.

The solution was applied to a column of D E A E cellulose D E 52 (25x17 cm, acetate form, equili-

1360

brated with 30% 2-propanol). The column was de-veloped with 50 ml of 30% 2-propanol and subse-quently with a linear gradient of 0—0.5 M triethyl-ammonium acetate pH 7 in 30% 2-propanol (total volume, 1.4 1). Fractions of 12 ml were collected. Starting material eluted in fraction 5 — 16 was recov-ered. A mixture of monophosphates was eluted in fractions 20—49. The fractions were combined and brought to dryness under reduced pressure. Triethyl-ammonium acetate was removed by subsequent lyophilization. Yield, 276 //mol, 31% (determined photometrically). The mixture contained 63% 5'-phosphate, 25% 4'-phosphate and 11% 3'-phos-phate as shown by HPLC.

Preparative HPLC

A solution of the isomeric phosphate mixture of 7 (35 //mol) in 5 ml of eluent (10 mM formic acid/ 10 mM ammonium formate) was applied in portions to a preparative HPLC-column (Lichrosorb RPjs, 16x250 mm). The effluent was monitored at 335 nm and fractions were collected and lyophilized. Yield: 1.8 //mol 3'-phosphate (7c, 97% pure), 5.1 //mol 4'-phosphate (7b, 98% pure), and 16.9 //mol 5'-phos-phate (7a, 97% pure).

2,5-Diamino-6-ribitylamino-4(3 H)-pyrimidinone 5'-phosphate (2a)

A suspension of palladium on charcoal (1 mg) in 2 ml of water was saturated with hydrogen at room temperature and atmospheric pressure. 7a (12 mg) was added, and the mixture was stirred under an atmosphere of hydrogen overnight. The catalyst was removed by filtration through a nitrocellulose filter. The solution was used immediately.

5-Amino-6-ribitylamino-2,4(l H,3H)-pyrimidine-dione 5'-phosphate (4a) was prepared from 8a as described for 2 a.

Results and Discussion

We have studied the direct phosphorylation of 2-amino-5-nitro-6-ribitylamino-4(3H)-pyrimidinone (7, Scheme I) with chlorophosphoric acid. This method has been used extensively with riboflavin and a variety of riboflavin derivatives where the yield of desired 5'-phosphate is in the range of 70% of total product [29, 30]. The crude product obtained by phos-phorylation of 7 contained starting material (A), sever-al monophosphates (B —D) and small quantities of bisphosphates (Fig. 2, top left). The main product, B. appeared identical with the compound obtained ear-

P. N i e l s e n - A . Bacher • Biosynthesis of Riboflavin 1360

0

HN N

~NH R 2

HN I

0

Y ^ N H

0

H 2 N Y ^ N

HN N ^

CH, i

CH2 CH2

H - C - 0 H H - C - O H H - C -OH

H - C - O H H -C - O H H - C -OH

H - C - 0 H H - C - O H H - C -OH

CH2OH CH 2 O® C H 2 0 ®

R, R2 R1 R2 Rt

7 NH2 NO2 7a NH2 NO2 2a NH2

8 OH NO2 8a OH NO2 4a OH

9 OH NO 9a OH NO

10 NH2 H 10a NH2 H

lier using the trityl derivative strategy. Ion exchange chromatography on D E A E cellulose (Fig. 2, top right) separated the monophosphates from the other com-ponents. Apparently pure monophosphates could be obtained by preparative HPLC on a reversed phase column using ammonium formate at pH 3 as eluent (Fig. 2. bottom). Amounts of about 1 mg can be run on a column of 16x250 mm. Each of the compounds B - D showed phosphate contents of 0.80—0.95 mol per mol of pyrimidine as detected after hydrolysis with alkaline phosphatase.

In an earlier study, we have analyzed the acid-catalyzed isomerization of riboflavin 5'-phosphate in considerable detail. These kinetic data can be sum-marized as follows:

k, k2 k3 5 ' - F M N ^ ^ 4 ' - F M N ^ 3 ' - F M N ^ 2 ' - F M N

k-i k_2 k_3

All kinetic constants were in a narrow range of 1 .5-9.5-10~ 6 sec - 1 . 5 ' -FMNhad the highest thermo-dynamic stability, and the 5'-isomer is formed pre-dominantly by treatment of unprotected riboflavin with chlorophosphoric acid [30]. We have utilized this experimental approach to characterize the py-rimidine phosphates B - D in closer detail. HPLC-purified compounds were treated with 0.1 M HCl at 50 °C, and the reaction mixtures were analyzed by HPLC at intervals (Fig. 3). Isomerization of com-pound B yields C in a relatively rapid first order reaction and D in a much slower reaction with sig-moidal kinetics. Isomerization of C yields B and D.

P. Nielsen —A. Bacher • Biosynthesis of Riboflavin 1361

25 20 15 Retention t ime

25 20 15 10 Retention t ime (min)

25 20 15 10 Retention time (min)

Fig. 2. HPLC chromatography of products obtained by phosphorylation of 2-amino-5-nitro-6-ribitylamino-4(3H)-pyrimidinone (7) with chlorophosphoric acid. Top left, crude product; top right, monophosphate mixture after chromatography on D E A E cellulose; bottom, 5'-phospha-te after preparative HPLC. Column, Nucleosil RPi8, 4 x 250 mm; eluent, 10 mM ammonium formate plus 10 mM formic acid; detection, f2 8 0 .

(x lO 6 s"1) Compound type k, k - , k2 k-2 k3 k—3

7 Phosphate 2.00 9.00 1.37 1.70 ND b ND Riboflavin phosphate3 1.60 9.41 2.21 3.06 2.92 1.55

Table I. Velocities of acid cata-lyzed isomerization of the mo-nophosphates of 2-amino-5-ni-tro-6-ribitylamino-4(3 H)-pyri-midinone (7) and of riboflavin in 0.1 M HCl at 50 °C.

a Data from Nielsen et al. [30]; b ND, not determined.

1362 P. Nielsen-A. Bacher • Biosynthesis of Riboflavin 1362

U u o - A n h R I V ^ N H a c V N v \

0 "NH

HN^N^NH i CH2

H-C -OH I

H-C -OH

H - C - O ® I CH,0H

R1 .. HN^N^NH, I

CH, I 1

H - C - O H

H - C - O ® l

H - C - O H I CH,0H

CH, I

H - C - O H

H -C -OH I

H - C - O H CH20R2

R1 R, R, R2

7b NO2 7c NO2 5a OH PO3H2

2b NH2 2c NH2 U NH2 OH

14a NH2 OPO3H2

time (h) Fig. 3. Acid-catalyzed isomerization of the 5'-phosphate (top) and 4'-phosphate (bottom) of 2-amino-5-nitro-6-ribi-tylamino-4(3H)-pyrimidinone. Samples in 0.1 M HCl were incubated at 50 °C. Aliquots were taken at intervals and analyzed by HPLC. O, 5'-phosphate (7a); • , 4'-phosphate (7b); A, 3'-phosphate (7c). Curves show computer fits cal-culated with the following parameters (xlO6 s"1): k,. 1.60; k_1? 9.41; k2, 2.21, k_2, 3.06.

bo th with first o rder kinetics. This suggests that B, C and D represent the structures 7 a (5 ' -phosphate) , 7 b (4 ' -phospha te ) , and 7 c (3 ' -phosphate) . Compute r analysis of these data gave the kinetic constants shown in Table I which are closely similar to the data for F M N which are shown for comparison. However , the kinetic character izat ion of the pyrimidine phos-pha te system is less comprehensive as compared to the F M N system because one of the isomerization products of compound D , presumably the 2 '-phos-pha te , migrated at the same velocity as B in H P L C .

The available evidence can be summarized as fol-lows:

B(7a) C(7b) ^ D(7c)

The c o m p o u n d s 7 a—7 c are fo rmed at an approxi-mate rat io of 5 : 2 : 1 by the react ion of 7 with ch lorophosphor ic acid. Thus , the regiospecificity is similar to that observed in the phosphoryla t ion of unpro tec ted r iboflavin.

Catalytic hydrogena t ion of 7 a yields the labile 2 a , the subst ra te of pyr imidine deaminase f rom Candida guilliermondii [13, 14]. T rea tmen t of this labile com-pound with diacetyl yielded the phosphoryla ted pter idine derivative 1 4 a . In o rder to examine the substra te specificity of pyrimidine deaminase f rom C. guilliermondii, the 5-ni t ropyrimidine derivatives 7 a—7 c were conver ted to the respective 5-amino c o m p o u n d s (2a—2c) by catalytic hydrogenat ion and incubated with partially purified pyrimidine deami-nase as descr ibed ear l ier [14]. Only the 5 ' -phosphate was deamina ted by the enzyme as shown in Table II. The enzymat ic deamina t ion of 2 a was not inhibited by the i somer 2 b .

The produc t 4 a of the pyrimidine deaminase was obta ined f r o m 8 or 9 by phosphoryla t ion and hydro-genat ion in analogy to the prepara t ion of 2 a . React ion of 2 a with diacetyl yielded the lumazine derivative 5 a . Phosphoryla t ion with chlorophosphor ic acid was also successful with a variety of substra te and product analogues of pyr imidine deaminase (Table III) . The isomeric phosphor ic acid esters could be separa ted by H P L C , but no deta i led investigation has been per-fo rmed . H P L C data are shown in Tab le III.

P. Nielsen-A. Bacher • Biosynthesis of Riboflavin 1363

Table I I . Substrate specificity of 2,5-diamino-6-ribityl-amino-4(3H)-pyrimidinone 5'-phosphate deaminase (DRPP-deaminase). Enzyme assays were performed as de-scribed earlier [14]. Assay mixtures contained pyrimidine substrate at the concentration indicated which had been freshly prepared by catalytic hydrogenation of the respec-tive precursor, 0.1 M phosphate buffer pH 7.0, 5 mM MgCl2, 10 mM dithioerythritol, and partially purified pyrimidine deaminase from C. guilliermondii in a total volume of 0.5 ml. Assay mixtures were incubated at 37 °C for 1 h, and 100 (A of a solution containing 8 ,«mol diacetyl, 200 U of riboflavin synthase from Bacillus subtilis and 0.2 U of alkaline phosphatase were added. The mixtures were again incubated at 37 °C for 3 h, and riboflavin was monitored by bioassay with Lactobacillus casei.

Substrate Riboflavin formed (uM)

None 0.07 2a (0.63 mM) 79 2b (0.58 mM) 1.3 2c (0.10 mM) 0.07 2a+2b (0.31 MM) 69 2a (1.0 mM) 0.07

2 = 2,5-Diamino-6-ribitylamino-4(3H)-pyrimidinone.

0

HN-^M^R

0

hn^-N^R, I R-»

Table I I I . Phosphorylation of 6-alditylaminopyrimidines and pteridines.

Product Yield

(%)

H P L C retention time (min)

Phosphateb

content (mol/mol)

7a 31.1 28.6 0.90 8a 23.5 13.0 1.05 9a 24.8 9.9 0.85

10 a 21.9 9.8 0.90 11a 43.8 8.9 1.15 12 a 35.3 12.6 0.95 13 a 37.0 11.2 0.80 5a 7.0 36.5 0.92

14 a 14.9 35.6 0.85

a Column, Nucleosil RP18, 4x250 mm; eluent: 10 mM ammonium formate/10 mM formic acid pH 3.7; flow rate, 0.7 ml/min; b determined after hydrolysis with alkaline phosphatase.

The reaction of the 5 ,6-diaminopyrimidine type compounds 2 and 4 with diacetyl yields the pter idine derivatives 5a and 14a [22], These compounds can also be prepared by direct phosphorylat ion of the corresponding pteridines 5 and 14 with chlorophos-phoric acid. However , the yield of the direct phos-phorylat ion reaction was relatively low.

R 1 R2 «3 R, R2 «3 11 OH NO arabi ty l 11a OH NO 5 - p h o s p h o a r a b i t y l

12 OH NO xylityl 12a OH NO 5 - p h o s p h o x y l i t y l

13 OH NO ethy l 13a OH NO p h o s p h o e t h y l

This work was suppor ted by the Deutsche For-schungsgemeinschaft and the Fonds der Chemischen Industr ie . We thank Angel ika Kohnle for expert technical and secretarial assistance and Dr . Peter Rauschenbach for help and advice.

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1364 P. N i e l s e n - A . Bacher • Biosynthesis of Riboflavin 1364

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