Phosphates of riboflavin and riboflavin analogs: A reinvestigation by high-performance liquid chromatography

ANALYTICAL

BIOCHEMISTRY

Phosphates

130,

359-368

(1983)

of Riboflavin and Riboflavin Analogs: A Reinvestigation by High-Performance Liquid Chromatography

PETER NIELSEN,~ETER RAUSCHENBACH,AND ADELBERT BACHER' Lehrstuhl

fir Organische Lichtenbergstrasse

Chemie und Biochemie der Technischen Universitiit 4, D 8046 Garching, German Federal Republic

Miinchen,

Received August 17, 1982 Phosphoric acid esters of riboflavin can be easily separated by reverse-phase high-performance liquid chromatography using eluants of 0.1 M ammonium formate in aqueous methanol. Commercial FMN preparations contained seven different flavin phosphates; the content of riboflavin S-phosphate was 70-7590 and is in agreement with previous studies. Millimole amounts of crude FMN can be processed by preparative HPLC. The method permits the preparation of >99%pure S-FUN. The following compounds were isolated in pure form and their structures determined: riboflavin 4’-phosphate, riboflavin 3’-phosphate, riboflavin 4,5’diphosphate; riboflavin 3’,4’diphosphate, and riboflavin 3’,5’-diphosphate. The latter compound binds tightly to apoflavodoxin from Megasphaeru elsdenii (&, = 9.7 X lo-’ M). The bound flavin has high catalytic activity, thus representing a novel type of FMN analog. A wide variety of structural analogs of FMN can be obtained in pure form by preparative HPLC. KEY WORDS: FMN; FMN analogs; riboflavin monophosphates: riboflavin diphosphates; highperformance liquid chromatography.

Nagelschneider and Hemmerich (8). It has been shown by various authors that 5’-FMN2 accounts only for about 70% of the total flavins in commercial FMN preparations. All published synthetic procedures lead to preparations with significant amounts of non-5’flavin phosphates. Considerable efforts have been made to purify the 5’-phosphate from these mixtures. By ion-exchange chromatography, the contaminating flavins can be reduced to about 15% (11). Preparative thinlayer chromatography yields preparations of about 95% purity, but the method is limited

The flavin coenzymes have a central role in biochemical redox reactions. FMN was discovered by Theorell (1) as the cofactor of old yellow enzyme. Soon afterward, the first synthetic preparation of FMN was reported by Kuhn, et al. (2). The interaction of FMN with apoenzymes has been studied in considerable detail. A valuable tool for the elucidation of flavin enzyme mechanisms was found in the replacement of FMN by a wide variety of structural analogs (3-5). For such investigations it is usually important to use the respective cofactor or cofactor analog in pure form. However, in spite of considerable efforts, the preparation of pure FMN and FMN analogs remained a problem. Phosphoric acid esters of riboflavin and its analogs can be prepared by chemical phosphorylation (6-8) or by enzymatic procedures (4,9,10). Both techniques are severely limited by inherent shortcomings. The chemical approach has been reviewed by Scola-

* Abbreviations used: 5’-FMN, riboflavin 5’-phosphate; 4’-FMN, riboflavin 4’phosphate; 3’-FMN, riboflavin 3’phosphate; 4’,5’-RDP, riboflavin 4’,5’-diphosphate; 3’,5’RDP, riboflavin 3’,5’-diphosphate; 3’,4’-RDP, riboflavin 3’,4’-diphosphate; 4’,5’-RCP, riboflavin 4’,5’-cyclophosphate; 5-deazaFMN, 5-deazariboflavin S-phosphate; 5deaza-3’,5’-RDP, 5deazariboflavin 3’,5’diphosphate; 5deaza-4’,5’-RDP, 5-deazariboflavin 4,Sdiphosphate; 5’LUP, 6,7-dimethyl-S-ribityllumazine S-phosphate; 5’-APP, 2-amino-5-nitro-6-ribitylamino-4(3H)-pyrimidinone 5’phosphate; 5’-OPP, 5-nitro-6-tibitylamino-2,4( 1H,3H)pyrimidine-dione S-phosphate.

’ To whom all correspondence should be addressed. 359

0003-2697183 $3.00 Copyright Q 1983 by Academic Press. Inc. All rights of reproductmn m any form reserved.

360

NIELSEN,

RAUSCHENBACH,

to small quantities (8). Purification of larger amounts by column chromatography on silica gel has been reported, but rigorous analytical evidence for the obtained purity is lacking (12). Highly purified S-FMN containing less than 1% contaminating flavins was obtained by affinity chromatography of crude FMN on immobilized apoflavodoxin from Megasphaera elsdenii (13). This method can also be applied to the purification of the 5’phosphate of riboflavin analogs. However, the method is limited to analogs which bind tightly to the immobilized protein. The problem of regiospecific synthesis can be solved by the use of enzymatic phosphorylation methods. Phosphorylation of riboflavin by immobilized flavokinase from liver has been described (9). However, the substrate specificity of the enzyme is limited. A second enzyme system from Brevibacterium ammoniogenes has a broader substrate specificity, but produces mainly FAD and the respective analogs (4); an HPLC method for the separation of FAD and FMN from the enzymatic reaction mixture was recently described (10). Quantitative analysis of the purity of FMN and FMN analogs provides considerable problems. So far the only sensitive and reliable method was the reverse titration of the cofactor with apoflavodoxin from M. elsdenii (11). The fluorescence of S-FMN is quenched to less than 1% upon binding to the apoprotein. When an impure cofactor preparation is titrated with an excess of apoflavodoxin, the residual fluorescence indicates the amount of contaminating flavin derivatives. Thin-layer chromatography also can resolve some impurities, but the sensitivity of the method is quite limited (8). Little is known about the by-products of the chemical FMN synthesis. Scola-NagelSchneider and Hemmerich (8) obtained 4’FMN by preparative thin-layer chromatography of a mixture obtained by partial hydrolysis of riboflavin 4’,5’-cyclophosphate. It was generally assumed that 4’-FMN is the major contamination in impure FMN preparations.

AND

BACHER

The present investigation was initiated in the course of attempts to prepare 5’-phosphates of ribitylaminopyrimidines involved in the biosynthesis of riboflavin (14). Using FMN as a model compound, we found that isomeric phosphates can be conveniently separated by reverse-phase HPLC. It was the aim of this study (i) to find simple and universal methods for the analytical and preparative separation of isomeric phosphate esters of riboflavin and riboflavin analogs and (ii) to assessthe structures of the isomers of FMN and analyze their possible role as cofactor analogs. Some of the results have been reported in abstract form (14). MATERIALS

AND

METHODS

Chemicals. The following materials were obtained from commercial sources: FMN from Sigma Chemical Company (Munich), E. Merck (Darmstadt, West Germany), Boehringer-Mannheim (Mannheim, West Germany), and Serva (Heidelberg, West Germany); tetrabutylammonium hydroxide from Fluka (Buchs, Switzerland); sodium periodate from E. Merck; metronidazole, 1-decanal, and Naja naja venom from Sigma; acid and alkaline phosphatase and Photobacterium jisheri luciferase from Boehringer-Mannheim. Methanol for HPLC was destilled over a long column. 5-Deazaflavin was a gift of Professor P. Hemmerich, Konstanz, West Germany, flavodoxin from M. elsdenii was a gift of Professor S. Ghisla, Konstanz, West Germany, and partially purified hydrogenase from Clostridium kluyveri was a gift of Dr. H. Sedlmaier, Munich, West Germany. The preparation of 6,7-dimethyl-S-ribityllumazine phosphate, 2-amino-5-nitro-6-ribitylamino4( 3H)-pyrimidinone phosphate, and 5-nitro6-ribitylamino-2,4( lH,3H)-pyrimidinedione phosphate will be described separately. HPLC. Analytical HPLC was performed with instruments from Waters or Hupe & Busch equipped with columns of Nucleosil 10 Cl* (Macherey and Nagel) 250 X 4 mm. The

PHOSPHATES

OF

RIBOFLAVIN

AND

ITS

TABLE

I II III IV V VI VII VIII IX

361

A REINVESTIGATION

IO-40 ml/min. The effluent was monitored photometrically. Fractions were collected and methanol was removed by evaporation under reduced pressure. The remaining solution was lyophilized. Thin-Ia-ver chromatography and electrophoresis. Thin-layer chromatography was performed with precoated silica gel plates (kieselgel 60 FZS4,E. Merck) or cellulose plates (cellulose FZS4,E. Merck). Solvent systems are described in Table 1. Electrophoresis was performed on cellulose acetate strips (Macherey and Nagel, Diiren, West Germany) in 0.1 M borate buffer, pH 8.2, or in 0.1 M diethylbarbiturate buffer, pH 9.0, at 6 V/cm. Periodate oxidation. An aqueous solution containing 35-50 PM flavin and 160 PM sodium periodate was incubated in a photometric cell at 22.5”C. The rate of periodate consumption was recorded at 230 nm (15).

following eluants were used: eluant 1, 0.1 M ammonium for-mate, pH 3.7, in 17% methanol; eluant 2, 5 mM tetrabutylammonium formate, pH 3.5, in 27% methanol; eluant 3, 10 mM ammonium formate, pH 3.7. The flow rates were 2 ml/min (eluants 1 and 2) and 1.O ml/min (eluant 3), respectively. The effluent was monitored by uv absorbance. Preparative separations were performed using a Liquid Chromatograph 830 from Dupont (Wilmington, Del.) with Lichrosorb RP18 columns ( 10 pm), 250 X 16 mm. Eluants contained 0.1 M ammonium formate, pH 3.7, in aqueous methanol. In order to keep separation times to a minimum, the methanol content of the eluant was adjusted for each compound under study as indicated in parentheses: riboflavin monophosphates (12.5%) riboflavin diphosphates (lO.O%), 5-deazaflavin phosphates (20.0%). The flow rates were

THIN-LAYER

ANALOGS:

CHROMATOGRAPHY

I OF COMPOUNDS

STUDIED

n-Butanol/ethanol/water (50/15/35, v/v)/cellulose n-Butanol/ethanol/water (50/15/35, v/v)/kieselgel t-Butanol/water (60/40, v/v)/cellulose t-Butanol/water (60/40, v/v)/kieselgel Collidine/water (75/25, v/v)/cellulose Collidine/water (75/25, v/v)/kieselgel n-Butanollacetic acid/water (50/20/30, v/v)/cellulose n-Butanollacetic acid/water (50/20/30, v/v)/kieselgel Chloroform/methanol/ethylacetate (50/50/20, v/v)/kieselgel

Compound Riboflavin 5’-FMN 4’-FMN 3’-FMN 4’,5’-RDP 3’,5’-RDP 3’,4’-RDP 4’,5’-RCP 5-DeazaFMN 5-Deaza-3’,5’-RDP 5-Deaza-4’,5’-RDP Note. Precoated y Trailing.

1 0.42 0.27 0.30 0.28 0.16 0.15 0.15 0.40 0.36

cellulose

II

III

IV

V

VI

VII

VIII

IX

0.54 0.1 I 0.12 0. IO 0.04 0.04 0.04 0.42 0.27 0.15 0. I 5

0.41 0.62 0.64 0.62 0.67 0.61 0.65 0.65

0.81 0.55 0.56 0.55 0.52” 0.52” 0.52” 0.72 0.54 0.42” 0.52”

0.74 0.14 0.21 0.21 0.05 0.05 0.04 0.43

0.93 0.82 0.82 0.84 0.46” 0.39” 0.37” 0.95 0.85 0.18” 0.18”

0.57 0.34 0.39 0.36 0.27 0.23 0.16 0.43 0.43

0.53 0.28 0.29 0.29 0.09 0.05 0.10 0.44 0.36 0.10 0.10

0.58 0 0 0 0 0 0 0.44 0 0 0

or silicagel

were developed

with

the solvent

systems

indicated.

0.22

362

NIELSEN,

RAUSCHENBACH,

F

c

2

A

B

A

7

40

30 retention

20 time

termined by titration of apoflavodoxin with an excess of the flavin phosphate at 23°C. Determination ofjlavodoxin activity. Apoflavodoxin (5.8 X 10-r’ mol) was reconstituted with the respective FMN analog (3 X 10m9mol) at pH 7.0. The flavodoxin activity was measured as described (16). Determination of luciferase activity. An aerobic solution (0.2 ml) containing 2.9 nM P. jisheri luciferase, 3 1 mM potassium phosphate, pH 7.0, and 0.1 mM dithioerythritol was mixed with 200 ~1 of a suspension containing 25 PM I-decanal, 0.01% Triton X- 100, and 30 IIIM phosphate, pH 7.0. The bioluminescence reaction was immediately initiated by the rapid injection of solution of reduced FMN or a reduced FMN analog. FMN and FMN analogs were reduced with a slight excess of sodium dithionite. Alternatively, the flavins were photoreduced in the presence of 10 tIIM EDTA (17). Bioluminescence activity was measured at 0°C with a Biolumat 9500 from Berthold Inc., Wildbad, West Germany. Miscellaneous methods. Organic phosphate was determined after enzymatic hydrolysis with alkaline phosphatase by the procedure of Eibl and Lands ( 18). Visible and uv absorbances were measured with a PM 6 photometer (Zeiss). The following extinction coefficients (at pH 7) were used in the calculations: riboflavin monophosphates and diphosphates, t445 = 12,500 M-’ cm-’ (19); 5-deazaflavin and the respective mono- and diphosphates, t396 = 12,000 M-’ cm-’ (20); 6,7-dimethyl-8-ribityllumazine and the respective monophosphates, t410 = 10,300 M-’ cm-’ (21).

LJULD

G

AND BACHER

10

0

(mini

FIG. 1. HPLC chromatogram of commercial FMN. Column, Nucleosil 10 &., 250 X 4 mm; eluant, 100 mM ammonium formate, pH 3.7, in 17% methanol.

Phosphorylation. Riboflavin analogs were phosphorylated according to Scola-NagelSchneider and Hemmerich (8). Typical preparations used 5 mmol of chlorophosphoric acid and 19 pmol of the respective flavin analog. The reaction was terminated by the addition of 5 ml of water. The pH of the solution was adjusted to 3 by the slow addition of 25% NH40H. This solution was directly applied to the preparative HPLC column. Fluorescence titration. Apoflavodoxin was prepared from A4. elsdenii flavodoxin by the method of Mayhew (11). Fluorescence titration was performed using a Farrand MKl spectrofluorometer under the following experimental conditions: FMN, excitation 452 nm, emission 5 13 nm; 5-deazaFMN, excitation 400 nm, emission 458 nm; and 6,7-dimethyl-8-ribityllumazine 5’-phosphate, excitation 410 nm, emission 482 nm. Aliquots of the titrant were added to the measuring cell by a precision syringe. Flavin phosphates were titrated with an excess of apoflavodoxin to monitor purity. Binding constants were de-

RESULTS

Figure 1 shows the chromatogram of a commercial sample of FMN on a reversephase HPLC column using an eluant of 0.1 M ammonium formate, pH 3.7, in 17% aqueous methanol (eluant 1). The chromatogram shows one major and six minor clearly resolved peaks (compounds A-G). The major peak (compound F) accounting for about 70%

PHOSPHATES

OF

TABLE RETENTION

RIBOFLAVIN

2

TIMES (mm) OF THE COMPOUNDS STUDY IN REVERSE-PHASE HPLC

Compound

Eluant

Riboflavin 5’-FMN 4’-FMN 3’-FMN 4’,5’-RDP 3’,5’-RDP 3’,4’-RDP Deazaflavin 5-DeazaFMN 5-Deaza-3’,5’-RDP 5’-LUP 5’-OPP 5’-APP

AND

1’

32.2 17.9 15.7 13.3 10.6 7.4 4.5 84.7 48.3 24.3

Eluant

2”

5.0 12.5 Il.3 10.2 31.6 25.2 17.8

UNDER

Eluant

3’

-

16.6 6.2 13.9

Note. Column, Nucleosil IO C,, (250 X 4 mm); eluant I, 100 mM ammonium formate buffer, pH 3.7, in 17% methanol; eluant 2, 5 mM tetrabutylammonium formate, pH 3.5, in 27% methanol; eluant 3, 10 mM ammonium formate, pH 3.7. a Flow 2.0 ml/mitt. ’ Flow 1.0 ml/min.

of the total flavin content represents S-FMN as shown by reverse fluorescence titration with apoflavodoxin from 44. elsdenii. Compounds D and E were identified as 3’-FMN and 4’FMN by experiments described below. Compounds A-C were identified as riboflavin diphosphates (for details see below). Compound G is riboflavin. Compound H (0.4% of the total flavin) comigrates with 4’-FMN under these experimental conditions, but can be sepTABLE COMPOSITION

OF COMMERCIAL

FMN-PREPARATIONS:

ITS

ANALOGS:

arated by ion-exchange chromatography (see below). The compounds under study could also be resolved by ion-pair chromatography on a reverse-phase HPLC column using an eluant of 5 mM tetrabutylammonium formate, pH 3.5, in 27% aqueous methanol (eluant 2). Under these experimental conditions, the sequence of elution is reversed, i.e., the diphosphates are eluted after the monophosphates (Table 2). The compositions of FMN samples from various commercial sources are summarized in Table 3. In agreement with earlier studies, we found substantial amounts of flavin impurities in all preparations tested. Each of compounds A-F can be separated on a semipreparative scale by reverse-phase HPLC on a column of Lichrosorb (250 X 16 mm) using eluant 1 with good yield. The eluant buffer can be removed by lyophilization. The results of the preparative HPLC separation could be further improved by prechromatography of crude FMN on a conventional ion-exchange column. Several procedures for separation of crude FMN by anion-exchange chromatography have been published (19,22). We have evaluated two methods by monitoring the compositions of individual fractions by analytical HPLC. Chromatography of crude FMN on a DEAEcellulose column with a gradient of O-O.5 M triethylammonium acetate, pH 7.0, in 30% aqueous 2-propanol separates the riboflavin monophosphates from the diphosphates (Fig. 3 ANALYSIS Molar

Compound

Riboflavin

Sigma Grade I Sigma Grade II Boehringer 15405 Serva 34360 y For details

see Table

0 4.5 4.5 9.6 2.

363

A REINVESTIGATION

BY REVERSE-PHASE ratio

5’-FMN

4’-FMN

3’-FMN

76.4 71.4 71.0 64.3

12.8 13.4 14.8 15.0

6.4 5.9 5.9 6.2

HPLC

(ELUANT

1)

(%) 4’,5’-RDP 2. I 2.1 1.6 2.0

3’.5’-RDP 2.0 2.3 1.7 2.3

3’,4’-RDP 0.3 0.3 0.5 0.6

364

NIELSEN,

RAUSCHENBACH,

60 < 0

AND BACHER

62

64 66 68 fraction number

70

OS

-i m 1

0.4 5 0 0.3 .zE : 0.2 E 3 0.1 f .2

60 fracton

80 number

FIG. 2. Chromatography of commercial F’MN on DEAE-cellulose. (A) Column of DEAE-cellulose Sephacel, acetate form, 2.5 X 2 1.5 cm; gradient O-O.5 M triethylammonium formate, pH 7, in 30% 2-propanol; fraction volume, 8.2 ml. (B) Column of DEAE-cellulose DE 52, carbonate form, 2.5 X 20.3 cm; elution with 0.1 M ammonium carbonate, pH 7.8; fraction volume, 1 I ml. Insets: Flavin monophosphate composition of individual fractions. 5’-FMN (O), 4’-FMN (m), 3’-FMN (Cl).

2A). The compounds under study were eluted in the following order (fractions are indicated in parentheses): riboflavin (lo-20), riboflavin 4’,5’-cyclophosphate (40-44), riboflavin monophosphates (50-7 l), and riboflavin diphosphates (100-140). The monophosphates are eluted as a single peak; however, the leading edge is enriched for 3’-FMN, whereas the trailing edge is enriched for 4’-FMN. HPLC chromatography of the fractions from the HPLC column yielded each of compounds A-F in pure form and in acceptable-yields.

While this work was in progress, van Schagen and Miiller (23) described the chromatography of FMN on DEAE-cellulose using an eluant of 100 mM ammonium carbonate. Under these conditions, the trailing edge of the monophosphate peak contains a limited amount of rather pure S-FMN, thus providing a good source for the final purification of S-FMN by HPLC (Fig. 2B). The procedure is less well suited for the preparation of compounds A-E. Using the combination of DEAE-cellulose chromatography

PHOSPHATES

OF

RIBOFLAVIN

AND

ITS

ANALOGS:

TABLE PROPERTIES

OF HPLC

365

A REINVESTIGATION

4

PURIFIED

PHOSPHORIC

ACID ESTERS Apoflavodoxin binding

Compound Riboflavin 5’-FMN 4’-FMN 3’-FMN 4’,5’-RDP 3’,5’-RDP 3’,4’-RDP 5-DeazaFMN 5-Deaza-3’,5’-RDP 5’-LUP 5’-OPP 5-APP

Purity” (%)

Periodate consumption’

99h 99n 98.5 99.0 99.0 95.0 90.0 98.0 95.0 97.0 96.5

Phosphate content’

2.80 2.12 I .02 I .07 0.89 0 0

1.04 0.91 I .03 1.98 1.87 2.03 -

Enzymatic hydrolysis‘+

R/’ (%10)

46.0 6.1 0.1 100.0 75.0 2.6

0.99 1.25 0.78

Structures of Isomeric Ribojlavin Monophosphates The structure of compound F is clearly established as S-FMN by the reported binding to apoflavodoxin. Each of compounds D-F produced 1 mol of inorganic phosphate per mole when treated with alkaline phosphatase. The velocity of enzymatic cleavage declines in the order F > E > D (Table 4). Compounds D and E consumed 1 mol of periodate per mole, whereas 5’-FMN (compound F) consumed 2 mol. Periodate cleavage of compounds E and F yields 7,8-dimethyl-lo-for-

0.9

0.1 I

4.6

9.1

6.3 17.9 2.6

0.16 2.6 15.0

Luciferase activityg

100.0 3.36 0.16 0.31 0.55 0.16 0.10 0.10 0.02 -

’ Determined by analytical HPLC. ’ Mole periodate consumed per mole flavin. ’ Mole phosphate per mole flavin. d Relative velocity of enzymatic hydrolysis with alkaline phosphatase from bovine ’ R,, residual fluorescence after titration with a twofold excess of apoflavodoxin. ’ KD, dissociation constant. y Relative activity, S-FMN = 100%. h No impurity detectable. ’ See Ref. (26).

and HPLC, it is routinely possible to prepare S-FMN of >99% purity. The fluorescence of this material is quenched to ~1% by reverse titration with an excess of apoflavodoxin from M. elsdenii.

KDJ (nM)

intestine.

mylmethylisoalloxazine. Cleavage of compound D yields a phosphorylated isoalloxazine derivative as shown by electrophoretic migration. A quantitative kinetic study of the acidcatalyzed isomerization of the compounds under study showed the following sequence of events (details to be published): FeEeD These data are consistent with the following assignments: D, 3’FMN; E, 4’-FMN; and F, S-FMN. Compound H was susceptible to cleavage by periodate. It could not be hydrolyzed by alkaline phosphatase. Treatment with hydrochloric acid yields a mixture of the monophosphates D-F and riboflavin. Electrophoretie mobility is smaller as compared to the monophosphates. These data suggest that the

366

NIELSEN,

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AND BACHER

compound is riboflavin 4’,5’-cyclophosphate, which has been previously described (8). Structures of Riboflavin Diphosphates The compounds A-C migrate faster than monophosphates in electrophoresis and contain approximately 2 mol of bound phosphate per mole (Table 4). They are not affected by phosphodiesterase. Alkaline phosphatase converts them to monophosphates and finally to riboflavin. In detail, compound C yields 4’FMN and riboflavin, whereas compounds A and B yield 3’-FMN and riboflavin. Partial hydrolysis of compound B to 3’-FMN occurs much faster as compared to compound A. Only compound C is susceptible to cleavage by periodate (1 mol of periodate per mole). The cleavage product contains no phosphate. These data are consistent with the following assignments: compound A, 3’,4’-riboflavin diphosphate; compound B, 3’,5’-riboflavin diphosphate; and compound C, 4’,5’-riboflavin diphosphate. HPLC Purification of Phosphates of Flavin Analogs We were able to purify several FMN analogs from crude mixtures by preparative HPLC. 5-Deazaflavin was phosphorylated with chlorophosphoric acid according to Scala-Nagelschneider and Hemmerich (8). The crude phosphorylation mixture could be directly applied to the HPLC column without prior removal of the inorganic phosphate. The elution pattern was similar to that of crude FMN. Other phosphoribityl compounds included in the study were 6,7-dimethyl-8-r& bityllumazine phosphate, 2-amino-5-nitro-6ribitylamino-4(3H)-pyrimidinone phosphate, and 5-nitro-6-ribityl-amino-2,4( lH,3H)-pyrimidinedione phosphate. Purity of the respective 5’-phosphate prepared by HPLC from the isomer mixture was monitored by analytical HPLC and, where possible, by reverse titration with apoflavodoxin (Table 4). It was possible in each case to purify milligram amounts of the respective 5-phosphate from the isomer mixture in high yields.

3’5.RDP

(uM)

FIG. 3. Binding of riboflavin 3’,5’diphosphate (3’,5’RDP) by apoflavodoxin from M. elsdenii. A solution containing 7.7 X lo-’ M apoflavodoxin, 10 mM sodium acetate, pH 6.0, and 0.2 M sodium chloride was titrated with 2.3 X 10m5M 3’,5’-riboflavin diphosphate at 23°C. After each addition of flavin, the system was allowed to reach equilibrium before reading.

Interactions

with Apoproteins

Riboflavin 3’,5’-diphosphate binds rather tightly to apoflavodoxin from M. elsdenii as shown by fluorescence titration (Fig. 3). The dissociation constant was 9.7 X 1OT9M, about 2 orders of magnitude larger as compared to the natural cofactor, 5’-FMN (&, = 1.1 X lo-” M; literature (24), 4.26 X 10-l’ M). Apoflavodoxin reconstituted with riboflavin 3’,5’-diphosphate catalyzed the transfer of reduction equivalents from H2 via hydrogenase from C. kluyveri to metronidazole with 107% efficiency as compared to native flavodoxin. Several other compounds studied could bind to apoflavodoxin, but had no detectable coenzyme activity. Some of the compounds studied had limited catalytic activity with luciferase from P. fisheri (Table 4). DISCUSSION

We describe HPLC techniques which can be used for the analytical and preparative separation of isomeric phosphoric acid esters of riboflavin and riboflavin analogs. Good analytical separation could be achieved both by reverse-phase HPLC and by ion-pair chro-

PHOSPHATES

OF RIBOFLAVIN

AND ITS ANALOGS:

A REINVESTIGATION

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matography. Riboflavin diphosphates showed date, and (iv) kinetic studies of the acid-catmore retention than monophosphates in ionalyzed isomerization. pair chromatography. The reverse was true in We have found that riboflavin 3’,5’-disimple reverse-phase chromatography. For phosphate binds tightly to apoflavodoxin from routine application and for preparative work, M. elsdenii and can act as a cofactor for the we found the simple reverse-phase HPLC enzyme with the same efficiency as 5’-FMN. preferable. In combination with conventional We conclude that a bulky substituent in the ion-exchange chromatography, preparative 3’-position but not the 4’-position is tolerated HPLC can provide millimole amounts of by the enzyme. Several of the compounds highly purified compounds. Whereas S-FMN studied have cofactor activity with luciferase and some of its analogs could previously be from P. fisheri (Table 4). obtained only by the use of sophisticated techThe isomers of FMN and the diphosphates niques using flavokinase or immobilized fla- are a new class of potential cofactor analogs vodoxin, the present techniques allow the which may be helpful in understanding the rapid preparation of the various phosphates interaction between the ribityl side chain of of riboflavin and its analogs. It is noteworthy FMN and a respective apoenzyme. that preparative HPLC can be successfully applied to crude mixtures after chlorophosACKNOWLEDGMENTS phoric acid phosphorylation without prior removal of inorganic phosphate. Thus, the The technical assistance of F. Wendling, M. Bunz, and method should be particularly useful in work G. Hansen is gratefully acknowledged. We thank Professor H. Simon for his continuous interest and support and with isotopically labeled material or with rare Professor S. Ghisla for a generous gift of flavodoxin. This analogs of riboflavin. It has also been shown work was supported by the Deutsche Forschungsgemeinthat the separation method can be applied schaft, Grant Ba 574/6-5. successfully to a wide variety of phosphoribityl compounds, whereas the best earlier REFERENCES methods, i.e., enzymatic phosphorylation or affinity chromatography on immobilized proTheorell, H. (1935) Biochem. Z. 275, 37. Kuhn, R., Rudy, H., and Weygand, F. (1936) Chem. tein, depended on the interaction of the flavin Ber. 69(B), 1543- 1547. analog under study with the respective proWalsh, C. (1980) Act. Chem. Rex 13, 148-155. tein. Walsh, C., Fisher, J., Spencer, R., Graham, D. W.. Little has been known so far about the isoAshton, W. T., Brown, J. E.. Brown, R. D., and mers of FMN. Scala-Nagelschneider and Rogers, E. F., (1978) Biochemistry 17, 1942-1951. 5. Massey, V., and Hemmerich, P. (1982) in Flavins Hemmerich (8) reported the purification of and Flavoproteins (Massey, V., and Williams, 4’-FMN by preparative thin-layer chromatogC. H., eds.), pp. 83-96, Elsevier, Amsterdam/New raphy. This compound was considered as the York. major contamination of commercial FMN. Forrest, H. S., and Todd, A. R. (1950) .I. Chem. Sot. Recently Miller and Moonen have studied 3295-3299. Flexser. L. A., and Farkas, W. G. (1952) U. S. Patent commercial FMN by “P NMR and observed 2610179; Chem. Abstr. 47, 8781g (1953). seven signals which were assigned to 5’-FMN, Scala-Nagelschneider, G., and Hemmerich, P. (1976) 4’-FMN, 3’-FMN, and 2’-FMN (25). HowEur. J. Biochem. 66, 567-577. ever, the compounds were not studied in pure Merrill, A. H., Jr., and McCormick, D. B. (1980) in form, and the assignments remain somewhat Methods in Enzymology, (McCormick, D. B., and Wright, L. D., eds.), Vol. 66, Part E. pp. 287-290, tentative. We have isolated in pure form five Academic Press, New York. riboflavin phosphates in addition to S-FMN. 10. Light, D. R., Walsh, C., and Marletta, M. A. (1980) The structures have been assigned on the basis Anal. Biochem. 109, 87-93. of(i) phosphate content, (ii) phosphatase-cat11. Wassink, J. H., and Mayhew, S. G. (1975) Anal. alyzed hydrolysis, (iii) reaction with perioBiochem. 68, 609-6 16.

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