Preparation of flavocoenzyme isotopologues by biotransformation of purines

Article abstract of DOI:10.1021/jo502480w

Isotope-labeled flavins are crucial reporters for many biophysical studies of flavoproteins. A purine-deficient Escherichia coli strain engineered for expression of the ribAGH genes of Bacillus subtilis converts isotope-labeled purine supplements into the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine, with yields up to 40%. The fermentation products can subsequently be converted into isotope-labeled riboflavin and the cognate flavocoenzymes, FMN and FAD, by in vitro biotransformation with better than 90% yield. Using this approach, more than 100 single or multiple ¹³C-, ¹⁵N-, ¹⁷O-, and ¹⁸O-labeled isotopologues of these cofactors and ligands become easily accessible, enabling advanced ligand-based spectroscopy of flavoproteins and lumazine receptor proteins at unprecedented resolution.

Full text of DOI:10.1021/jo502480w

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Article
Preparation of flavocoenzyme isotopologues by biotransformation of purines
Boris Illarionov, Feng Zhu, Wolfgang Eisenreich, Adelbert Bacher, Stefan Weber, and Markus Fischer
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/jo502480w • Publication Date (Web): 30 Jan 2015
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Preparation of flavocoenzyme isotopologues by biotransformation of
purines
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Boris Illarionov,‡ Feng Zhu,‡ Wolfgang Eisenreich,ǁ Adelbert Bacher, ǁ,‡ Stefan Weber § and
Markus Fischer ‡*
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‡ Hamburg School of Food Science, University of Hamburg, Grindelallee 117, D-20146
Hamburg, Germany
ǁ Department Chemie, Technische Universität München, Lichtenbergstraße 4, D-85748,
Germany;
§ Institut für Physikalische Chemie, Albert-Ludwigs-Universität Freiburg, Albertstraße 21
D-79104 Freiburg, Germany
Markus.Fischer@chemie.uni-hamburg.de
* To whom correspondence should be addressed
Table of Contents Graphic
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Abstract
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Isotope-labeled flavins are crucial reporters for many biophysical studies of flavoproteins. A
purine-deficient Escherichia coli strain engineered for expression of the ribAGH genes of
Bacillus subtilis converts isotope-labeled purine supplements into the riboflavin precursor,
6,7-dimethyl-8-ribityllumazine, with yields up to 40 %. The fermentation products can
subsequently be converted into isotope-labeled riboflavin and the cognate flavocoenzymes,
FMN and FAD, by in vitro biotransformation with better than 90 % yield. Using this
approach, more than 100 single or multiple 13C, 15N, 17O and 18O-labeled isotopologues of
these cofactors and ligands become easily accessible enabling advanced ligand-based
spectroscopy of flavoproteins and lumazine receptor proteins at unprecedented resolution.
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Introduction
Flavocoenzymes are absolutely required in all organisms where they serve as cofactors
for a wide variety of redox reactions involving one-electron and two-electron reactions.1
Moreover, various non-redox proteins including certain dehydrogenases, DNA repair
enzymes, optical transponders and blue light receptors depend on flavin cofactors.2-4
Various spectroscopic techniques used for the investigation of flavoproteins, such as
NMR, EPR and IR spectroscopy benefit from the introduction of stable isotope labels into the
flavin cofactors. Specifically, 13C and 15N labelings result in substantial sensitivity gains for
13C and 15N NMR, and may be exploited for signal enhancement in photochemically-induced
dynamic nuclear polarization NMR (photo-CIDNP NMR).5-7 To probe electronic structures of
organic molecules via the detection of hyperfine couplings using EPR-derived hyperfine
spectroscopies such as ENDOR8,9, ESEEM10, or HYSCORE11, (i) 12C-to-13C replacement is
indispensable because of these two isotopes only 13C carries a nuclear spin, and (ii) 14N-to-15N
substitution provides spectral simplification due to the lack of a quadrupole moment of 15N,
and means to unambiguously assign nitrogen hyperfine couplings due to the different strength
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of the interaction of the unpaired electron spin with 14N and 15N. Both labelings are
furthermore prerequisite for vibrational assignments in IR spectroscopy.
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Methods for chemical and for enzyme-assisted synthesis of flavocoenzymes with site-
specific stable-isotope labeling have been developed over a period of several decades (for
review see12). Notably, certain isotopologues carrying 13C and/or 15N in the pyrimidine and/or
pyrazine rings of the isoalloxazine chromophore can be obtained with relative ease by
chemical synthesis using isotopologues of barbituric acid as synthones.13,14 The benzenoid
ring of flavins can be labeled with 13C by enzyme-assisted synthesis using commercially
available isotopologues of glucose as synthones.15 More recently, a recombinant Escherichia
coli strain that had been engineered to express the ribA, ribG and ribH genes of Bacillus
subtilis was used to convert universally or specifically 13C-labeled glucose into 6,7-dimethyl-
8-ribityllumazine, the direct biosynthetic precursor of riboflavin.16 Depending on the label
distribution in the proffered glucose, fermentation afforded the lumazine derivative either
with universal 13C labeling or as a mixture of different isotopologues that can be transformed
into riboflavin with near-quantitative yield by enzyme-mediated biotransformation in vitro
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(cf. for details). However, riboflavin isotopologues carrying single C labels in either the 4
or 10a position and isotopologues carrying single 15N labels in either the 1 or 3 position of the
isoalloxazine chromophore are not accessible by current technology but could be particularly
useful for certain spectroscopic experiments. In this paper, we describe the in vivo
biotransformation of isotope-labeled purines into isotope-labeled 6,7-dimethyl-8-
ribityllumazine (6, cf. Scheme 1) resp. riboflavin. Since selective labeling is technically more
easily achieved for purines than for isoalloxazines, this opens an elegant and affordable
approach to the synthesis of numerous single- or multiple-labeled flavin isotopologues that
can now be exploited as spectroscopic probes for investigation of a large group of structurally
and functionally diverse flavoproteins.
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For practical reasons, the in vivo biotransformation of purines in this study was designed
to yield the riboflavin precursor, 6,7-dimethyl-8-ribityllumazine (6, cf. Fig. 1) (rather than
riboflavin) as fermentation product, which is subsequently converted into riboflavin, FMN or
FAD by enzyme-mediated in vitro biotransformation. Notably, however, 6 is not only an
intermediate of riboflavin biosynthesis but has also been found to serve as protein cofactor in
its own right. Specifically, 6 is used as chromophore by lumazine protein, which acts as
optical transponders in bacterial luminescence and modulates both the frequency range and
the quantum yield of emitted light (for review see 18). More recently, 6 has been shown to
serve as antenna chromophore in certain DNA photolyases and as a ligand for receptors used
by the subclass of human mucosa-associated T-cells (MAIT cells).19,20 Hence, isotopologues
of 6 which are generated in the present work have the potential to contribute to the
investigation of these more recently discovered receptor proteins. Notably, the present method
can afford isotopologues of 6 which are not accessible by prior technology. It also provides
convenient access to many multiply labelled isotopologues of 6 as well as riboflavin, FMN
and FAD.
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Results and Discussion
The biosynthesis of riboflavin starts from GTP (for review see 21). By a sequence of six
enzyme-catalyzed reactions, the carbon and nitrogen atoms of GTP except C-8 and the
carbonyl oxygen in the 4 position (designated a – i in Fig. 1) are incorporated into the vitamin,
where they constitute the pyrimidine ring, the pentityl side chain and parts of the pyrazine
ring, whereas the carbon atoms of the xylene moiety are derived from the pentose phosphate
pool via 3,4-dihydroxy-2-butanone 4-phosphate (8, Scheme 1). In order to recruit that
pathway for the preparative conversion of isotope-labeled purines into the riboflavin precursor
6, a purine-deficient E. coli mutant (carrying a purF deletion) was endowed with a plasmid
directing the expression of the ribAGH genes of the Bacillus subtilis rib operon under the
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control of a T5 promoter and lac operator (the B. subtilis rib operon used comprises the
ribABGH genes, but the ribB gene specifying riboflavin synthase has been inactivated by an
F2A mutation conducive to the production of a full length protein devoid of catalytic
activity).22 Whereas the recombinant rib operon does not supply catalytically active riboflavin
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synthase,
a
limited amount of active riboflavin synthase is provided by
transcription/translation of the chromosomal ribC gene of the E. coli host under the control of
its natural promoter (somewhat unfortunately, the gene specifying riboflavin synthase is
designated ribB in B. subtilis but ribC in E. coli). When grown with purine supplements
(hypoxanthine (1), xanthine (3) or adenine), 6 is accumulated in the culture medium reaching
concentrations up to 0.3 mM (Fig. 2); riboflavin is also accumulated, albeit in lower
concentration, up to about 50 µM.
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Fig. 1. Bioconversion of purine bases by the recombinant E. coli strain PL919. Due to
deletion of the purF gene, the strain is unable to produce purines de novo.
Biosynthetically equivalent atoms are marked with letters a – i.
Since the recombinant E. coli strain has an absolute requirement for a purine source due
to deletion of the purF gene, isotope-labeled purines are biosynthetically diverted to 6 and to
riboflavin without dilution. Due to the enhanced expression of riboflavin biosynthesis
enzymes, labeled purines are efficiently converted into 6, with yields up to 40 % based on
purine. Concentrations up to 0.3 mM 6 (i.e. about 100 mg of 6 per liter of culture medium)
can be obtained in shaking cultures with glucose as carbon source (Fig. 2). However, it should
be noted that the concentration of 6 declines rapidly after reaching its maximum at early
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stationary phase; timely termination of the fermentation is therefore essential. Besides 6, the
fermentation broth also contains a minor amount of isotope-labeled riboflavin (generated by
the activity of the chromosomally encoded riboflavin synthase). Riboflavin and 6 can be
harvested easily by adsorption to Florisil® which retains both compounds with remarkable
selectivity. Subsequent to separation by cation exchange chromatography, riboflavin and 6
can be crystallized from water. Using various 13C-, 15N-, 17O- or 18O-labelled xanthine or
hypoxanthine substrates (Table 1), the resulting 6 and riboflavin isotopologues were
characterized by NMR and LC-MS techniques as shown in the Supporting Information
(Tables S1-S6).
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Fig. 2. (A) Growth of E. coli PL919 in minimal medium with supplements (80 mg/L) of
adenine ( ▲); OD600, optical density at 600 nm. (B)
■), xanthine (●) or hypoxanthine (
accumulation of 6,7-dimethyl-8-ribityllumazine (open symbols) and riboflavin (closed
symbols).
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The riboflavin precursor 6 can be converted into riboflavin with near-quantitative yield
by enzyme catalysis under strictly anaerobic conditions (required to protect the oxygen-
sensitive intermediate 9). Briefly, as shown in Scheme 1, 6 is converted into a mixture of
riboflavin and 9 by riboflavin synthase, and 6 is then regenerated from 9 by lumazine
synthase using 8 (prepared from ribose 5-phosphate) as second substrate. Riboflavin can be
recovered directly in crystalline form from the reaction mixture. Residual product can be
recovered from the mother liquor by cation exchange chromatography. Alternatively, the
crude riboflavin obtained from 6 can also be used directly as substrate for the enzyme-assisted
preparation of FMN or FAD.
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We also report optimized protocols for the conversion of isotope-labeled riboflavin into
the cognate FMN or FAD isotopologues. Near-quantitative yields can be obtained by
recycling the ATP serving as phosphate donor (Scheme 1). Moreover, the formation of FAD
is facilitated by the inclusion of inorganic pyrophosphatase in the reaction mixture. Due to the
use of the auxiliary enzymes, the spent reaction mixture contains only pyruvate, FMN or
FAD, catalytic amounts of adenosine nucleotides, and protein, which can be removed by
ultrafiltration. In many cases, the crude reaction mixtures can be used directly for the
reconstitution of apo-flavoproteins in order to generate samples for physical studies, without
prior purification. Alternatively, FMN resp. FAD can be purified by hydrophobic
chromatography.
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Scheme 1.
Methods for the synthesis of xanthine and hypoxanthine are very well documented in
the literature and enable the preparation of virtually all conceivable single-labeled and
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multiple-labeled isotopologues from simple isotope-labeled synthones such as cyanide,
bromoacetate, ammonia and/or nitrite that are all commercially available at moderate costs.23-
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Hence, numerous isotopologues of 6 and of riboflavin carrying single or multiple labels in
8
9
the heterocyclic part of the respective chromophores can now be prepared as desired for
specific spectroscopic problems (cf. Tables 1 and 2). Moreover, it is possible to predetermine
the label distribution of the heterocyclic chromophore moiety by the labeling pattern of a
purine precursor and to simultaneously introduce 13C labeling to the carbocyclic part of the
chromophore by culturing the recombinant E. coli strain on 13C-labeled glucose in
conjunction with a labeled or unlabeled purine precursor. As an example, we demonstrate the
preparation of a riboflavin isotopologue with comprehensive 13C labeling of the benzenoid
ring but with an unlabeled pyrimidine ring by growing the recombinant E. coli strain with
unlabeled hypoxanthine as purine source and [U-13C6]glucose as carbon source; the yield of
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[
6α,6,7,7α,1’,2’,3’,4’,5’-13C9]-6, based on [U-13C6]glucose, is about 1.0 %. Notably, multiple-
labeled “designer isotopologues” can open new approaches to the investigation of polarization
transfer pathways in free radical states of flavoproteins. Using this combinatorial approach,
more than 100 of single or multiple 13C, 15N, 17O and 18O-labeled isotopologues of these
cofactors and ligands become easily accessible enabling advanced ligand-based spectroscopy
of flavoproteins and lumazine receptor proteins at unprecedented resolution.
Table 1. Conversion of purine isotopologues into 6,7-dimethyl-8-ribityllumazine (6).
Purine
Supplement
Isotope
enrichment b
Carbon source
Product
Yield % a
[4-13C1]xanthine
[6-13C1]xanthine
[9-15N1]xanthine
[2-13C1]xanthine
[1-15N1]xanthine
[6-17O1]hypoxanthine
glucose
glucose
glucose
glucose
glucose
glucose
[8a-13C1]-6
[4-13C1]-6
[8-15N1]-6
[2-13C1]-6
[3-15N1]-6
[4-17O1]-6
11
8
> 97
> 97
94
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24
10
32
> 97
> 97
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[6-18O1]hypoxanthine
glucose
[4-18O1]-6
41
1 c
89 e
[6,6α,7,7α,1’,2’,3’,4’,5’-
13C9]-6
hypoxanthine
[U-13C6]glucose
>97 f
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b
Based on proffered purine, mol/mol. Isotope enrichment was determined as described in
Supporting Information, pp. 40-44. c Based on proffered [U-13C6]glucose. d Isotope enrichment of
H217O used as starting material for hypoxanthine production was 75 %. Isotope enrichment of
e
H218O as starting material for hypoxanthine production was 95 %. Isotope enrichment of [U-
f
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13C6]glucose used for fermentation was 99 atomic %.
Experimental Section
Materials. Xanthine isotopologues were obtained by published procedures.23-34 [6-
17O1]Hypoxanthine and [6-18O1]hypoxanthine were obtained from 6-bromopurine as
described in Supporting Information.
Recombinant riboflavin synthase (E. coli), lumazine synthase (B. subtilis), 3,4-
dihydroxybutanone 4-phosphate synthase (E. coli), riboflavin kinase (Schizosaccharomyces
pombe), and flavokinase/FAD-synthetase (Corynebacterium ammoniagenes) were prepared
by published procedures.17,35,36
Bacterial strain. A purine-deficient E. coli strain JC182 (λ−, e14−, purF1, thi
−
1) (E. coli
Genetic Stock Center, Yale University, New Haven, CT) was transformed with plasmids
pRFN4 16 and pREP4 37 affording the recombinant strain PL919.
[8a-13C1]6,7-Dimethyl-8-ribityllumazine. E. coli strain PL919 was grown in LB medium
with ampicillin (50 mg/L) and kanamycin (15 mg/L) overnight. The cells were harvested and
resuspended in 1 L of culture medium containing 12 g of Na2HPO4, 3 g of KH2PO4, 0.5 g of
NaCl, 2 g of NH4Cl, 60 mg of MgSO4, 30 mg of CaCl2, 5 g of glucose, 5 g of Casamino
Acids (Difco Laboratories GmbH, Augsburg, Germany), 30 mg of ampicillin, 80 mg of [4-
13C1]xanthine, 4 mL of vitamin solution (containing, per ml, pyridoxamine hydrochloride,
0.08 mg, thiamine hydrochloride, 0.04 mg, p-aminobenzoate, 0.08 mg, calcium pantothenate,
0.08 mg, biotin, 0.02 mg, folic acid, 0.04 mg, nicotinamide, 0.06 µg, cyanocobalamine, 0.4
mg) and 1 mL of trace element solution (containing, per mL, FeSO4, 4.5 mg, MnCl2, 8.5 mg,
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ZnSO4, 0.5 mg, CuCl2, 0.15 mg, NiCl2, 0.1 mg, CoCl2, 0.05 mg, H3BO3, 0.1 mg). The
suspension was incubated overnight with shaking at 30 °C. The cells were pelleted, and the
supernatant was passed through a column of Florisil (3.0 × 10 cm) that was subsequently
washed with 0.4 L of water and developed with 50 % aqueous acetone containing 10 mM
NH4OH. The effluent was concentrated to dryness under reduced pressure. The residue was
dissolved in 50 mL of water. The solution was centrifuged, and the supernatant was passed
through a column of AG 50 WX8 (200 – 400 mesh, H+ form, 1 × 20 cm) that was then
developed with water. Green-fluorescent fractions were combined and concentrated to
dryness under reduced pressure affording 22.4 mg (69 µmol) of 6,7-dimethyl-8-
ribityllumazine (yield, 11.4 % based on proffered xanthine). Yellow-fluorescent fractions
were also combined and concentrated affording 1.3 mg (3.5 ꢀmol) of riboflavin. The products
were crystallized from water. 13C enrichment at position 8a carbon atom was > 97 % (LC-
MS).
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[4-13C1]6,7-Dimethyl-8-ribityllumazine. The compound was prepared from 100 mg of [6-
13C1]xanthine as described above. Yield, 20 mg, 8.2 % based on proffered xanthine. 13C
enrichment at position 4 carbon atom was >97 % (LC-MS).
[2-13C1]6,7-Dimethyl-8-ribityllumazine. The compound was prepared from 100 mg of [2-
13C1]xanthine as described above. Yield, 20 mg, 8.2 % based on proffered xanthine. 13C
enrichment at position 4 carbon atom was >97 % (LC-MS).
[8-15N1]6,7-Dimethyl-8-ribityllumazine. The compound was prepared from 95 mg of [9-
15N1]xanthine as described above. Yield, 22 mg, 10 % based on proffered. N enrichment at
15
position 8 nitrogen atom was 94 % (LC-MS).
[3-15N1]6,7-Dimethyl-8-ribityllumazine. The compound was prepared from 16 mg of [1-
15N1]xanthine as described above. Yield, 3.3 mg, 10 % based on proffered xanthine. 15N
enrichment at position 3 nitrogen atom was 96 % (LC-MS).
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[4-17O1]6,7-Dimethyl-8-ribityllumazine. The compound was prepared from 43 mg of [6-
17O1]hypoxanthine as described above. Yield, 33 mg, 32 % based on proffered hypoxanthine.
17O enrichment at position 4 carbonyl group was 67 % (LC-MS).
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[4-18O1]6,7-Dimethyl-8-ribityllumazine. The compound was prepared from 60 mg of [6-
18O1]hypoxanthine as described above. Yield, 59 mg, 41 % based on proffered hypoxanthine
(mol per mol). 18O enrichment at position 4 carbonyl group was 87 % (LC-MS).
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[6α,6,7,7α,1’,2’,3’,4’,5’-13C9]6,7-Dimethyl-8-ribityllumazine. Fermentation was performed
as described above using [U-13C6]glucose (3 g) and unlabeled hypoxanthine (100 mg, Sigma-
Aldrich, Taufkirchen, Germany) as supplement, per liter. Yield, 55 mg, 1.03 % based on
proffered [U-13C6]glucose.
3,4-Dihydroxy-2-butanone 4-phosphate. The following procedure is based on a method
described earlier.38 A reaction mixture (40 mL) containing 100 mM Tris hydrochloride, pH
8.0, 5 mM MgCl2, 5 mM DTT, 10 mM D-ribose 5-phosphate, 25 units of
phosphoriboisomerase from spinach (Sigma, Taufkirchen, Germany) and 30 units of 3,4-
dihydroxybutanone 4-phosphate synthase was incubated at 37 °C. The pH value of the
reaction mixture was kept at 8.0 by adding aliquots of 5 M NaOH. After six h, the reaction
mixture was passed through an Ultracell ultrafiltration disc NMWL 10 kDa (Merck-Millipore,
Darmstadt, Germany). Ethanol (96%, 200 ml) was added to the filtrate, and the mixture was
centrifuged (4500 × g, 4 °C, 30 min). Barium acetate (690 mg) was added to the supernatant.
After incubation at -20 °C overnight, the sample was centrifuged (4500 × g, 4 °C, 30 min).
The pellet was resuspended in 5 ml of water. Sodium sulfate (57 mg) was added, and the
mixture was centrifuged (1000 × g, 4 °C, 10 min). The supernatant containing 3,4-dihydroxy-
o
2-butanone 4-phosphate was stored in 0.5 ml aliquots at -80 C. Universally 13C-labeled 3,4-
dihydroxy-2-butanone-4-phosphate was prepared as described earlier.38
[10a-13C1]Riboflavin. A reaction mixture containing 50 mM Tris hydrochloride, pH 7.0, 100
mM NaCl, 5 mM dithiothreitol, 60 mg of [8a-13C1]6,7-dimetyl-8-ribityllumazine, 36 mg of
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3,4-dihydroxy-2-butanone-4-phosphate, 33 units of riboflavin synthase and 33 units of
lumazine synthase in a total volume of 25 ml of was incubated at 37 °C. The formation of
riboflavin was monitored photometrically at 470 nm (ε470 = 9,600 M−1 cm−1). The yellow
precipitate was harvested by centrifugation, washed with water and dried over phosphorus
pentoxide. Yield, 62.4 mg, 92 % based on proffered 6.
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Other riboflavin isotopologues ([2-13C1]-7, [4-13C1]-7, 10-15N1]-7, [3-15N1]-7, [4-17O1]-7, [4-
18O1]-7, [5a,6,7,7α,8,8α,9,9a,1’,2’,3’,4’,5’-13C13]-7) were synthesized using the same
protocol. The starting amounts of the 6 and 8 as well as the yield of 7 are shown in Table 2.
Table 2. Synthesis of riboflavin isotopologues.
Isotope
enrichment b
Substrate A
Substrate B
Product
Yield, % a
[8a-13C1]-6 (60 mg)
[2-13C1]-6 (10 mg)
[4-13C1]-6 (10 mg)
[8-15N1]-6 (10 mg)
[3-15N1]-6 (3.0 mg)
[4-17O1]-6 (4.5 mg)
[4-18O1]-6 (5.0 mg)
8 (36 mg)
8 (6.0 mg)
8 (6.0 mg)
8 (6.0 mg)
8 (1.8 mg)
8 (2.7 mg)
8 (3.0 mg)
[10a-13C1]-7 (62.4 mg)
[2-13C1]-7 (12.0 mg)
[4-13C1]-7 (12.2 mg)
[10-15N1]-7 (12.5 mg)
[3-15N1]-7 (3.3 mg)
[4-17O1]-7 (5.4 mg)
[4-18O1]-7 (5.5 mg)
92
96
96
96
96
95
95
> 97
> 97
> 97
94
> 97
67
89
[6,6α,7,7α,1’,2’,3’,
4’,5’-13C13]-6
(5.0 mg)
Based on proffered 6, mol/mol.
Supporting Information, pp. 40-44.
[5a,6,7,7α,8,8α,9,9a,1’,
2’,3’,4’,5’-13C13]-7
(5.6 mg)
[U-13C4]-8
(3.0 mg)
95
>97
a
b
Isotope enrichment was determined as described in
[10a-13C1]-FMN from [10a-13C1]riboflavin. [10a-13C1]Riboflavin (54 mg) was suspended in
22 ml of 100 mM Tris hydrochloride, pH 8.0, containing 5 mM MgCl2, 5 mM DTT, 0.02%
NaN3, 0.5 mM ATP, 8.5 mM sodium phosphoenol pyruvate, 55 units of pyruvate kinase from
rabbit muscle and 20 units of riboflavin kinase. The mixture was incubated at 37 °C for 12 h.
The pH value of the reaction mixture was kept at 8.0 by the addition of 5 M NaOH as
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required. The progress of the reaction was monitored by thin layer chromatography
(microcrystalline cellulose plates, Macherey-Nagel; eluent, 350 mM phosphate, pH 7.0).
Typically, riboflavin was completely converted to FMN after 12 h. Yield, 62 mg, 94 % based
on proffered riboflavin. The solution was passed through an Ultracell ultrafiltration disc
NMWL 10 kDa (Merck-Millipore, Darmstadt, Germany) and stored at -80 °C. Other FMN
isotopologues ([2-13C1]-10, [4-13C1]-10, 10-15N1]-10, [3-15N1]-10, [4-17O1]-10, [4-18O1]-10,
[5a,6,7,7α,8,8α,9,9a,1’,2’,3’,4’,5’-13C13]-10) were synthesized using the same protocol. The
starting amounts of 7 were between 2 and 3 mg. The yield was between 95% and 97%.
[10a-13C1]-FMN from [8a-13C1]6,7-dimethyl-8-ribityllumazine. [8a-13C1]-6 (10 mg) was
suspended in 5 ml of 100 mM Tris hydrochloride, pH 7.6, containing 5 mM MgCl2, 5 mM
DTT, 0.02% NaN3, 6.5 mM 3,4-dihydroxy-2-butanone 4-phosphate, 0.5 mM ATP, 7.5 mM
sodium phosphoenol pyruvate, 10 units of lumazine synthase, 10 units of riboflavin synthase,
11 units of pyruvate kinase from rabbit muscle and 4 units of riboflavin kinase. The mixture
was incubated at 37 °C for 12 h. The pH value of the reaction mixture was kept at 8.0 by the
addition of 5 M NaOH as required. The progress of the reaction was monitored by thin layer
chromatography (microcrystalline cellulose plates, Macherey-Nagel; eluent, 350 mM
phosphate, pH 7.0). Typically, 6 was completely converted to FMN after 12 h. The solution
was passed through an Ultracell ultrafiltration disc NMWL 10 kDa (Merck-Millipore,
Darmstadt, Germany) and stored at -80 °C. Yield, 13 mg, 92 % based on proffered 6.
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[10a-13C1]-FAD. [10a-13C1]Riboflavin (44 mg) were resuspended in 18 ml of 100 mM Tris
hydrochloride, pH 8.0, containing 5 mM MgCl2, 5 mM DTT, 0.02% NaN3, 8.5 mM ATP and
9 mM sodium phosphoenol pyruvate. Pyruvate kinase from rabbit muscle (15 units), 20 units
of inorganic pyrophosphatase from baker’s yeast and 12 units of bifunctional
flavokinase/FAD synthetase were added. The suspension was incubated at 37 °C 12 h. The
pH value of the reaction buffer was kept at 8.0 by addition of 5 M NaOH as required. The
progress of the reaction was monitored visually (disappearance of riboflavin crystals) as well
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as by thin layer chromatography (microcrystalline cellulose plates, Macherey-Nagel; eluent,
350 mM phosphate, pH 7.0). Typically, riboflavin was completely converted to FAD within
12 h. Yield, 86 mg, 95 % based on proffered riboflavin (mol per mol). The solution was
passed through an Ultracell ultrafiltration disc NMWL 10 kDa (Merck-Millipore, Darmstadt,
Germany) and stored at -80 °C. Other FAD isotopologues ([2-13C1]-11, [4-13C1]-11, 10-15N1]-
11, [3-15N1]-11, [4-17O1]-11, [4-18O1]-11, [5a,6,7,7α,8,8α,9,9a,1’,2’,3’,4’,5’-13C13]-11) were
synthesized using the same protocol. The starting amounts of 7 were between 2 and 3 mg. The
yield was between 95% and 97%.
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Alternatively, FAD could be prepared directly from 6 as follows. [8a-13C1]-6 (10 mg) was
suspended in 5 ml of 100 mM Tris hydrochloride, pH 7.6, containing 5 mM MgCl2, 5 mM
DTT, 0.02% NaN3, 6.5 mM 3,4-dihydroxy-2-butanone 4-phosphate, 6.5 mM ATP and 7.5
mM sodium phosphoenol pyruvate. Lumazine synthase (10 units), 10 units of riboflavin
synthase, 11 units of pyruvate kinase from rabbit muscle and 10 units of flavokinase/FAD
synthetase were added and the mixture was incubated at 37 °C for 12 h. The pH value of the
reaction mixture was kept at 8.0 by addition of 5 M NaOH as required. The progress of the
reaction was monitored by thin layer chromatography (microcrystalline cellulose plates,
Macherey-Nagel; eluent, 350 mM phosphate, pH 7.0). Typically, 6 was completely converted
to FAD after 12 h. The solution was passed through an Ultracell ultrafiltration disc NMWL 10
kDa (Merck-Millipore, Darmstadt, Germany) and stored at -80 °C. Yield, 21.5 mg, 90 %
based on proffered 6 (mol per mol).
Purification of FMN and FAD. The purification procedure is based on a method described
earlier.39 Aliquots (200 µL) containing 6 mg of the FMN or FAD were applied to a reverse
phase column (Nucleosil C18, Macherey-Nagel, Düren, Germany, 250 × 21 mm) that had
been equilibrated with 35% aqueous methanol containing 0.1 M ammonium formate. The
column was developed with the same eluent at a flow rate of 15 ml/min (22 oC). The retention
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times for FMN and FAD were 6.4 min and 5.3 min, respectively. Fractions were combined
and methanol was removed by evaporation. The residue was lyophilized and stored at -20 °C.
Acknowledgements
9
This work was supported by the Deutsche Forschungsgemeinschaft (grants FI 824/6-1 and
WE 2376/4-1), and by the Hans-Fischer-Gesellschaft e.V.
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Supporting Information. LC-MS and NMR data of labeled riboflavin and 6,7-dimethyl-8-
ribityllumazine samples (including signal assignments and numerical values for chemical
shifts and coupling constants) are available free of charge via the Internet at
http://pubs.acs.org
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