A two-step synthesis of 7,8-dichloro-riboflavin with high yield

Abstract

We report a two-step protocol for the synthesis of 7,8-dichloro-riboflavin with a 2.5-fold increase in yield compared to previous five-step protocol methods.

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Flavoproteins are one of the major classes of oxidoreductases that catalyze a large variety of reactions.¹ Extensive structural and mechanistic studies have been performed to understand the basis of such versatility. Flavoenzymes can catalyze either single or double electron transfers. Depending on their ability to react with molecular oxygen, flavoenzymes can be found in dehydrogenase, monooxygenase and oxidase subclasses. In particular, oxidases and monooxygenases play a fundamental role in aerobic metabolism. Light absorption in the visible spectrum has also been correlated to photoreceptor function of some flavoproteins.^2–4

Above understanding fundamental biological processes, interest in flavoenzymes is further increasing due to multiple applications in the pharmaceutical, fine chemical, biotechnological and food industries. For the last decades, potential applications for cheap and easily renewable biocatalysts that work in mild conditions have been continuously described.⁵

Centered around its flavin cofactor, usually FAD or FMN, reactions catalyzed by a given flavoenzyme depend on the protein folding and enzyme-cofactor interaction. In nature, only a few examples of biosynthesis and biological characterization of flavin analogs have been observed.⁶ One of the most described is cofactor F420 (8-hydroxy-5-deazaflavin) and its catalytic function in several dehydrogenases.⁷ Several natural and synthesized flavin analogs have been described as inhibitors of flavoenzymes, affecting their metabolic pathways. In particular, antibiotic activity has been studied for analogs that induce growth inhibition for several microorganisms.^8–10 Flavin analogs are also extensively used to alter or enhance molecular processes of catalysis by flavoproteins. More precisely, those variants are used to analyze cofactor-enzyme covalent binding, molecular oxygen activation, enantioselectivity, semiquinone intermediate formation or photochemical properties of flavins or flavoproteins.^6,11–16

Dichloro-riboflavin (3) is one of the commonly used riboflavin analog, and it can easily be converted in FMN and FAD derivatives. Like various other riboflavin analogs that are substituted in positions 7 or 8, it is classically synthesized as described by Kuhn and coworkers.⁸ This synthesis is a five-step protocol with a rather low yield, based on monoribitylation of a di-substituted dinitrophenylene with ribamine, followed by catalyzed hydrogenation of the remaining nitro group. Ribamine must first be synthesized by reacting ribose with ammonia at high pressure and temperature. An alternative five-step synthesis of 3 has been described by Fujita and coworkers.¹⁷ In this case, ribose was directly coupled to dichloro-aniline, before nitration and catalyzed hydrogenation in the ortho position of the dichloro-phenyleneribamine to obtain 2. In both protocols, the dichloro-riboflavin was obtained by condensation of 2 with alloxan.

Commercially available substituted phenylenediamine, like 4,5-dichloro-1,2-phenylenediamine (1), potentially allows for the direct coupling with ribose under mild conditions, as described for 7,8-dimethylriboflavin or 1-deazariboflavin synthesis.^18,19 However, these previous studies pointed out that protection of one of both amine groups by tert-butyloxycarbonyl was necessary to achieve monoribitylation of the corresponding phenylenediamine. Following this idea, we used similar conditions to synthesize 3 from 1 (Scheme 1). We observed that ribitylation of Boc-protected aromatic reactant (1a) was less than 10% when we used 1a:D-ribose in a 1 : 3 molar ratio. For comparison, 92% yield was observed by using 4,5-dimethyl-1,2-phenylenediamine to synthesize the ribitylated aniline under identical conditions.¹⁶ A 1 : 6 molar ratio was necessary to maximise the yield. This indicated that dichloro substitution decreases the ribitylation efficiency, probably by decreasing the nucleophilic property of amine groups. Even though synthesis of 3 is achievable by this protocol, it requires 5 steps and leads to low yield that maybe not be appropriate for industrial applications. Therefore, we tried to obtain direct monoribitylation without Boc protection.

Scheme 1 Synthesis of the dichloro-riboflavin using Boc protection.

Our second approach is described in Scheme 2. Monoribitylation of 1 is achieved by reaction with D-ribose and sodium cyanoborohydride in methanol, at 65 °C, for 48 h. Crude product was washed with dichloromethane to remove unreacted 1. Typically, 1 : 1.5 : 3 molar ratio of 1 : D-ribose : sodium cyanoborohydride allowed synthesis of 2 in 33% yield. Higher yield could be achieved by increasing D-ribose concentration.

Scheme 2 Direct synthesis of 7,8-dichloro-riboflavin.

Condensation of 2 with alloxan was performed as previously described⁸ by using 1 : 1.5 molar ratio of 2 : alloxan, in the presence of 0.4 M boric acid in glacial acetic acid, giving 3 in only two steps with a global yield of 25% while it was only 10% for the first method.

As shown in Fig. 1, to further characterize 3, we performed spectroelectrochemical experiments. The UV spectrum of the oxidized 3 is similar to the classical dimethyl-riboflavin spectrum with maximal peaks at 341 nm and 445 nm. The redox potential of 3 was −0.30 V vs. Ag/AgCl and was determined by following the potential dependence of absorbance at 450 nm. This value is in agreement with published data.^8,20

Fig. 1 (A) The background-corrected absorption spectra of 0.33 mg mL⁻¹ 7,8-dichloro-riboflavin solution at pH 7.0 equilibrated stepwise at −0.60, −0.50, −0.45, −0.40, −0.35, −0.30, −0.25, −0.2 to −0.15 V. The background spectra was obtained during the electrolysis at −0.6 V. (B) Potential dependence of the spectral change at 450 nm, the dotted curve representing a regression line according to Nernstian equation.

In summary, we have described a more efficient and faster protocol than previous methods for the synthesis of 7,8-dichloro-riboflavin.

This work was financed in part by a European Young Investigator Award (EURYI), la Région Aquitaine and an Excellent Young Researchers Overseas Visit Program (S.T).

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Footnote

† Electronic Supplementary Information (ESI) available: Experimental details and copies of ¹H and ¹³C NMR spectra. See DOI: 10.1039/c2ra01211a/

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