Synthesis of Isoflavone Conjugates

Abstract

During this study different approaches were studied to obtain isoflavone sulphates, glucuronides and sulphoglucuronides. Three isoflavone disulphates (daidzein-di-O-sulphate, genistein-di-O-sulphate and glycitein-di-O-sulphate) and three isoflavonoid disulphates (dihydrodaidzein-di-O-sulphate, dihydrogenistein-di-O-sulphate and equol-di-O-sulphate) were synthesised in moderate yields by using in situ prepared pyridine sulphur trioxide complex, made from chlorosulphonic acid and pyridine. These disulphated compounds can be used to develop analytical procedures and study the biological activity of disulphated products.

As the use of the HPLC-MS methods in the field of isoflavones has increased its popularity, deuterated isoflavone disulphates were synthesised. A new microwave assisted deuteration method, using CF3COOD, was developed for this purpose. Three polydeuterated isoflavone disulphates (daidzein-d6-di-O-sulphate, genistein-d4-di-O-sulphate and glycitein-d6-di-O-sulphate) were obtained in moderate yields with high isotopic purity.

A synthetic method was developed for daidzein sulphoglucuronide (daidzein-7-O-b-D-glucuronide-4´-O-sulphate), which is a major metabolite in rat bile. By using protection/deprotection steps, the desired product was finally obtained in moderate yield. The method developed can be used in further studies of synthesis of isoflavonoid mixed conjugates. As a part of this study, the structure of naturally occurring daidzein-4´-O-b-glucoside was verified. Different glycosidation methods are reviewed and possible factors affecting the stereoselectivity are discussed.

The study of the selective chlorination of isoflavones was a consequence of the observed unexpected chlorination during the synthesis of isoflavone acid chlorides by thionyl chloride. This fascinating phenomenon was investigated further with various isoflavones and as a result a method for producing isoflavone chlorides (8-chlorogenistein, 6,8-dichlorogenistein and 6,8-dichlorobiochanin A) was developed.

Protecting groups played a great role during this study, which led to an intensive study on them. A regioselective protection method was developed by using direct introduction of the protecting group (Benzyl and Benzoyl) to positions 7-O or 4´-O in daidzein, genistein and glycitein with t-BuOK as a base in DMF in moderate yields. The possibility of exploiting the transesterification was also investigated. It was observed that by using K2CO3 as a base in DMF, daidzein, genistein and glycitein could be benzoylated at position 4´-O selectively, in the presence of the more acidic 7 hydroxy group. Transesterification also proved to be useful in the glycosidation of isoflavones at position 7-O, starting from 7-O-benzoylated isoflavones.

Different carboxylic acid derivatives were synthesised for use either in the development of radioimmunoassay (7-O-carboxymethylglycitein and 4´-O-carboxymethylglycitein) or synthesis of daunorubicin isoflavone derivative for biological testing (7-O-carboxypropylbiochanin A and 7-O-carboxypropylgenistein).

Tutkimuksessa tutkittiin ja kehitettiin synteesireittejä erilaisten isoflavonijohdannaisten valmistamista varten. Isoflavonit ovat polyfenoleita, joita esiintyy kasvikunnan tuotteissa, pääasiallisesti palkokasveissa. Tutkimuksessa syntetisoitiin isoflavonien metaboliitteja kuten, isoflavoni-disulfaatteja sekä tutkittiin isoflavonisulfoglukuronidien synteesiä valmistamalla daitseiinisulfoglukuronidi. Näitä kyseisiä yhdisteitä voidaan käyttää malliaineina tutkittaessa isoflavonien metaboliaa ihmiselimistössä sekä selvitettäessä syntetisoitujen metaboliatuotteiden mahdollisia biologisia aktiivisuuksia. Tutkimuksen aikana kehitettiin myös menetelmä deuteroitujen isoflavoni-disulfaattien syntetisoimiseksi. Leimattuja isoflavoni-disulfaatteja voidaan käyttää kehitettäessä analyyttisiä menetelmiä isoflavoni-disulfaattien määrittämiseen fysiologisista matriiseista. Mielenkiintoinen ja harvinainen aromaattisen renkaan klooraus tionyylikloridilla onnistuttiin näyttämään todeksi, ja näin mahdollistamaan bakteeriperäisten isoflavonikloridien syntetisointi. Lisäksi tutkittiin useiden eri suojaryhmien soveltuvuutta isoflavonijohdannaisten syntetisoimiseksi.