Cis-isohumulone

Isohumulone. The mixture of cis- and /rcw -isohumulone. Similarly iso-cohumulone refers to the mixture of cw- and /r /i -isocohumulone and isoadhumulone refers to the mixture of cis- and treats- isoadhumulone. cis-Isohumulone. The iso-a-acid with empirical formula C21H30O5. It is an oil with the higher partition coefficient in a phase system of a hydrocarbon and a buffer, and contains an isovaleryl side-chain. Cis means that the 3-methyl-2-butenyl side-chain and the tertiary hydroxyl group are on the same side of the ring. 

Preparative isolation of the two isohumulones was achieved by reversed phase partition chromatography (chloroform as stationary phase on Hyflo Super Cel buffers with 25% methanol as mobile phase) (12) and by CCD (3000 transfers in the two-phase system iso-octane aqueous buffer pH 5.5) (13). Originally, the compound in the band with K 0.61 was called isohumulone A, the compound of the band with K 0.784 isohumulone B. This was unfortunate, considering the possible confusion with Weichharz A and Weichharz B of Windisch (5). Afterwards, the compounds were appropriately named trans and cis isohumulone (see 5.1.2.). The isohumulones have also been separated on a preparative scale by thin layer chromatography (Kieselgel H benzene ether 16 1 as eluent) (14). 

The NMR spectra of both isomers display the appropriate signals for the protons in the 3-methylbutanoyl group, one 3-methyl-2-butenyl side chain and one 4-methyl-3-pentenoyl entity. Distinction can be made between the epimeric isohumulones. The least hindered, more stable cis compound clearly shows a more simple pattern (21). The geminal dimethyl groups of the alkenyl side chain at C-5 occur as a singlet in the cis form and as two singlets in the trans form. The ring proton at C-5 is a distinct triplet for cis isohumulone and a doublet of doublets for trans isohumulone. 

Also, the methylene protons of the alkenyl side chain at C-5 absorb at different S values in trans isohumulone, forming an AB-spin system. In cis isohumulone a simple triplet signal is observed. 

The isomer with the highest distribution coefficient occurs usually as a light-yellow oil. By avoiding acidification of the aqueous phase upon isolation of the relevant CCD band, cis isohumulone or cis 2-(3-methylbutanoyl)-5-(3-methyl-2-butenyl)-3,4-di-hydroxy-4-(4-methyl-3-pentenoyl)-2-cyclopentenone (65, Fig. 36) can be obtained in... 

The isohumulones are racemized to the extent of about 15%, which may be due to a partly non-stereoselective protonation upon formation of the intermediate or to epimerization after formation of the isohumulones. Indeed, trans isohumulone can be converted to cis isohumulone at a pH value higher than 9. A true thermodynamic equilibrium can not be reached, because cis isohumulone degrades faster with increasing pH. 

Apparently, the composition of the isohumulone mixture depends on the medium in which it is formed. This conclusion contradicts previous ideas (45). Table 9 displays the relative percentages of the isohumulones and the corresponding reaction conditions. The ratio trans isohumulone cis isohumulone of 32 68 is the composition in normal brewing conditions. Cis isohumulone is the thermodynamically most stable compound, since the two large vicinal side chains are in the trans configuration. 

The considerable increase of the isomerization rate, caused by a divalent cation such as magnesium (46) or by strong alkali, is due to the formation of the di-anion (47). The isomerization, catalyzed by magnesium(ll) ions, is very remarkable. The conversion proceeds with a yield of at least 90% within 10 min at 70°C and the ratio cis isohumulone trans isohumulone is 55 45 (48). 

Epimerization of the isohumulones, i.e. the interconversion of cis into trans isohumulone and vice versa, proceeds via inversion of one chiral centre. This process can only be studied in aqueous alkaline medium, since interfering reactions occur in acidic conditions. As the base becomes stronger, degradation also increases (see 8.4.1.4.). The optimum pH value is around 11.0. In these conditions the degradation rate of cis isohumulone is faster th n the epimerization rate. In trans isohumulone the epimerization rate (half-lifetime 380 min) is faster than the degradation rate. 

Epimerization at C-5 can occur directly via the anion in a-position with respect to the carbonyl group. In this case the enantiomer of the commonly found cis isohumulone is formed. On the other hand, epimerization via the quaternary ring carbon atom C-4 can only occur via ring expansion to humulone, which then gives rise to the mixture of... 

In practice, the isohumulones can indeed be converted to humulone, since the compounds all possess an acyloin entity and the acyloin rearrangement is reversible. Up to 2% humulone can be isolated by isomerization of trans isohumulone in aqueous buffers of pH 10.0 and 11.0 (80). The yield of humulone would even amount to 10%, when cis isohumulone is shaken in the two-phase system iso-octane aqueous buffer pH 5.0 (81,82). It has been found that the conversion of the isohumulones to humulone occurs between pH values of 2.5 and 12.0 (66,83). The same phenomenon is found upon heating to 100°C with a maximum yield of 15% after 20 h. 

Further heating causes decomposition. Consequently, the isohumulones can never be obtained in pure form by distillation because of humulone impurities. It can not be concluded whether traces of humulone, present in beer (84), are due to non-reacted humulone from hops or to conversion of the isohumulones. The ring expansion most probably follows the same stereochemical pathway as the ring contraction, which is reflected in the degree of racemization (about 15%) for both reactions. It is found that cis isohumulone, obtained from trans isohumulone, is racemized for 45% in agreement with the reaction sequence humulone - trans isohumulone - humulone - cis isohumulone. Trans isohumulone obviously epimerizes to cis isohumulone via humulone in aqueous buffer solutions up to pH 12.0. Isomerization via the carbanion at C-5, which accounts for the epimerization of the humulinic acids (see 8.4.1.4.), may occur in stronger alkaline medium, but it is then competitive with degradation reactions. 

When the humulinic acids are formed either from humulone or from the isohumulones, the ratio of (+) cis (-) trans is 7 3 (35). The percentage of cis humulinic acid decreases as a function of time to ca. 10% of the mixture. Conversion of cis to trans humulinic acid may occur via proton abstraction or via enolizatlon at both chiral centres. This means that the four stereo-isomers may be interconverted. When only one chiral carbon atom is epimerized, isomerization is the result. There is no loss of optical activity when the epimerization occurs at C-4, while inversion at C-5 causes racemization. Isomerization at both centres will be accompanied by racemization. Humulone must be converted first to the isohumulones, which further may be deacylated to the humulinic acids (36). In principle, this reaction can start from both epimeric isohumulones, but cis isohumulone is hydrolyzed much faster than trans isohumulone. In fact, hydrolysis of trans isohumulone is only observed in conditions, whereby trans isohumulone is epimerized to cis isohumulone. This phenomenon does not exclude direct hydrolysis, but the fraction of trans isohumulone, that is converted via the cis epimer, will probably be much higher. Up to pH 12 hydrolysis is slower than epimerization, while above pH 12 the formation of humulinic acids increases much faster than epimerization. As a consequence, it is possible to obtain a rather high conversion rate of trans to cis isohumulone without appreciable degradation. 

In NaOH 2 N at 50°C, little cis isohumulone is obtained, since it is almost immediately converted to the humulinic acids. This indicates that the formation rate of the humulinic acids is determined by the epimerization from trans to cis isohumulone, which is the slowest step in the reaction sequence. In these conditions trans isohumulone reacts 3.6 times slower than cis isohumulone. It may thus be concluded that only cis isohumulone is deacylated to the humulinic acids. 

This may be explained by the favourable steric conditions for reaction of the base with the carbonyl function of the 4-methyl-3-pentenoyl side chain, which in cis isohumulone is in the trans position relative to the vicinal 3-methyl-2-butenyl side chain. Through a retro carbanion addition to the carbonyl group a stabilized carbanion is generated at C-4. Inversion should occur smoothly giving a cis-trans mixture of humulinic acids in a ratio of 7 3 by a kinetically controlled protonation. 

The formation of (-i-) trans humulinic acid from (+) cis humulinic acid causes obviously strong racemization of (-) trans humulinic acid, which had originally been formed from cis isohumulone. Depending on the reaction conditions and the racemization time, an excess of (+) trans humulinic acid may result. As proof, (-i-) cis humulinic acid has been epimerized in 2 N NaOH at 50°C during 3 h, leading to (+) trans humulinic acid. 

Fig.119. Chromatograms for the magnesium salt mentioned in Table 24 on the two discussed columns. BHT was added to the sample solution. The first doublet is for the trans and cis isocohumulones, the next triplet is for the trans and cis isohumulones and the isoadhumulones. Varian 5040 LC with Varian detector at 270 nm. 1 ml solvent per minute. 5 pm packing particles. Left (A) RoSiL-C18 for "Hop Acids Analysis", right (B) Nucleosil-CI 8 for "Hop Analysis". The IS is 2,6-di-t.butylphenol.

Fig. 120. Micro-LC of an iso-octane extract of lager beer. Column 210 mm x 0.32 mm i.d., 5 pm RoSiL-C18 (for "Hop Acids Analysis"). Mobile phase as described before, except for 5 % Cetavlon hydroxide added. Flowrate 6.4 pl.min. Pressure 104 kg.cm 2. Detection at 270 nm with Varian 2050 UV detector provided with a miniaturized detection cell (320 pm capillary). Sample loop injection of 200 pi of the iso-octane extract. Peak 1 trans and cis isocohumulones. Peak 2 trans and cis isohumulones + isoadhumulones Peak 3 2.6-di-t.butylphenol (IS).

We have assumed that isomerization is accompanied by about 15% racemization. Equilibration of either trans or cis isohumulone would proceed via humulone and be accompanied by 15% racemization on each conversion from five- to six-membered ring structures and vice versa. This assumption has never been proved... 

Cis or trans isohumulone (1.5 g 4.14 x 10 mol) in iso-octane (50 ml) is added, under stirring, to sodium borohydride in water (50 ml) during 18 h at ambient temperature. For cis isohumulone four equivalents of sodium borohydride (0.175 g) are used, for trans isohumulone eight equivalents (0.35 g). The reaction mixture is subsequently acidfied to pH 1 (HCI), extracted with iso-octane, dried and concentrated to a viscous oil (1.4 g), which is further separated by CCD in the two-phase system iso-octane aqeous buffer pH 5.4. After 450 transfers the K values for trans... 

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