Absolute Configuration of Beer's Bitter Compounds**

The science and art of making beer, likely the oldest liquid fermented by humans, stretches over millennia. Production typically involves boiling beer wort together with hops, which acts as a natural preservative,1 but the generated iso-α-acids are known to be prone to decomposition,2, 3 and consequently, more stable reduced hops extracts, such as the tetrahydro-iso-α-acids, have been developed. These latter compounds are separately produced and frequently added to beer to achieve a consistent level of bitter taste. Scheme 1 gives an overview of the iso-α-acids formed by heat-induced isomerization. 
 
 
 
Scheme 1 
 
Hops bitter acids. Humulones: R=isobutyl, cohumulones: R=isopropyl, adhumulones: R=sec-butyl. 
 
 
 
Herein, we determined the absolute configuration of several cis and trans iso-α-acids by X-ray crystallography. We show how we unequivocally assigned the chiral center in (−)-humulone to be (6S) and give absolute structures for several of its derivatives, most of which contradict the general perception circulating through the literature since 1970.4 
 
Typically, the predominant α-acid is humulone, a phloroglucinol derivative with two prenyl groups and one isovaleryl group as side chain. The process of isomerization involves contraction of the six-membered α-acid ring (through acyloin rearrangement) to form the five-membered iso-α-acid ring with two chiral centers,5 resulting in cis and trans diastereomers. Photo-induced isomerization is stereospecific and can be used to produce pure trans iso-α-acids. 
 
The isomerization process has been recognized for over 80 years,6–8 yet the absolute configuration of carbon atoms 4 and 5 of the iso-α-acids (Scheme 1) have remained speculative. For the most predominant member of the iso-α-acid family, isohumulone, the cis and trans isomerization products, differing at carbon atom 4, were described preliminarily almost 50 years ago (Scheme 1).9 Ultimately, the absolute configuration of (4R, 5S) for (+)-cis-isohumulone was inferred according to Horeau’s method of partial decoupling. The absolute configuration of (−)-tetrahydrohumulone was inferred using the Cotton effect, which relates spectral details in an optical rotary dispersion curve to the configuration of a molecule, among other things.4, 10 However, contradictive and predominantly unsupported or inconclusive configurational assignments are reported as well.11–13 
 
Claims that beer and the bittering acids found in beer are beneficial when consumed in moderation have accumulated over time, including positive effects on diabetes,14–16 forms of cancer,17–20 and inflammation,21 and even linking reduced iso-α-acid derivatives to weight loss.22–24 Some of these derivatives affect one illness,21, 25–28 whereas others, differing only in the configuration of carbon atoms 4 and 5 of the iso-α-acids (Scheme 1), are ineffective.29 In addition, it was discovered that different grades of bitterness can be related to opposite enantiomers of tetrahydro-iso-α-acids.30 
 
However, the degree of bitterness of the isomerized bittering acids has yet to be related to a specific structure–function relationship after decades of confusion over the configuration of iso-α-acids, and in general, specifics of the isomerization process need to be resolved. Moreover, preservation of configuration during thermal isomerization has in the past been assigned to the ring carbon atom with the 3-methylbutyl side chain (see Scheme 1, C5 of the iso-α-acids, derived from C4 of the α-acids),6, 7, 10 which was opposed by other reports,11, 31, 32 thus demonstrating the state of confusion about the underlying hops chemistry. 
 
In order to unequivocally derive the absolute configuration of the iso-α-acids, we sought to use X-ray diffraction on a salt of (+)-cis-isohumulone or a suitable derivative and record the optical rotation of the compounds to ensure accurate literature comparison. 
 
Please note that the isomerization process results in a multitude of very similar compounds, which need to be separated from each other, purified, and unambiguously characterized, requiring an elaborate procedure (for details see the Supporting Information). 
 
As crystal salts we chose those containing either a heavy atom (to enable anomalous X-ray scattering for adequate phase resolution to define the configuration of a structure) or a compound of known absolute configuration to serve as an internal reference. 
 
Empirically, the vast number of useful salt combinations was greatly reduced by the inability to grow X-ray quality crystals. The initial success in elucidating the absolute configuration occurred with a purified ‘tetrahydro’ derivative (compound 1, (+)-cis-tetrahydroisohumulone), synthesized from (+)-cis-isohumulone through heterogeneous catalytic hydrogenation.29 
 
The absolute configuration, which was determined by X-ray analysis on a potassium salt (+)-1 a), indicates that the configuration of compound 1 (4S, 5R) is opposite to the originally reported configuration of the unsaturated precursor (+)-cis-isohumulone.10 Considering that the inversion of both asymmetric centers (C4 and C5 in Figure 1) is unlikely to occur during mild catalytic hydrogenation, the initial assignment found in the literature would appear to be incorrect. In an attempt to verify this observation, additional crystallization experiments were undertaken with compounds related to 1, resulting in the (−)-cinchonidine crystal salts of (+)-cis-tetrahydroisocohumulone (2), (+)-cis-tetrahydroisoadhumulone (3), and (+)-cis-isohumulone (4; Table 1, salts 2 b, 3 b, 4 b). 
 
 
 
Fig 1 
 
Structural details of (+)-cis-tetrahydroisocohumulone (2). The conserved stereocenter during isomerization is C4, while cis and trans iso-α-acids differ stereochemically at C5, which is nonchiral in the precursor α-humulone molecule. Related ... 
 
 
 
 
 
Table 1 
 
Crystallized compounds suitable for X-ray structure determinations and details on measurement of specific rotation. 
 
 
 
As expected, the absolute configurations of these compounds were consistent with the one we determined for compound 1 (4S, 5R). For comparison, we added the unnatural enantiomer of 1 to this series, (−)-cis-tetrahydroisohumulone ((−)-1), characterized here as (4R, 5S). 
 
Encouraged by these findings, we focused on the stereochemical assignment of the α-acid precursor (−)-humulone. Although neither a potassium nor (−)-cinchonidine salt were amenable to crystallization, we discovered that (−)-humulone (5) formed a small and weakly diffracting crystal (5 c) with trans-(1R, 2R)-(−)-diaminocyclohexane, thus enabling determination of the configuration of (−)-humulone as (6S). This finding is consistent with our initial assignment of (+)-cis-tetrahydroisohumulone, which again is opposite to the configuration found in most references. 
 
Finally, we were able to crystallize (−)-trans-tetrahydroisohumulone (6) with (+)-cinchonine (salt 6 d). The resulting structure has unequivocally a (4S, 5S) configuration, and anomalous X-ray diffraction (enabled through the presence of chloroform solvent molecules) confirmed the result. Table 1 summarizes all findings. 
 
In light of these discoveries, the isomerization of humulone to isohumulones proceeds with a net retention of configuration from the tertiary alcohol in (6S)-(−)-humulone to the α-hydroxy ketone (C4) in (4S, 5R)-(+)-cis-isohumulone, while configuration at C5 (Figure 1) is not determined. These results are in contrast with the proposed isomerization mechanism found in most reports, which assume that the conserved stereocenter is on C5, while cis and trans differ stereochemically at C4. Considering that the oxygen atoms possess negative charges during the isomerization, one might imagine the chelation of two vicinal oxygen atoms to a divalent cation, a process that is known to accelerate the rate of isomerization, while limiting decomposition. 
 
Excessive beer consumption cannot be recommended to propagate good health, but it has been demonstrated that isolated humulones and their derivatives can be prescribed with documented health benefits.21 The absence of correct stereochemical assignment for these compounds has prevented verification of the actual species responsible for biological activity. Now that the stereochemistry for these compounds has been confirmed and methods have been developed to substantiate the configuration of new entities in this series (see the Supporting Information), future work on their biological activities should be greatly accelerated. 
 
The utility of X-ray diffraction as the ultimate and preferred method to obtain unequivocal answers regarding absolute stereochemical questions (such as those above) has once more been demonstrated. One question remains: to what extent can one trust those assignments derived indirectly through Horeau’s method of partial decoupling and the Cotton Effect in optical rotary dispersion, now that we have discovered a case where these methods have failed the scientific community?


Supplementary Information
All solvents and buffers used for HPLC analyses were purchased through VWR International and were HPLC or ACS grade. Chemical Water was obtained from a Barnstead Nanopure Infinity Ultrapure system maintained within our laboratories. Commercially available hops extracts (Hopsteiner) were used for the isolation/preparation of individual hop acids. The chemicals used as bases for crystallization were purchased from Sigma Aldrich Chemical and used without further purification. Authentic hops standards were provided by the American Society of Brewing Chemists. Further structural confirmation was provided via specific rotation, melting point, NMR, UV, and UPLC-MS/MS when compared to preceding literature reports as available.

SI1.2 Analytical Instrumentation
NMR spectra were recorded at 298 K on a Bruker AV300, AV500, DRX500 and/or Varian INOVA 500, or 600 spectrometers. 1 H-NMR chemical shifts are reported versus TMS and referenced to residual solvent. Elemental analyses and specific rotation measurements were performed by Robertson Microlit Laboratories (Madison, NY) utilizing a Perking-Elmer 341-Polarimeter. All reactions/purifications were monitored by HPLC and/or UPLC-MS/MS. HPLC analyses were performed using a Shimadzu Prominence HPLC system (Shimadzu Scientific Instruments, Inc.). Suitable non-chiral reverse-phase chromatography and chiral normal-phase chromatography methods used for chemical and/or reaction evaluation by HPLC are presented in Tables SI-1 and SI-2 respectively. UPLC-MS/MS analyses were performed using a Shimadzu Nexera UPLC system (Shimadzu Scientific Instruments, Inc.) connected to an API-2000 MS/MS (ABSciex). All UPLC conditions can be seen in Table SI-3. All fragmentation information was acquired using information dependent acquisition (IDA) in negative mode (M-H) and the fragmentation methods can be provided upon request. A representative chromatogram for the UPLC-MS/MS analysis of the ASBC tetrahydro iso-α acid standard (ICS-T2) can be seen in Figure SI-1, with a table of fragmentations and relative retention times (Table SI-4) for the corresponding structures ( Figure  SI-2).   All countercurrent chromatography (CCC) experimentation was conducted using a J-type synchronous three-coiled planetary motion CCC-1000 (PharmaTech Research Corp.) as the column; this instrument possesses 810 mL of total column volume. This CCC column was coupled with Shimadzu preparative liquid chromatography instrumentation. Standard protocols for CCC were used over the course of the experimentation , [S4-S6] and specific details of the methods used can be found in Table SI-5. [S7,S 9] Further information is available upon request. Hops isomerized resin extract (45g, 0.12 mol) was mixed with 200mL of water and following the addition of 13mL (0.12 mol) of 9.2M KOH over 5 minutes, this formed a homogeneous solution.
In a separate flask, 141g (0.12 mol) of β-cyclodextrin were mixed with 1.1L of water and heated to 70 °C with mixing until the solution became homogenous/ translucent. The two solutions were slowly combined using a dropping funnel over 30 minutes. Following full addition, the mixture was removed from heat and allowed to come to room temperature at which point it was transferred to 4 °C storage for a period of 3 days. A precipitate formed during chill stabilization, and the supernatant was separated using centrifugation (1000 g, 10 minutes, 4 °C). The pellet was washed with 500 mL of water and the centrifugation was repeated; both aqueous supernatants were combined in a 4L separatory funnel. Ethanol (800 mL) was added to the supernatant, followed by 100mL of 1N HCl to bring the pH to ~1; without the ethanol a difficult emulsion forms. The supernatant was extracted three times with 200mL of hexanes and the extract was subsequently washed with water and brine to remove any residual β-cyclodextrin. Water (50mL) and 9.2M KOH (0.08mol, 10mL) were added in order to adjust to pH >8, followed by extraction of the organic layer three times with water (50 mL each time). The aqueous extract was collected in a 500mL Erlenmeyer flask and with vigorous stirring 12.4g (0.10 mol) of magnesium sulfate were slowly added to form a shelf-stable magnesium salt precipitate with the cis iso-α acids. The precipitate was collected via vacuum filtration, further dried via lyophilization and 21g were stored for future use. The yield and diastereoselectivity can be adjusted based on the equivalents of cyclodextrin used. [S8] SI2.1.2 Catalytic Hydrogenation Isohumulone (~700 mg, 2 mmol) was mixed with water and one equivalent of KOH (1.25 M aq. solution); a clear solution formed. To this solution was added an aqueous solution of magnesium sulfate (1.2 eq). A precipitate formed immediately, which was filtered, washed with water and dried on high vacuum overnight. This material was dissolved in methanol, 10% Pd on carbon was added (0.1 eq) and the reaction mixture was stirred under hydrogen (1 atm) until reaction completion (typical reaction time of 2 hours). After reaction completion, the catalyst was filtered off and the solvent was evaporated; this magnesium salt is well suited for storage. To liberate the free acid, material was suspended in 1M aq. HCl and the free acid was extracted to dichloromethane, the resulting dichloromethane extract was dried with sodium sulfate and evaporated.
Alternatively, isohumulone was dissolved in methanol and 1 equivalent of magnesium oxide was added. The resulting mixture was stirred for 30 minutes at which time most of the magnesium oxide dissolved. To this mixture was added Pd catalyst and the same steps as in the previous procedure were followed. The yield for either hydrogenation reaction typically ranges between 80-95%.

SI2.1.3 Differential pH Extraction
Hops CO 2 extract (101g,~0.16 mol of alpha acids) was dissolved in 100mL of dichloromethane in a 250mL Erlenmeyer flask. When the solution was homogeneous, it was transferred to a 1L separatory funnel and the flask was washed with an additional 100mL of dichloromethane. Water (200mL) was added to the separatory funnel along with 10mL (0.09mol) of 9.2 M KOH to bring the aqueous solution to pH 8. The organic layer was extracted twice with 200mL of water. An additional 200mL of water and 4mL (0.04mol) of 9.2M KOH were added and the pH was maintained at approximately 8.5. The final 200mL aqueous extraction used 5mL (0.05mol) of 9.2M KOH, for a total of 0.18 moles of KOH or approximately 1.1eq relative to the alpha acids presumed present in the extract. The aqueous extracts were combined and back-extracted one time to remove any excess beta acids. The dicholoromethane solutions were combined and concentrated in vacuo to produce a mixture of beta acids and essential hop oils. The aqueous extract was acidified to pH 2 using sulfuric acid and extracted three times with 100mL of dichloromethane, concentrated in vacuo to isolate 70g of alpha acids in ≥98% homogeneity in an apparent quantitative yield.

SI2.2.1 Preparation of 1:
Cis tetrahydro isohumulone was purified using countercurrent chromatography (CCC) according to a reported procedure from a commercially available hops extract. [S7] Analytical data confirmed ≥95% homogeneity following CCC purification.  36, 21.41, 21.42, 21.45, 21.49, 24.36, 25.68, 27.32, 27.95, 31.77, 35.30, 36.37, 45.80, 50.42, 87.28, 110.34, 196.81, 199.57, 204.78, 210.78; The purified material was converted to the potassium salt by reaction with 1 equivalent of potassium hydroxide, and was recrystallized from water to increase purity and remove discoloration. After achieving sufficient homogeneity, the potassium salt was further recrystallized to render crystals of sufficient dimension for X-ray diffraction experiments. Potassium salt (+1a): Anal. Found: C, 61.79, H, 8.18, K 9.28. C16H27O43 K8 requires C, 61.23, H, 8.27, K, 9.50. m.p. 141 °C, optical rotation +71.5, c=1.0, MeOH Water (450 µL) and (+)-cis tetrahydro isohumulone potassium salt (49.5 mg) were added to a 4 mL vial, and the vial was sealed with a cap and heated using a thermal heating block (70°C) until dissolution occurred. The solution was allowed to cool to 35 °C over a two hour period. During this time, in situ formation of crystals was observed. The mixture was allowed to stand at room temperature (22 °C) for eight hours, at which point the formation of additional crystals was observed. An individual crystal suitable for X-ray analysis was identified, carefully removed from solution, mounted, and submitted for X-ray diffraction analysis.

SI2.2.2 Preparation of (-1):
(-)-cis tetrahydro isohumulone: ((4R,5S)-3,4-dihydroxy-2-(3methylbutanoyl)-5-(3-methylbutyl)-4-(4-methylpentanoyl)cyclopent-2-en-1-one). A mixture of alpha acids (65 g) was dissolved in 200ml of limonene, bubbled with Ar for 30 min and heated at 145 °C for 120 minutes, at which point the reaction mixture was cooled to 50 °C, solvent was evaporated and the degree of racemization was evaluated and found to be 3% ee (enantiomeric excess). The racemic alpha acids were isolated using lead-acetate precipitation procedure [S3] and further purified by CCC using the procedure outlined in Table S-5. 4 (±)-Humulone (4.34 g, 12.0 mmol) was dissolved in 200mL of isopropanol and stirred. (1R,2R)-4-Cyclohexene-1,2-diamine (1.41g , 12.6 mmol) was added slowly to the racemic mixture. Crystals began forming within 1 hour at room temperature, and the mixture was placed in the refrigerator overnight (4 °C chill-stabilization). The crystals were collected via filtration, and were found to be enriched in (-)-humulone (35% ee). The mother liquor was concentrated in vacuo, followed by the addition of 20mL of isopropanol for further recrystallization in order to recover additional (-)-humulone (35% ee) and enrich the mother liquor in (+)-humulone (85% ee). (+)-Humulone (1.43g , 4.0mmol) was combined with 1.0 mL of water and heated to 81 °C. While the mixture stirred, 273mg (2.3mmol) of magnesium sulfate were added followed by the slow addition of 430µL (4.0mmol) of 9.2M KOH to form a hard resin precipitate. The mixture was heated and stirred for a total of 7 hours at approximately 80 °C. Upon completion, the water was removed via pipette from the vial (leaving the resinous precipitate), and 10mL of methanol was used to dissolve the remaining material. Magnesium oxide (256mg) was added and the mixture was stirred and filtered on a 0.2µm syringe filter into 2 separate and equal volume stocks. Stock 1 had 34mg of additional magnesium oxide and 200mg of 10% Pd/C added, and it was stirred under hydrogen (1 atm) for 1 hour. Stock 2 had 47mg of magnesium oxide and 187mg of 10% Pd/C added, and it was stirred for 2.5 hours under hydrogen (1 atm). The pH was brought to 1-2 using sulfuric acid, and the resulting mixture was filtered to remove any precipitates. Following CCC purification, [S7] 351mg (1.0 mmol, 25% yield from (+)-humulone) of (-)-cis tetrahyrdo isohumulone) was isolated for crystallization. Analytical data confirmed the purified material to be ≥95% (-)-cis tetrahydro isohumulone. The purified material was converted to a potassium salt by reacting with 1 equivalent of potassium hydroxide, and was recrystallized from water to increase purity. After achieving sufficient homogeneity, the potassium salt was further recrystallized to render crystals of sufficient dimension for X-ray diffraction experiments. and the potassium salt of (-)-cis tetrahydro isohumulone (11.7 mg) were added to a 4 mL vial, the vial was sealed with a cap and heated using a thermal heating block (56°C) until dissolution occurred. The solution was allowed to cool to 43 °C for a few hours and a seed crystal was used to induce crystallization. The mixture was allowed to stand at room temperature (22 °C) until taken for X-ray analysis. An individual crystal suitable for X-ray analysis was identified, carefully removed from solution, mounted, and submitted to X-ray diffraction analysis.

SI3. X-RAY STRUCTURES Methodology and instrumentations
Colorless crystals of about one-third of a mm in diameter were mounted on glass capillaries with oil. Data were collected on a Bruker APEX II single crystal X-ray diffractometer, Mo-radiation (collection temperature, see Table SI6). Crystal-to-detector distance was 40 mm and exposure time was 10 to 120 seconds per degree for all sets. The scan width was 0.5 o . Data collection completeness to 25 o in ϑ was above 98% in all cases. Further details are given in Table SI-6 below. The data were integrated and scaled using SAINT, SADABS within the APEX2 software package by Bruker . [S12] Solution by direct methods (SHELXS, SIR97) [S13,S14] produced a complete heavy atom phasing model consistent with the proposed structure. The structures were completed by difference Fourier synthesis with SHELXL97 [S15,S16] Scattering factors are from Waasmair and Kirfel. [S17] Hydrogen atoms were placed in geometrically idealised positions and constrained to ride on their parent atoms with C---H distances in the range 0.95-1.00 Angstrom. Isotropic thermal parameters (U eq ) were fixed such that they were 1.2U eq of their parent atom's Ueq for CH's and 1.5U eq of their parent atom's U eq in the case of methyl groups. All nonhydrogen atoms were refined anisotropically by full-matrix least-squares. The weighting schemes were optimized by the values suggested by SHELXL after each refinement. CCDC 913165 -913171 contains the supplementary crystallographic data for this paper. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif. SI3.1 X-ray structure of Compound +1a, potassium salt of (+)-cis n-tetrahydro iso α α α α-acid This structure, described in [S2] is added for completion. The asymmetric unit for this structure consists of 4 negatively charged molecules of (1) coordinating with 4 potassium cations. Two potassiums are bridged by a single water molecule, another disordered water coordinates with the remaining two cations. Ketones of the organic moieties coordinate with the cations leading to a close packing pattern. The absolute configuration was obtained from anomalous scattering (Absolute structure parameter = 0.04(4)) Figure SI-3. ORTEP representations of the asymmetric unit in the structure for compound 1 with displacement ellipsoids at the 50% probability level. Orthorhombic Monoclinic Space group P 2 1 2 1 2 P 2 1 2 1 2 P 2 1 P 2 1 P 2 1 P 2 1 2 1 2 1 P 2 1 Unit cell dim. a ( Å) 23.3110 (9) 23.2614 (16) 17.3908 (6) 17.4871 (6) 14.474(2) 6.6424 (12) 13.6570(12) b ( Å) 28.9052 (12) 28.8798 (22) 11.8605 (4) 12.2205(4) 9.5817 (13) 17.292 (4) 10.4637(9) c ( Å) 13.6845 (5) 13.6741 (11) 20.4103 (7) 20.0825 (7) 14.579 (2) 24.070 (6) 14.6463 (14)   SI3.2 X-ray structure of Compound -1a, potassium salt of (-)-cis n-tetrahydro iso α α α α-acid Details of the structure for compound (2) are similar to that of (1), except that it possesses the opposite handedness. SI3.3 X-ray structure of Compound 2b, (+)-cis co-tetrahydro iso α α α α-acid * cinchonidine One disordered t BuOMe molecule was found in addition to the four molecules shown below, the disorder of which causes further disorder in the structure (not demonstrated in Figures SI-7 or SI-8). Table SI-7 summarizes the hydrogen bonds.
Figure SI-13. ORTEP representations of the asymmetric unit in the structure for compound 6d with displacement ellipsoids at the 50% probability level. Disorder of solvent omitted for clarity.