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Synthesis, structure and tandem mass spectrometrical characterization of the diastereoisomers of quinic acid Sagar Deshpande, Marius Febi Matei, Rakesh Jaiswal, Bassem S. Bassil, Ulrich Kortz, and Nikolai Kuhnert J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b02472 • Publication Date (Web): 11 Aug 2016 Downloaded from http://pubs.acs.org on August 11, 2016

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Journal of Agricultural and Food Chemistry

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Synthesis, Structure and Tandem Mass spectrometric Characterization of the

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Diastereomers of Quinic Acid

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Sagar Deshpande,a Marius Febi Matei,a Rakesh Jaiswal, a Bassem S. Bassil,a,b Ulrich Kortza

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and Nikolai Kuhnerta*

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a)Department of Life Sciences and Chemistry, Jacobs University Bremen, 28759 Bremen,

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Germany

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b) Current address: Department of Chemistry, Faculty of Sciences, University of Balamand, PO Box 100, Tripoli, Lebanon

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*Author to whom correspondence should be addressed:

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Telephone: 0049 421 200 3120

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Fax: 0049 421 200 3229

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Email: [email protected]

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ABSTRACT

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(-)-Quinic acid possess eight possible stereoisomers, which occur both naturally and as

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products of thermal food processing. In this contribution, we have selectively synthesized four

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isomers namely, epi-quinic acid, muco-quinic acid, cis-quinic acid and scyllo-quinic acid in

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order to develop a tandem LC-MS method identifying all stereoisomeric quinic acids. Four

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derivatives have been unambiguously characterized by single crystal X-ray crystallography.

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The missing diastereomers of quinic acid were obtained by non-selective isomerization of (-)-

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quinic acid using acetic acid/conc. H2SO4 allowing chromatographic separation and

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assignment of all diastereomers of quinic acid. We report for the first time that a full set of

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stereoisomers are reliably distinguishable on the basis of their tandem mass spectrometric

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fragment spectra as well as their elution order. A rational for characteristic fragmentation

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mechanisms is proposed. In this study, we also observed that muco-quinic acid , scyllo-quinic

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acid and epi-quinic acid are present in hydrolyzed Guatemala roasted coffee sample as

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possible products of roasting.

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INTRODUCTION:

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(-)-Quinic acid 1 is distributed naturally in a variety of plant materials ranging from coffee to

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cinchona bark to tobacco leaves and cranberries in its free form or in the form of its depsides,

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chlorogenic acids. It was first isolated in 1790 and was given an empirical formula of

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C7H12O6 in 1838.1 In a single coffee bean, up to 4.0 mg free (-) quinic acid is found.2 In

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Colombian Arabica green coffee, up to 7.0 g/kg of quinic acid is present, which increases up

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to 10.0 g/kg upon roasting.3 Quinic acid provides characteristic astringent taste to the

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beverage, hence it is also used as a flavor enhancer in certain beverages.4 The esters of the 3R,

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4S, 5R isomers of the quinic acid, also referred to as chlorogenic acids, constitute the most

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important quinic acid derivatives in nature and in our diet.5,6 For quinic acid diastereomers

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the carbon numbering is retained with respect to the parent compound.7

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Quinic acid possesses eight possible stereoisomers: four meso forms and two pairs of

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enantiomers (Figure 1). Formally the diastereomers 2-6 can be derived from the parent

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compound (-)-quinic acid 1 by inversion of one or two of the four stereogenic centres as

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demonstrated in Figure 1. As reported by Kuhnert and co-workers, 80 different chlorogenic

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acid derivatives have been identified in green coffee beans.7 After roasting and brewing, this

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number is increased to a minimum of 120 derivatives identified on the basis of the presence of

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characteristic fragment ions corresponding to quinic acid and quinic acid lactones in MSn

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chromatograms.8,9 Products of epimerization of Chlorogenic acids for example muco-caffeoyl

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and feruloyl esters were reported in roasted coffee formed at elevated temperatures.8 After

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ingestion of the foods containing quinic acid esters, metabolism in humans may also give rise

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to the esters of the diastereomers of quinic acid.8,10 This fact supports the assumption that the

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roasting or food processing in general facilitates the isomerization at the stereogenic centers in

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3R, 4S, 5R esters of the quinic acid. 11,12

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A number of esters of the diastereomers of quinic acid have been previously reported in the

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literature as plant secondary metabolites. For example, in Lactuca indica L., Asimina triloba

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and Aster scaber 3,5-Dicaffeoyl-muco-quinic acid was identified. In Asimina triloba 3-

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caffeoyl-muco-quinic acid was also identified.

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dicaffeoyl-epi-quinic acid and 1,3-dicaffeoyl-epi-quinic acid was identified.16 3,5-dicaffeoyl-

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epi-quinic acid esters were also reported in Ilex kudingcha.17 These muco, epi and scyllo

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esters of diastereomers of quinic acid are reported to show important biological activities like,

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hepatoprotectivity, antioxidant activity and anti HIV-1 integrase activity.13-17 Considering the

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fact that regiosomers as well as the esters of the diastereomers of quinic acids are readily

13-15

In Chrysanthemum morifolium 3,5-

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distinguishable by their fragmentation pattern in tandem MS experiments, it is very important

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to acquire authentic synthetic standards with defined stereochemistry by organic synthesis.

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Development of the tandem mass spectrometric methods for the identification of

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diastereomers of quinic and chlorogenic acids will enable us to identify novel chlorogenic

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acid derivatives present in the biological samples even in very low concentrations. Since,

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these biological samples emerge either biosynthetically, through man-induced processes such

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as, roasting, brewing, cooking etc. or as products of metabolism, their mass spectrometric

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study will help to improve understanding of the changes in the chlorogenic acid profile

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through biosynthetic processes in great detail.

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MATERIALS AND METHODS

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Chemicals

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All the chemicals (analytical grade) were purchased from Sigma-Aldrich (Taufkirchen,

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Germany) and were used without further purification.

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LC/MSn

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The LC equipment (Agillent 1100 series, Karlsruhe, Germany) comprised a binary pump, an

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auto sampler with a 100 µL loop, and a DAD detector with a light-pipe flow cell (recording at

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254 and 320 nm and scanning from 200 to 600 nm). This was interfaced with an HCT Ultra

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ion-trap mass spectrometer fitted with an ESI source (Bruker Daltonics HCT Ultra, Bremen,

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Germany) operating in full scan, auto MSn mode to obtain fragment ion m/z. As necessary,

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MS2, MS3, and MS4 fragment-targeted experiments were performed to focus only on

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compounds producing a parent ion at m/z 191, 173. Tandem mass spectra were acquired in

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Manual-MSn mode using fixed collision energy. The fragmentation amplitude was set to 0.75

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V. Also, direct injection experiments targeting the fragments in MS2, MS3, and MS4 were

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performed on all diastereomers of quinic acid keeping the fragmentation amplitude constant at

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1.0 volts. MS operating conditions (negative mode) was optimized using (-)-quinic acid with a

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capillary temperature of 365 oC, a dry gas flow rate of 10 L/min, and a nebulizer pressure of

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10 psi. 4 ACS Paragon Plus Environment

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HPLC

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Separation was achieved on a 250mm × 4.6 mm i.d. column containing diphenyl 5 µm and

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5mm × 4.6 mm i.d. guard column of the same material (Varian, Darmstadt, Germany). The

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elution solvent was water: formic acid (1000:0.05 v/v) delivered at a total flow rate of 800

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µL/min by 30 min isocratically.

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NMR

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1

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400 MHz for 1H NMR and 100 MHz for 13C NMR at room temperature using a 5 mm probe.

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The chemical shifts (δ) are reported in parts per million and were referenced to

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Tetramethylsilane (TMS at 0 ppm). The coupling constants (J) are quoted in hertz. The

123

following abbreviations are used: s, singlet; d, doublet; t, triplet; m, multiplet; bs, broad

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signal; bd, broad doublet; dd, doublet of doublets, ddd, doublet of doublet of doublets.

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Synthesis of the mixture of the epimers of (-)-quinic acid

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The mixture of the epimers of (-)-quinic acid was obtained non-selectively by the process

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previously described by Maier and co-workers.6

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Synthesis of the epi-quinic acid (2)

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Methyl quinate was prepared by refluxing quinic acid 1 (5g, 26.02 mmol) with MeOH (100

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mL) and Amberlite IR120 acidic resin (5g) for 12 h. The reaction mixture was then filtered

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and concentrated in vacuo. The product was obtained in more than 95% yield and the purity

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of the product was confirmed by NMR. It was then subjected to selective silyl protection on

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C3 and C5 of the methyl quinate by tert-butyldimethylsilyl chloride (TBDMSCl).18

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Methyl quinate (2650 mg, 12.86 mmol) was stirred with triethylamine (5 mL) in anhydrous

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dimethylformamide (26 mL) in an inert atmosphere and to this solution, tert-

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butyldimethylsilyl chloride (5034 mg, 33.436 mmol) was added and the mixture was stirred

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for 2 h at 0 0C and 16 h at room temperature. EtOAc was added and the residue was filtered.

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Concentrated filtrate was purified using flash chromatography (gradient eluent: 20% EtOAc

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in

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butyldimethylsilyl)quinate 7 in 75% yield, which was confirmed by single crystal X-ray

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diffraction have free C-4 on quinic acid skeleton. Compound 7 was used as a precursor for

H NMR and 13C NMR spectra were acquired on a JEOL ECX-400 spectrometer operating at

petroleum

ether)

to

afford

white

crystalline


methyl

3,5-Di-O-(tert-


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the synthesis of epi-quinic acid 2 and scyllo-quinic acid 5 as shown in the supplementary

143

information.

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Dess-Martin periodinane (390 mg, 0.92 mmol) was added to the solution of compound 7 (200

145

mg, 0.46 mmol) dissolved in dichloromethane (15 mL) at room temperature. Reaction was

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stirred overnight and diluted with Et2O. Then to this 20 ml of a 1:1 saturated mixture of

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Na2S2O3 and NaHCO3 solution was added until the reaction became clear. Organic layer was

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collected and the aqueous layer was extracted with EtOAc (3× 20 mL). Combined organic

149

layers were collected, dried and concentrated in vacuo. Crude product was subjected to flash

150

chromatography (eluent: 24% EtOAc in petroleum ether), which afforded methyl 3,5-Di-O-

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(tert-butyldimethylsilyl)-4-oxoquinate 12 in the form of sticky colorless solid in 87%.

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NMR (100 MHz, CDCl3): 206.51, 173.15, 75.98, 75.33, 69.81, 52.94, 46.71, 41.22, 25.79,

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25.61, 18.45, 17.88, -4.73, -5.04, -5.25, -5.30.

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Compound 12 was subjected to reduction at C-4 by the action of NaBH4 and L-selectride. Out

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of these two reduction procedures, reduction with NaBH4 showed higher selectivity towards

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the formation of epi- derivative as compared to L-selectride. 177 mg of compound 12 (0.4074

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mmol) was dissolved in ethanol and the reaction flask was immersed in an acetone bath.

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Liquid nitrogen was slowly added until the bath temperature reached -30 0C. NaBH4 (23 mg,

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0.6111 mmol) was added and the mixture was stirred for 40 min. Solvent was removed

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immediately under reduced pressure at 30 0C in a rotary evaporator. The residue was then

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extracted with water and EtOAc mixture three times. Organic layers were collected and dried

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over Na2SO4 and concentrated.

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indicated by the loss of a peak at 206 ppm. The crude product was directly subjected to

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hydrolysis by 2M HCl and water without further purification. Product of the hydrolysis was

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diluted with water and extracted with water and EtOAc mixture thrice. Aqueous layers were

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collected and concentrated in vacuo. Resulting white product was used in HPLC-MS analysis.

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Synthesis of the muco-quinic acid (3)

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muco-Quinic acid 3 was obtained from the methyl TMB-muco-quinate (TMB Ley’s

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tetramethoxybutane), which was synthesized by the procedure previously reported by Jaiswal

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et al..8, Methyl TMB-muco-quinate (500 mg, 1.56 mmol) was subjected to hydrolysis by 70%

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aqueous trifluoroacetic acid for 1 hour. Resulting methyl muco-quinate was stirred with 2M

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KOH for 20 min followed by neutralization by Amberlite IR 120 acidic resin for 10 min. The

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mixture was filtered and concentrated under reduced pressure. The resulting yellowish solid

13

13

C

C NMR of the crude product confirmed reduction at C4


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was analyzed by NMR. 1H NMR (400 MHz, D2O): 1.73 (dd, 1H, H6ax, 3JHH 12.4), 1.75 (dd,

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1H, H2ax, 3JHH 12.4), 1.86 (dd, 1H, H6eq, 2JHH 4.1, 3JHH 11.2), 1.89 (dd, 1H, H2eq, 2JHH 4.6, 3JHH

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11.4), 3.21 (t, 1H, H4, 3JHH 9.6), 3.62 (ddd, 2H, 2JHH 4.6, H3 and H5, 3JHH 11.9, 9.1). 13C NMR

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(100 MHz, D2O): 180.9, 79.5, 74.7, 69.8, 69.7, 40.5, 40.4

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Synthesis of the cis-quinic acid (4)

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3,4-O-Cyclohexylidene-1,5-quinide 13 was synthesized by adding quantities of 10.00 g

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(52.04 mmol) of quinic acid and 200 mg (1.05 mmol) of p-toluenesulfonic acid monohydrate

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(PTSA·H2O) to 100 mL of cyclohexanone to give a white suspension. The reaction was then

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refluxed for 24 h to give a yellow solution which was cooled to 50 ºC and neutralized with a

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solution of NaOEt (71.5 mg) in EtOH (5 mL) to give a yellow clear solution. The solvents

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were removed under reduced pressure and to the resulting yellow viscous liquid a volume of

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100 mL of EtOAc was added. The organic phase was washed with 50 mL of H2O and the

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aqueous phase was back-extracted with 30 mL EtOAc. The combined organic layers were

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washed with a half-saturated NaHCO3 solution, dried on Na2SO4, filtered and evaporated. The

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resulting yellow viscous liquid was recrystallized from a 1:1 n-heptane:EtOAc solution to

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afford white crystals of compound 13 (9.26 g, 36.43 mmol, 70%, mp. 172-174 °C); 1H-NMR

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(CDCl3): δH 4.73 (dd, 1H, J = 5.6, 2.8 Hz), 4.48 (td, 1H, J = 6.8, 2.8 Hz), 4.29 (ddd, 1H, J =

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6.4, 2.8, 1.4 Hz), 2.66 (d, 1H, J = 11.9 Hz), 2.38-2.31 (ddd, 1H, J = 14.7, 7.8, 2.3 Hz), 2.31-

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2.25 (ddt, 1H, J = 11.9, 6.4, 2.3 Hz), 2.17 (dd, 1H, J = 14.7, 3.2 Hz), 1.73-1.68 (m, 2H), 1.67-

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1.60 (m, 2H), 1.57-1.51 (m, 4H), 1.43-1.36 (m, 2H);

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110.7 (C-1'), 76.1 (C-4), 71.8 (C-1), 71.6 (C-3), 71.2 (C-5), 38.6 (C-6), 37.0 (C-2), 34.5 (C-

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6'), 33.7 (C-2'), 25.1 (C-5'), 24.0 (C-3'), 23.6 (C-4').

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3,4-O-Cyclohexylidene-1,5-quinide 13 (8.75 g, 34.41 mmol) was dissolved in 100 mL MeOH

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and a 21% solution NaOMe/MeOH was added (187 mg NaOMe). The clear solution was

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stirred overnight, the mixture was then quenched with glacial acetic acid (232 µL) and the

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volatile components were removed under vacuum. The resulting mixture was dissolved in

200

EtOAc and washed 3 times (3x40 mL). The organic layer was dried over Na2SO4, filtered and

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the solvent was removed under low pressure. The crude product was purified by column

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chromatography on silica gel (20-50% EtOAc/petroleum ether) to give methyl 3,4-O-

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cyclohexylidene-quinate 14 as a white solid (5.87 g, 20.51 mmol, 60%). 1H-NMR (CDCl3):

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δH 4.46 (dt, 1H, J = 5.9, 3.7 Hz), 4.14-4.07 (m, 1H), 3.97 (t, 1H, J = 5.9 Hz), 3.79 (s, 3H),

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2.26 (m, 1H), 2.25 (d, 1H, J = 4.1 Hz), 2.08 (ddd, 1H, J = 13.4, 4.1, 3.0 Hz), 1.86 (dd, 1H,

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13.7, 11.0 Hz), 1.74-1.68 (m, 2H), 1.68-1.52 (m, 6H), 1.44-1.33 (m, 2H); 13C-NMR (CDCl3):

13

C-NMR (CDCl3): δC 178.9 (-COOR),



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δC 175.6 (-COOCH3), 110.1 (C-1'), 79.4 (C-4), 74.1 (C-1), 73.1 (C-3), 68.7 (C-5), 53.0 (-

208

CH3), 39.0 (C-6), 38.0 (C-2), 34.9 (C-6'), 34.8 (C-2'), 25.0 (C-5'), 24.1 (C-3'), 23.7 (C-4').

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To a suspension of Dess-Martin periodinane (2.96 g, 6.99 mmol) in anhydrous CH2Cl2 (65

210

mL) compound 14 (1.82 g, 6.36 mmol) was added. The reaction mixture was stirred at room

211

temperature for 18 h, was then diluted with Et2O (100 mL) and 50 mL of a saturated aqueous

212

Na2S2O3 and 50 mL of a saturated NaHCO3 solution (100 mL) were added. The mixture was

213

stirred until the solids were dissolved (20 min). The aqueous layer was extracted with Et2O

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(2× 50 mL) and the combined organic layers were dried over Na2SO4, filtered and

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concentrated in vacuum. Product methyl 3,4-O-cyclohexylidene-5-oxoquinate 15 (1.81 g,

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6.36 mmol, 100%) was used for the next step without further purification. 1H-NMR (CDCl3):

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δH 4.72 (m, 1H), 4.41 (d, 1H, J = 5.5 Hz), 3.81 (s, 3H), 2.89 (d, 1H, J = 14.2 Hz), 2.80 (dd,

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1H, J = 14.7, 2.3 Hz), 2.56 (t, 1H, J = 2.3 Hz), 2.55 (d, 1H, J = 4.1 Hz), 1.74-1.64 (m, 2H),

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1.64-1.56 (m, 4H), 1.56-1.47 (m, 2H), 1.43-1.32 (m, 2H); 13C-NMR (CDCl3): δC 204.4 (C-5),

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173.0 (-COOCH3), 111.7 (C-1'), 78.2 (C-4), 76.9 (C-1), 75.9 (C-3), 53.5 (-CH3), 49.1 (C-6),

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37.0 (C-2), 35.3 (C-6'), 34.7 (C-2'), 24.9 (C-5'), 23.9 (C-3'), 23.8 (C-4').

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To obtain methyl 3,4-O-cyclohexylidene-epi-quinate 16, compound 15 (1.53 g, 5.33 mmol)

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was dissolved in a 1:1 mixture (v/v) MeOH/THF (100 mL) and was cooled to -30 ˚C with an

224

acetone/liquid nitrogen bath. NaBH4 (222 mg, 5.86 mmol) was added and the mixture was

225

stirred at -30 ˚C for 1 h. The solvents were removed in vacuum and the residue was extracted

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three times with a water/EtOAc mixture. The organic layers were dried over Na2SO4, filtered

227

and the solvent was removed under reduced pressure. The product was purified by column

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chromatography (40% EtOAc/petroleum ether). 1H-NMR (CDCl3): δH 4.52 (dt, 1H, J = 7.6,

229

5.0 Hz), 4.30 (dd, 1H, J = 7.6, 4.1 Hz), 3.90 (dt, 1H, J = 9.6, 4.1 Hz), 3.78 (s, 3H), 2.18-2.06

230

(m, 3H), 2.02 (dd, 1H, J = 14.2, 10.1 Hz), 1.75 (m, 2H), 1.69-1.59 (m, 4H), 1.59-1.51 (m,

231

2H), 1.44-1.35 (m, 2H);

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73.4 (C-1), 72.5 (C-3), 66.1 (C-5), 53.0 (-CH3), 38.1 (C-6), 36.4 (C-2), 35.6 (C-6'), 34.0 (C-

233

2'), 25.2 (C-5'), 24.1 (C-3'), 23.7 (C-4').

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Crystals of 4 suitable for single crystal X-ray diffraction were obtained in an NMR tube

235

containing demethylated 16 dissolved in CDCl3 by removal of the acid-labile cyclohexylidene

236

protection promoted by the trace amounts of HCl present in the deuterated solvent. 1H-NMR

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(D2O): δH 3.76 (br, 1H), 3.67 (t, 1H, J = 3.2 Hz), 3.64 (t, 1H, J = 3.2 Hz), 1.97 (dd, 2H, J =

238

12.4, 4.1 Hz), 1.62 (t, 2H, J = 12.4 Hz); 13C-NMR (D2O): δC 177.1 (-COOCH3), 72.4 (C-4),

239

71.1 (C-1), 66.9 (C-3, C-5), 35.9 (C-2, C-6).

13

C-NMR (CDCl3): δC 175.4 (-COOCH3), 110.0 (C-1'), 73.8 (C-4),


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Synthesis of the scyllo-quinic acid (5)

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Methyl 3,5-Di-O-(tert-butyldimethylsilyl)quinate (7) (500 mg, 1.15 mmol) was mixed and

242

stirred with 12 mL of anhydrous pyridine and cooled to 0 0C. Methanesulfonyl chloride was

243

added drop wise to the mixture. Reaction was stirred at room temperature for overnight. 10

244

mL NaHCO3 added and after stirring for 10 min, aqueous phase was washed two times with

245

Et2O. Organic layers were collected, dried over Na2SO4 and concentrated. Crude product was

246

subjected to flash chromatography (eluent: 22% EtOAc in petroleum ether), which afforded

247

methyl 3,5-di-O-(tert-butyldimethylsilyl)-4-O-methanesulfonylquinate 8 in 95% yield. 1H

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NMR (400 MHz, CDCl3): 0.06 (s, 3H, MeSi), 0.08 (s, 3H, MeSi), 0.11 (s, 3H, MeSi), 0.12 (s,

249

3H, MeSi), 0.86 (s, 9H, Me3CSi), 0.88 (s, 9H, Me3CSi), 1.94 (dd, 1H, H6ax, 2JHH 13.74, 3JHH

250

10.07, Hz), 2.00-2.10 (m, 1H, H2ax), 2.12-2.31 (m, 2H, H6eq, H2eq), 3.04 (s, 3H, MeS), 3.77 (s,

251

3H, -OMe), 4.25 (dd, 1H, H4, 3JHH 8.7, 2JHH 2.3 Hz), 4.34 (m, 1H, H5) and 4.57 (m, 1H, H3).

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Compound 8 (520 mg, 1.014 mmol) was dissolved in 5 mL anhydrous dimethylformamide

253

and to this was added cesium fluoride (790 mg, 5.2 mmol). The mixture was stirred for 30

254

min and cinnamic acid (770 mg, 5.2 mmol) was added. The reaction was heated to 90 0C and

255

stirred for 36 h. DMF was removed in vacuum and the obtained crude product was subjected

256

to column chromatography (eluent: 45% EtOAc, 55% petroleum ether). One fraction

257

collected from the column chromatography confirmed the formation of the desired product in

258

the NMR analysis and formed a crystal in the NMR tube suitable for single crystal X-ray

259

diffraction.

260

The mixture of compound 9 and compound 10 was subjected to hydrolysis through the same

261

procedure as described earlier. Product of the hydrolysis was diluted with water and extracted

262

with water and EtOAc mixture three times. Aqueous layers were collected and concentrated

263

in vacuo. Resulting white product was used in HPLC/MS analysis.

264

Hydrolysis of the chlorogenic acids in roasted coffee

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10 grams of finely grounded roasted Arabica coffee from Guatemala was boiled in distilled

266

water for 1 hour. The mixture was then filtered and the water in the filtrate was removed

267

under reduced pressure. Resulting residue was subjected to base hydrolysis by treatment of 25

268

mL 2M NaOH for 2 h. The reaction was neutralized by addition of 2M HCl. The mixture was

269

diluted with 25 mL water and the aqueous layer was extracted with EtOAc (3 × 20 mL). The

270

aqueous layers were collected and concentrated in vacuo. Resulting yellowish product was

271

used directly in HPLC-MS analysis. 9 ACS Paragon Plus Environment


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Synthesis of the methyl esters of epi-, muco-, cis-, scyllo-quinic acids and (-)-quinic acid

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As described earlier, methyl quinates of all the synthesized diastereomers except the methyl

274

cis-quinate 17 were prepared by refluxing respective diastereomers in MeOH with equal

275

amount of Amberlite IR120 acidic resin for 12 h. The reaction mixture was then filtered and

276

concentrated in vacuo. The resulting product was used directly in HPLC/MS analysis in case

277

of methyl epi-quinate, methyl scyllo-quinate and methyl muco-quinate. Crystals of methyl cis-

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quinate 17 suitable for single crystal X-ray diffreaction (Figure 4) were obtained in an NMR

279

tube containing methyl 3,4-O-cyclohexylidene-epi-quinate 16 dissolved in CDCl3 by removal

280

of the acid-labile cyclohexylidene protection promoted by the trace amounts of HCl present in

281

the deuterated solvent. The methyl quinate of (-)-quinic acid was confirmed by the NMR and

282

then utilized in HPLC/MS.

283

X-ray crystallography

284

Crystals were mounted on a Hampton cryoloop in light oil for data collection at 100 K.

285

Indexing and data collection were performed on a Bruker D8 SMART APEX II CCD

286

diffracto-meter with κ geometry and Mo Kα radiation (graphite mono- chromator, λ =

287

0.71073 Å; Karlsruhe, Germany). Data integration was performed using SAINT. Routine

288

Lorentz and polarization corrections were applied. The SHELX package was used for

289

structure solution and refinement. Refinements were full-matrix least-squares against F2 using

290

all data. In the final refinement, all non-hydrogen atoms were refined anisotropically and

291

hydrogen atoms were either found directly and refined isotropically or placed in calculated

292

positions. Crystallographic data (CIF files) are available at www.ccdc.cam.ac.uk.

293 294

RESULTS AND DISCUSSIONS

295

Synthesis of quinic acid diastereomers

296

Several methods for the synthesis of the epi-quinic acid 2 have been reported,

297

formation of a 1, 5-quinide followed by selective protection of C-3-OH by silyl protecting

298

group and subsequent inversion of configuration at C4 by oxidation-reduction sequences. This

299

approach was found to be unreliable in our hands, since silyl-protection was observed to be

300

non-selective and separation low yielding. In the present work, once the methodology to

301

obtain 7 in pure form was developed, the ambiguity in the selective protection was eliminated

302

in order to leave C-4-OH free for further treatment (Figure 2). Methyl 3,5-di-O-(tert10 ACS Paragon Plus Environment

18

exploiting

Page 11 of 29


303

butyldimethylsilyl) quinate 7 served as a precursor for both of the diastereomers, epi-quinic

304

acid 2 and scyllo-quinic acid 5. Of these, diastereomer 2 was obtained by oxidation-reduction

305

pathway and 5 was synthesized by incorporating SN2 reaction strategy, using cinnamic acid as

306

a nucleophile leading to inversion of configuration at C4. However, in fact the actual product

307

isolated, was found to have rearranged to yield 3-O-cinnamoyl-1,4-scyllo-quinide 9 possibly

308

due to the traces of acid present in the work-up procedure. 3-O-Cinnamoyl-1, 4-scyllo-quinide

309

9 and 3-O-cinnamoyl-1,5-quinide 10 were co-crystallized and were obtained in very low

310

yield. Hence, attempts towards the isolation of compound 9 were avoided and the crystals

311

were directly hydrolyzed to obtain a 9: 1 mixture of scyllo-quinic acid 5 and quinic acid as

312

judged by NMR and LC-MS data.

313

Cis-Quinic acid 4 was obtained starting from 1,3-cyclohexylidene protected quinide

314

(Figure 3). Opening of the lactone ring using methoxide furnished methylester 14, which was

315

subsequently oxidized to ketone 15 using the Dess-Martin periodinane reagent.

316

Diastereoselective reduction using NaBH4 yielded a 4:1 mixture of diastereomers, from,

317

which methyl ester 17 could be obtained in pure form by crystallization. Hydrolysis yielded

318

the target compound 4 in poor yield, however in crystalline form suitable for single crystal X-

319

ray diffraction studies, hence confirming its structure.

320

Discussion of single crystal X-ray structures

321

For five synthetic intermediates crystals suitable for single crystal X-ray diffraction could be

322

obtained. The resulting X-ray structures confirm the relative stereochemistry of the quinic

323

acid scaffold as diastereomers of quinic acid and therefore support the relative

324

stereochemistry of the final quinic acid diastereomer products. In the crystal of cis-quinic acid

325

4 (Figure 4), we observed two molecules in the asymmetric unit representing the same

326

structure. Compounds 3-O-cinnamoyl-1,4-scyllo-quinide 9 and 3-O-cinnamoyl-1,5-quinide 10

327

co-crystallized along with one molecule of chloroform in one asymmetric unit. The crystal

328

structure of 3,5-di-O-(tert-butyldimethylsilyl)quinate 7 showed conformational disorder in

329

one of the silyl groups (Si1A and Si1B), both conformations are shown in Figure 4. The

330

disorder was modeled and showed a value of 50% for each conformation. Table 2 illustrates

331

the crystal data and structure refinement for compounds methyl cis-quinate 17, cis-quinic acid

332

4,

333

Cyclohexylidene-1,5-quinide (13) and methyl 3,5-Di-O-(tert-butyldimethylsilyl)quinate 7.

3-O-cinnamoyl-1,4-scyllo-quinide(9),

3-O-cinnamoyl-1,5-quinide

334 11 ACS Paragon Plus Environment

10,

19

13

3,4-O-


335

LC-MS analysis of quinic acid diastereomers

336

With authentic standards in hand for four quinic acid diastereoismers from this work and

337

muco quinic acid from previous work,8 a HPLC method was developed allowing separation

338

of quinic acid diastereomers for future food analysis and tandem MS investigations. All

339

quinic acid diastereomers can be readily detected in negative ion mode as pseudomolecular

340

ions ([M-H], C7H11O6) at m/z 191. 0550 +/- 0.0003 using high resolution MS or at 191.1 using

341

an ESI ion trap mass spectrometer. Using Maier’s protocol for unselective isomerization using

342

concentrated acid

343

arguments of exclusion. Observation the retention times of all the diastereomers, shows that

344

they elute very close to each other . The elution order can be given as, 1>5>2>3=4. Where 1

345

(quinic acid) elutes at 3.9 min and 3/4 muco- and/or cis-quinic acid elute at 3.4 minutes (Table

346

1). Although diastereomers 3 and 4 partially co-elute at the same retention time, their tandem

347

MS shows clear distinction as can be seen in Figure 7 and 8. All tandem MS data for quinic

348

acid diastereomers 1-5 with the precursor ion at m/z 191.0 are shown in Figures 5-9.

349

An analysis of a roasted and subsequently hydrolyzed Arabica coffee extract shows that in an

350

extracted ion chromatogram at m/z 191 four diastereomers of quinic acid can be observed and

351

assigned (Figure 10) The identified compounds are epi-quinic acid 2, muco-quinic acid 3 and

352

cis-quinic acid 4 along with quinic acid 1. Following an earlier report we assume that these

353

compounds are formed at elevated temperature during coffee roasting, since they are absent

354

from the corresponding green coffee bean material.

355

acid secondary alcohols are prone to epimerization under thermal conditions, whereas

356

structurally related carbohydrates are not. 24-26

357

Tandem mass spectrometry of quinic acid diastereomers

358

Recent advances in tandem mass spectrometry have shown that certain classes of isomeric

359

compounds can be reliably distinguished by tandem mass spectrometry based on significant

360

differences in their tandem mass spectra. In particular regioisomers have been shown to

361

display characteristic fragmentation mechanisms allowing a reliable assignment of structure.

362

27-29

363

the compounds available here allowing a proof of principle study that diastereomeric

364

structures can be distinguished and assigned based on their tandem MS spectra.

365

Fragment spectra were obtained in negative ion mode using collision induced dissociation

366

(CID) using an ion trap mass spectrometer. MS2, MS3 and in some cases MS4 spectra were

6

the missing isomer neo-quinic acid 6 could be tentatively assigned by

6, 20-23

It is surprising to note that quinic

Much less is known about differences in fragment spectra of sets of diastereomers with


Page 12 of 29

Page 13 of 29


367

recorded. The MS2 fragmentation all of the diastereomers was investigated under constant

368

collision energy in the LC-MS runs at 0.75 volts (see Table 1). Optimum value for the

369

collision energy was obtained by taking (-)-quinic acid 1 as a standard. All diastereomers

370

show characteristic fragmentations including neutral loss of 46 Da (-CH2O2) and sequential

371

neutral loss of 18 Da (-H2O). Comparison of MSn spctra of fragment ions with MS2 spectra of

372

reference compounds the structure of the fragment ions resulting from water loss can differ

373

leading either to olefinic fragment ions or to epoxide fragment ions. The diastereomers differ

374

however significantly in the intensity of fragment ions and the order of fragmentation events

375

in MSn.

376

Diastereomers 2 and 5 elute very close to each other and they fragment almost identically in

377

MS2, however, in MS3, scyllo-quinic acid 5 produces a base peak at m/z 93 (C6H5O) whereas

378

epi-quinic acid 2 gives a base peak at m/z 111 (C6H7O2) in MS3. In another case, (-)-quinic

379

acid 1 and muco-quinic acid 3 show similar fragmentation patterns giving base peaks at m/z

380

127 (C6H7O3) and m/z 109 (C6H5O2) in MS2 and MS3 respectively but, they differ in the

381

retention times with compound 3 eluting 0.5 min earlier than compound 1. In another case

382

distinction between diastereomers 1 and 3 on the basis of their tandem MS, fragmentation had

383

to be carried out in MS4.

384

As shown in scheme A in Figure 11, (-)-quinic acid (1) with a pseudomolecular ion at m/z

385

191.1 (C7H11O6) as precursor ion undergoes concomitant dehydration and deformylation to

386

produce a base peak at m/z 127 (C6H7O3) corresponding to fragment Q1 in MS2. Loss of 46

387

Da and 18 Da takes place simultaneously as no secondary peak at m/z 145, corresponding to

388

deformylated quinic acid can be detected. Interestingly the carboxyl moiety on C-1 is

389

formally fragmented as formic acid reminiscent of an ester elimination or Tchugaev

390

elimination in mechanistic organic chemistry. Presumably dehydration takes place in between

391

C-2 and C-3 due to the fact that the proton on C-2 and hydroxyl group on C-3 share an anti-

392

periplanar arrangement. The precursor ion at m/z 127 further fragments in MS3 to give base

393

peak at m/z 109, which could suggest the presence of dihydroxybenzene-type structure.

394

However, since C-4-OH and C-5-OH do not possess the anti-periplanar arrangement between

395

either protons and hydroxyl groups required for stereoelectronic reasons for an E2 type

396

elimination a different pathway is taken. Comparison of MS2 spectra of all three

397

dihydroxybenzenes (1,3-, 1,4- and 1,2-C6H6O2) shows that none of them is identical to the

398

MS4 spectrum of 1 with m/z 127 as precursor ion (see Figure S5 in supplementary

399

information). The fragmentation of an epoxide such as cyclohexene oxide in MS2 gives only a 13 ACS Paragon Plus Environment


400

peak at m/z 81 with a neutral loss of 16 Da reminiscent of the MS4 of (-)-quinic acid 1. Hence

401

an epoxide fragment ion Q3 is proposed as product of fragmentation of Q1.

402

Scheme B in Figure 11 shows the proposed fragmentation pathway for epi-quinic acid 2. The

403

fragmentation can be explained if elimination of water through an E2 route takes place

404

between C-1 and C-2 to give epi-shikimic acid with a base peak at m/z 173 (C7H9O5) (Q3).

405

Indeed the MS3 with m/z 173 as precursor ion is almost identical to the MS2 spectrum of

406

shikimic acid.29 A direct infusion experiment shows that m/z 111 (C6H7O2) is a primary peak

407

in the MS3 of epi-quinic acid (2) (Figure 11). It can be explained through the assumption of

408

the presence of Q4 arising through the dehydration in between C-4 and C-5, which possess

409

the required anti-periplanar arrangement of a proton and –OH group as can be seen in the

410

Figure 11.

411

The suggested fragmentation of muco-quinic acid 3 is shown in scheme C. The carboxyl

412

group on C-1 is cleaved as a formic acid and the dehydration takes place in between C-2 and

413

C-3 due to anti-periplanar arrangement similar to the fragmentation in 1 to give base peak in

414

MS3 at m/z 127 (Q5). As shown in the scheme, when the cyclohexane ring is inverted, an

415

anti-periplanar geometry between C-2-H and C-3-OH arises suitable for dehydration between

416

C-2 and C-3. As can be observed in the direct infusion experiment in Figure 13, m/z 109

417

fragments further to give m/z 81.2 exactly similar to the MS4 of (-)-quinic acid (1). This

418

means that the fragments Q2 and Q6 are fragment ions of identical structure. Diastereomers 1

419

and 3 can be distinguished in MS2 of 3, as a secondary peak at m/z 145 was observed, which

420

signifies the decarboxylated molecule (Figure 10). In the structural arrangement of the

421

diastereomer 3, absence of the hydrogen bonding between 1,3-diaxially positioned oxygen

422

anion on C1 and hydrogen on C-3-OH does not facilitate the elimination of C-3-OH as

423

observed in case of (-)-quinic acid 1.

424

MS2 of the cis-quinic acid 4 gives base peak at m/z 93 along with the secondary peaks at m/z

425

173 and m/z 111. Base peak at m/z 93 suggests that decarboxylation and three dehydration

426

processes had taken place simultaneously to obtain the stable aromatic phenolate fragment ion

427

Q9. Unfortunately, phenol as an external standard and m/z 93 in compound 4 do not fragment

428

further as observed in direct infusion experiments, even at high collision energies. However

429

mechanistic arguments show that the phenolate structure of Q9 is reasonable if not expected,

430

since in intermediate Q7 all C-OH moieties show an anti-periplanar arrangement with Q8 at

431

m/z 111 as a secondary peak and reaction intermediate in MS2. 14 ACS Paragon Plus Environment

Page 14 of 29

Page 15 of 29


432

scyllo-Quinic acid 5 shows unique fragmentation pattern if compared to the other

433

diastereomers in present work. In MS2 it shows the pattern similar to the MS2 of the epi-

434

quinic acid 2 and in MS3, m/z 173 directly fragments into m/z 93 similar to the MS2 of the cis-

435

quinic acid 4 as can be seen in Figure 10. It is unclear at this point of time how the

436

fragmentation scheme in case of 5 can be explained mechanistically.

437

In conclusion, we have selectively synthesized all the diastereomers of the quinic acid except

438

for the neo-quinic acid 6 to prove that they can be chromatographically resolved and can be

439

identified by their characteristic fragmentation behavior in tandem MS spectra. This study

440

also illustrates the importance of the tandem mass spectrometry in the area of identification

441

and confirmation of the stereochemistry of comparatively small molecules like the

442

diastereomers of the quinic acid. It is also worth mentioning that it is the first time the crystal

443

structures of 3-O-cinnamoyl-1,4-scyllo-quinide 9 , 3-O-cinnamoyl-1,5-quinide 10, cis-quinic

444

acid 4, methyl cis-quinate 17 and 3,4-O-Cyclohexylidene-1,5-quinide 13 have been reported

445

(Figure 4). The fragmentation mechanisms are explained on the basis of the conformational

446

differences between the diastereomers of the quinic acid. Presence of the diastereomers like 3,

447

5 and 2 in hydrolyzed roasted coffee sample proves the existence of selected diastereomers of

448

chlorogenic acids in roasted coffee. However, it still remains unclear if the epimerization

449

occurs after the esterification with cinnamoyl functionality or the cinnamoyl group esterifies

450

with epimerized quinic acid during the food processing.

451 452

Supplementary informations are available containing crystallographic data, further synthetic

453

details and chromatograms and spectral data for all quinic acid diasteromers discussed.

454 455

ACKNOWLEDGEMENTS

456

The authors gratefully acknowledge the financial support from Kraft Food (now Mondelez)

457

and Jacobs University Bremen gGmbH. Furthermore, excellent technical support from Ms.

458

Anja Mueller and use of the X-ray facility of Prof. Dr. Kortz laboratory is acknowledged.

459 460 461 15 ACS Paragon Plus Environment


462

References

463

(1)

descriptors. Tetrahedron-Asym. 1997, 8, 3551-3554.

464 465

Eliel, E. L.; Ramirez, M. B. (-)-Quinic acid: configurational (stereochemical)

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Koshiro, Y.; Jackson, M. C.; Katahira, R.; Wang, M.; Nagai, C.; Ashihara, H.

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Biosynthesis of chlorogenic acids in growing and ripening fruits of Coffea arabica and

467

Coffea canephora plants. Z. Naturforsch., C: J. Biosci. 2007, 62, 731-742.

468

(3)

capillary electrophoresis. Colloq. Sci. Int. Cafe, [C. R.]. 1995, 16, 218-220.

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Weers, M.; Balzer, H.; Bradbury, A.; Vitzthum, O. G. Analysis of acids in coffee by

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Frank, O.; Zehentbauer, G.; Hofmann, T. Bioresponse-guided decomposition of roast

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coffee beverage and identification of key bitter taste compounds. Eur. Food Res.

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Technol. 2006, 222, 492-508.

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bioavailability and effects on health. Nat. Prod. Rep. 2009, 26, 1001-1043.

474 475

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Scholz-Boettcher, B. M.; Ernst, L.; Maier, H. G. New stereoisomers of quinic acid and their lactones. Liebigs Ann. Chem. 1991, 1029-1036.

476 477

Crozier, A.; Jaganath, I. B.; Clifford, M. N. Dietary phenolics: chemistry,

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Jaiswal, R.; Patras, M. A.; Eravuchira, P. J.; Kuhnert, N., Profile and Characterization

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of the Chlorogenic Acids in Green Robusta Coffee Beans by LC-MSn: Identification

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of Seven New Classes of Compounds. J. of Agric. Food Chem. 2010, 58, 8722-8737.

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caffeoyl- and feruloyl-muco-quinates. Org. Biomol. Chem. 2012, 10, 5266-5277.

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Jaiswal, R.; Dickman, M. H.; Kuhnert, N. First diastereoselective synthesis of methyl

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Matei, M. F.; Jaiswal, R.; Kuhnert, N. Investigating the Chemical Changes of

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Chlorogenic Acids during Coffee Brewing: Conjugate Addition of Water to the

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Olefinic Moiety of Chlorogenic Acids and Their Quinides. J. Agric. Food Chem. 2012,

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60, 12105-12115.

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Crozier, A. Recent studies on the bioavailability of dietary (poly)phenolics. Abstracts

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of Papers, 245th ACS National Meeting & Exposition, New Orleans, LA, United

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States, April 7-11, 2013. 2013, AGFD.

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Bennat, C.; Engelhardt, U. H.; Kiehne, A.; Wirries, F. M.; Maier, H. G.. HPLC

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analysis of chlorogenic acid lactones in roasted coffee. Zeitschrift für Lebensmittel-

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Untersuchung Forschung. 1994, 199, 17-21.

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Schrader, K.; Kiehne, A.; Engelhardt, U. H.; Maier, H. G. Determination of

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chlorogenic acids with lactones in roasted coffee J. Sci. Food Agric. 1996, 71, 392-

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Kim, K. H.; Kim, Y. H.; Lee, K. R. Isolation of quinic acid derivatives and flavonoids

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from the aerial parts of Lactuca indica L. and their hepatoprotective activity in vitro

497

Bioorg. Med. Chem. Lett. 2007, 17, 6739-6743.

498

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derivative from Asimina triloba Phytochemistry. 1998, 49, 103-108.

499 500

Haribal, M.; Feeny, P.; Lester, C. C. A caffeoylcyclohexane-1-carboxylic acid

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Kwon, H. C.; Jung, C. M.; Shin, C. G.; Lee, J. K.; Choi, S. U.; Kim, S. Y.; Lee, K. R.

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A new caffeoyl quinic acid from Aster scaber and its inhibitory activity against human

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immunodeficiency virus-1 (HIV-1) integrase. Chem. Pharm. Bull. 2000, 48, 1796-

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Kim, H. J.; Lee, Y. S. Identification of new dicaffeoylquinic acids from

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Chrysanthemum morifolium and their antioxidant activities Planta Med. 2005, 71,

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Thuong, P. T.; Su, N. D.; Ngoc, T. M.; Hung, T. M.; Dang, N. H.; Thuan, N. D.; Bae,

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K.; Oh, W. K. Antioxidant activity and principles of Vietnam bitter tea Ilex kudingcha

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Food Chem. 2009, 113, 139-145.

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Sanchez-Abella, L.; Fernandez, S.; Armesto, N.; Ferrero, M.; Gotor, V. Novel and

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Efficient Syntheses of (-)-Methyl 4-epi-Shikimate and 4,5-Epoxy-Quinic and -

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Shikimic Acid Derivatives as Key Precursors to Prepare New Analogues. J. Org.

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Chem. 2006, 71, 5396-5399.

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Fernandez, S.; Diaz, M.; Ferrero, M.; Gotor, V. New and efficient enantiospecific

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synthesis of (-)-methyl 5-epi-shikimate and methyl 5-epi-quinate from (-)-quinic acid.

516

Tetrahedron Lett. 1997, 38, 5225-5228.

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Kuhnert, N.; Dairpoosh, F.; Golon, A.; Yassin, G. H.; Jaiswal, R. Lessons from the

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analysis of complex dietary mixtures by mass spectrometry – understanding the

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chemistry of black tea thearubigins, coffee melanoidines and caramel. Food and

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Function, 2013, 4, 1130-1147.

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Jaiswal, R.; Matei, M. F.; Witt, M.; Kuhnert, N. Understanding the fate of chlorogenic

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acids in coffee roasting using mass spectrometry based targeted and non-targeted

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analytical strategies. Food Func. 2012, 3, 976-984.

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Jaiswal, R.; Kuhnert, N. Hierarchical scheme for the identification of 3,4,5 triacyl

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chlorogenic acids in Robusta green coffee beans. Rapid Commun. Mass Spectrom.

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2010, 24, 2283-2294.

527 528

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Jaiswal, R.; Kuhnert, N. Identification and characterization of five new classes of chlorogenic acids containing aliphatic side chains in Burdock (Arcticum lappa L.) 17 ACS Paragon Plus Environment


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roots by liquid chromatography tandem mass spectrometry. Food and Function 2011,

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2, 63-71.

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Moreira, A. S. P.; Coimbra, M. A.; Nunes, F. M.; Simoes, J.; Domingues, M. R. M.,

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Evaluation of the Effect of Roasting on the Structure of Coffee Galactomannans Using

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Model Oligosaccharides. Journal of Agricultural and Food Chemistry 2011, 59,

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10078-10087.

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Nunes, F. M.; Reis, A.; Domingues, M. R. M.; Coimbra, M. A., Characterization of

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galactomannan derivatives in roasted coffee beverages. Journal of Agricultural and

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Food Chemistry 2006, 54, 3428-3439.

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Agricultural and Food Chemistry 2012, 60, 3266-3274.

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Clifford, M. N.; Knight, S.; Kuhnert, N. Discriminating between the six isomers of dicaffeoylquinic acid by LC-MSn. J. Agr. Food Chem. 2005, 53, 3821-3832.

541 542

Golon, A.; Kuhnert, N., Unraveling the Chemical Composition of Caramel. Journal of

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Jaiswal, R.; Matei, M. F.; Glembockyte, V.; Patras, M. A.; Kuhnert, N., Hierarchical

543

Key for the LC-MSn Identification of All Ten Regio- and Stereoisomers of

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Caffeoylglucose. Journal of Agricultural and Food Chemistry 2014, 62, 9252-9265.

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Jaiswal, R.; Sovdat, T.; Vivan, F.; Kuhnert, N., Profiling and Characterization by LC-

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MSn of the Chlorogenic Acids and Hydroxycinnamoylshikimate Esters in Mate (Ilex

547

paraguariensis). J. Agric. Food Chem. 2010, 58, 5471-5484.

548 549 550 551 552 553 554 555 556 557


Page 18 of 29

Page 19 of 29


558

TABLE LEGEND

559

Table 1. MS2 data of quinic acid diastereomers in negative ion mode at 75% collision energy

560 561

FIGURE LEGEND

562

Figure 1. Stereoisomers of quinic acid

563

Figure 2. Reaction scheme for obtaining scyllo-quinic acid 5 and epi-quinic acid 2

564

Figure 3: Reaction scheme for the synthesis of cis-quinic acid 4

565

Figure 4: Single crystal X-ray structures of cis-quinic acid (4) , 3,4-O-Cyclohexylidene-1,5-

566

quinide (13), 3-O-cinnamoyl-1,4-scyllo-quinide (9), 3-O-cinnamoyl-1,5-quinide (10), methyl

567

cis-quinate (17), methyl 3,5-Di-O-(tert-butyldimethylsilyl)quinate (7) Conformer A and of

568

methyl 3,5-Di-O-(tert-butyldimethylsilyl)quinate (7) Conformer B.

569

Figure 5: MS2 and MS3 spectra of (-) quinic acid 1 in negative ion mode

570

Figure 6: MS2 and MS3 spectra of epi- quinic acid 2 in negative ion mode

571

Figure 7: MS2 and MS3 spectra of muco-quinic acid 3 in negative ion mode

572

Figure 8: MS2 spectrum of cis- quinic acid 4 in negative ion mode

573

Figure 9: MS2 and MS3 spectra of scyllo- quinic acid 5 in negative ion mode

574 575 576

Figure 10: Extracted ion chromatogram in negative ion mode of methanolic roasted coffee bean extract at m/z 191.1 showing quinic acid diastereomers (for chromtaograms of individual quinic acids diastereomers please ssee supplementary information)

577 578

Figure 11: Proposed fragmentation scheme of quinic acids diastereomers in negative ion mode

579 580 581 582 583



Page 20 of 29

Tables Table 1. Chromatographic and MS2 data of quinic acid diastereomers 1-5 in negative ion mode at 75% collision energy MS2 (m/z 191.0 as precursor ion)

Compound

No.

Retention Time [min] Base peak m/z

m/z

int%

m/z

Secondary peak int% m/z

int%

m/z

int%

1 2

3.9 3.6

127 173

173 127

82 86

85 145

56 30

93 85

51 24

111 111

23 17

3 4

3.4 3.4

127 93

173 173

64 37

85 111

45 34

109 155

20 7

145 61

19 5

5

4.2

173

127

91

85

50

111

48

93

46


Page 21 of 29


Figures HO

COOH

HO

OH OH

2

epi-quinic acid

C-1

HO

HO

COOH C-3

COOH

3

HO

OH

OH

OH

cis-quinic acid

(-) quinic acid 1 parent compound

3

OH

HO

OH

muco-quinic acid

COOH

5

HO

OH

HO

C-5

1

4

C-4 HO

COOH HO

C-5 HO

COOH

HO

C-3

OH HO

OH

HO

OH

5

OH neo-quinic acid

(+) quinic-acid

OH

6

OH OH

O OH

HO OH

OH

(-) quinic acid 1

OH OH OH

OH

epi-quinic acid 2

O

OH

O HO

HO OH

muco-quinic acid 3

OH

O

OH OH

O

OH

HO OH

HO

OH

OH

scyllo-quinic acid

O

COOH

HO OH

cis-quinic acid 4

OH

HO

OH

HO OH

scyllo-quinic acid 5

OH neo-quinic acid 6



Page 22 of 29

Figure 1. Stereochemical relationship of diastereomers of quinic acid (arrows indicating inversion at stereogenic centre) with trivial names and diastereomers of quinic acid 1-6 in chair conformation O HO

OH

HO

Si O

OH

O

O

O

HO

f

O HO

O

d

HO

OH

HO

O HO O

5

+

2

O

O

O

OH

O 8

HO

12

O

Si

Si

O

OH

O S

O

Si O

g

OH

O

11

Si

OH

HO

Si O

O O

Si O

Si

7

e

O

c

OH

1 HO

HO

O

a, b

OH

HO

O

O

HO

+1

g

O

+

OH

O

O

O OH

O

OH OH

1

9

10

Reagents and conditions: (a) MeOH, reflux, 12 h, Amberlite IR 120, 100%; (b) 2.6 equiv TBDMSCl, 2.6 equiv Et3N, DMF 0 0C, 2 h and then to RT, 16 h 75%; (c) 1.5 equiv MsCl, Py, RT, overnight, 95%; (d) 11, CsF, DMF, 90 0C, 24 h 2% 9 and 10; (e) 2 equiv DessMartin periodinane, CH2Cl2 , overnight, RT, 87%; (f) 1.5 equiv NaBH4, Ethanol, -30 0C, 40 min.; (g) 2M HCl, H2O, 1 h. Figure 2. Reaction scheme for obtaining scyllo-quinic acid (5) and epi-quinic acid (2)


Page 23 of 29


O

O HO

O

HO OH

O

HO

a

HO O

b

O

c

O HO

OH OH

1

O

O

OH

O

O

O

O

13

O

14

15

O

O

HO

HO OH HO

d

OH

O

f O

OH O

OH

4

16

e

O HO O HO

OH OH

17

Reagents and conditions: (a) cyclohexanone, PTSA·H2O, reflux, 24 h; (b) 21% NaOMe/MeOH, MeOH, rt, overnight; (c) Dess-Martin periodinane, DCM, rt, 18 h; (d) NaBH4, MeOH/THF (1:1), -30 ˚C, 1 h; (e) KOH, THF, rt, 45 min; (f) HCl (trace amounts in CDCl3). Figure 3: Reaction scheme for the synthesis of cis-quinic acid 4



4

Page 24 of 29

10

17

7 conformer A

9

7 conformer B

Figure 4: Single crystal X-ray structures of cis-quinic acid (4) , 3,4-O-Cyclohexylidene-1,5quinide (13), 3-O-cinnamoyl-1,4-scyllo-quinide (9), 3-O-cinnamoyl-1,5-quinide (10), methyl


Page 25 of 29


cis-quinate (17), methyl 3,5-Di-O-(tert-butyldimethylsilyl)quinate (7) Conformer A and of methyl 3,5-Di-O-(tert-butyldimethylsilyl)quinate (7) Conformer B.

-MS2(190.7), 3.9min

[%] 100

(1)

126.8 85.2

172.7 188.6

93.0 110.9

61.5

154.9 -MS3(190.8>126.9), 4.0min

0 108.9

100

0

85.5 40

60

80

100

120

140

160

180

200

m/z

Figure 5: MS2 and MS3 spectra of (-) quinic acid 1 in negative ion mode

-MS2(190.7), 3.6min

[%] 100

(2)

126.8 144.8

85.1 0

59.5

172.7

108.8 -MS3(190.8->172.5), 3.7min 154.8

100 111.0 0

50

100

150

200

250

m/z

Figure 6: MS2 and MS3 spectra of epi- quinic acid 2 in negative ion mode



-MS2(190.7), 3.4min

[%] 126.8

(3)

100

172.7

144.8

85.1 108.9 0

154.7 -MS3(190.8->126.8), 3.5min

108.8

100

0

Page 26 of 29

40

60

85.1 80 100

120

140

160

180

200 m/z

Figure 7: MS2 and MS3 spectra of muco-quinic acid 3 in negative ion mode

-MS2(190.7), 3.4min

[%] 100

(4)

93.1

80 60

172.7

110.9 40 20 59.1 0

40

60

136.8 154.7

86.1 80

100

120

140

160

180

200

Figure 8: MS2 spectrum of cis- quinic acid 4 in negative ion mode


m/z

Page 27 of 29


-MS2(190.7), 3.7min

[%]

(5)

100

172.6

126.8 85.1 110.9

154.7 188.7

71.3

0

-MS3(190.8->173.3), 3.8min 93.0

100

127.8 110.9 152.7 0

50

100

150

200

250

300

m/z

Figure 9: MS2 and MS3 spectra of scyllo- quinic acid 5 in negative ion mode

Intens. x106 1.0

EIC 191.0 Hydrolysed roasted coffee

0.8 0.6 0.4 1

0.2 352 0.0

1

2

3

4

5

6

Time [min]

Figure 10: Extracted ion chromatogram in negative ion mode of methanolic roasted coffee bean extract at m/z 191.1 showing quinic acid diastereomers (for chromtaograms of individual quinic acids diastereomers please see supplementary information)



O

A

OH

O

OH OH

O

H OH -HCO2H H O

H

OH H OH

O

B

O OH

-HCO2

O

OH

OH

C

OH -HCO2H OH

HO OH

O

D

H OH

H OH

O

H O

2

O

OH OH OH

H

5 6 3 2

-H2O

OH

OH

m/z 111 Q4

O

O

OH

OH

OH

-H2O O

O

O

OH

HO

4

OHH

-H2O

3

H

O

H OH H OH O OH

H

m/z 109 Q2

OH

1

OH OH

O

OH OH

4

OH

m/z 173 Q3

O

O

m/z 127 Q1

3

2

1

OH

OH OH

5

6

-H2O

O

OH

OH OH

O -H2O

-H2O

OH O

O

O

H O

OH OH

Page 28 of 29

m/z 109

m/z 127 Q5

H

H

OH OH OH

O OH

m/z 173 Q7

m/z 111 Q8

Q6

O

m/z 93 Q9

Figure 11: Proposed fragmentation scheme of quinic acids diastereomers in negative ion mode


Page 29 of 29


TOC Graphic

OH

O

OH OH

O OH

HO

HO OH

OH

OH

O

OH OH

O OH

HO OH

HO

HO OH

OH

OH

O

OH OH

HO OH

HO

OH

OH OH

O

OH

HO OH


Synthesis, Structure, and Tandem Mass ... - ACS Publications

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