Synthesis, Structure, and Tandem Mass Spectrometric Characterization of the Diastereomers of Quinic Acid

Article abstract of DOI:10.1021/acs.jafc.6b02472

(-)-Quinic acid possess eight possible stereoisomers, which occur both naturally and as products of thermal food processing. In this contribution, we have selectively synthesized four isomers, namely, epi-quinic acid, muco-quinic acid, cis-quinic acid, and scyllo-quinic acid, to develop a tandem LC-MS method identifying all stereoisomeric quinic acids. Four derivatives have been unambiguously characterized by single-crystal X-ray crystallography. The missing diastereomers of quinic acid were obtained by nonselective isomerization of (-)-quinic acid using acetic acid/concentrated H₂SO₄ allowing chromatographic separation and assignment of all diastereomers of quinic acid. We report for the first time that a full set of stereoisomers are reliably distinguishable on the basis of their tandem mass spectrometric fragment spectra as well as their elution order. A rationale for characteristic fragmentation mechanisms is proposed. In this study, we also observed that muco-quinic acid, scyllo-quinic acid, and epi-quinic acid are present in hydrolyzed Guatemalan roasted coffee sample as possible products of roasting.

Full text of DOI:10.1021/acs.jafc.6b02472

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Article
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
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Journal of Agricultural and Food Chemistry
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Synthesis, Structure and Tandem Mass spectrometric Characterization of the
Diastereomers of Quinic Acid
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Sagar Deshpande,a Marius Febi Matei,a Rakesh Jaiswal, Bassem S. Bassil,a,b Ulrich Kortza
and Nikolai Kuhnerta*
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a)Department of Life Sciences and Chemistry, Jacobs University Bremen, 28759 Bremen,
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:
Telephone: 0049 421 200 3120
Fax: 0049 421 200 3229
Email: n.kuhnert@jacobsꢀuniversity.de
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ABSTRACT
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(ꢀ)ꢀQuinic acid possess eight possible stereoisomers, which occur both naturally and as
products of thermal food processing. In this contribution, we have selectively synthesized four
isomers namely, epiꢀquinic acid, mucoꢀquinic acid, cisꢀquinic acid and scylloꢀquinic acid in
order to develop a tandem LCꢀMS method identifying all stereoisomeric quinic acids. Four
derivatives have been unambiguously characterized by single crystal Xꢀray crystallography.
The missing diastereomers of quinic acid were obtained by nonꢀselective isomerization of (ꢀ)ꢀ
quinic acid using acetic acid/conc. H2SO4 allowing chromatographic separation and
assignment of all diastereomers of quinic acid. We report for the first time that a full set of
stereoisomers are reliably distinguishable on the basis of their tandem mass spectrometric
fragment spectra as well as their elution order. A rational for characteristic fragmentation
mechanisms is proposed. In this study, we also observed that mucoꢀquinic acid , scylloꢀquinic
acid and epiꢀquinic acid are present in hydrolyzed Guatemala roasted coffee sample as
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
cinchona bark to tobacco leaves and cranberries in its free form or in the form of its depsides,
chlorogenic acids. It was first isolated in 1790 and was given an empirical formula of
C7H12O6 in 1838.1 In a single coffee bean, up to 4.0 mg free (ꢀ) quinic acid is found.2 In
Colombian Arabica green coffee, up to 7.0 g/kg of quinic acid is present, which increases up
to 10.0 g/kg upon roasting.3 Quinic acid provides characteristic astringent taste to the
beverage, hence it is also used as a flavor enhancer in certain beverages.4 The esters of the 3R,
4S, 5R isomers of the quinic acid, also referred to as chlorogenic acids, constitute the most
important quinic acid derivatives in nature and in our diet.5,6 For quinic acid diastereomers
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
enantiomers (Figure 1). Formally the diastereomers 2-6 can be derived from the parent
compound (ꢀ)ꢀquinic acid 1 by inversion of one or two of the four stereogenic centres as
demonstrated in Figure 1. As reported by Kuhnert and coꢀworkers, 80 different chlorogenic
acid derivatives have been identified in green coffee beans.7 After roasting and brewing, this
number is increased to a minimum of 120 derivatives identified on the basis of the presence of
characteristic fragment ions corresponding to quinic acid and quinic acid lactones in MSn
chromatograms.8,9 Products of epimerization of Chlorogenic acids for example mucoꢀcaffeoyl
and feruloyl esters were reported in roasted coffee formed at elevated temperatures.8 After
ingestion of the foods containing quinic acid esters, metabolism in humans may also give rise
to the esters of the diastereomers of quinic acid.8,10 This fact supports the assumption that the
roasting or food processing in general facilitates the isomerization at the stereogenic centers in
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
literature as plant secondary metabolites. For example, in Lactuca indica L., Asimina triloba
and Aster scaber 3,5ꢀDicaffeoylꢀmucoꢀquinic acid was identified. In Asimina triloba 3ꢀ
13ꢀ15
caffeoylꢀmucoꢀquinic acid was also identified.
In Chrysanthemum morifolium 3,5ꢀ
dicaffeoylꢀepiꢀquinic acid and 1,3ꢀdicaffeoylꢀepiꢀquinic acid was identified.16 3,5ꢀdicaffeoylꢀ
epiꢀquinic acid esters were also reported in Ilex kudingcha.17 These muco, epi and scyllo
esters of diastereomers of quinic acid are reported to show important biological activities like,
hepatoprotectivity, antioxidant activity and anti HIVꢀ1 integrase activity.13ꢀ17 Considering the
fact that regiosomers as well as the esters of the diastereomers of quinic acids are readily
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distinguishable by their fragmentation pattern in tandem MS experiments, it is very important
to acquire authentic synthetic standards with defined stereochemistry by organic synthesis.
Development of the tandem mass spectrometric methods for the identification of
diastereomers of quinic and chlorogenic acids will enable us to identify novel chlorogenic
acid derivatives present in the biological samples even in very low concentrations. Since,
these biological samples emerge either biosynthetically, through manꢀinduced processes such
as, roasting, brewing, cooking etc. or as products of metabolism, their mass spectrometric
study will help to improve understanding of the changes in the chlorogenic acid profile
through biosynthetic processes in great detail.
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MATERIALS AND METHODS
Chemicals
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All the chemicals (analytical grade) were purchased from SigmaꢀAldrich (Taufkirchen,
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
auto sampler with a 100 µL loop, and a DAD detector with a lightꢀpipe flow cell (recording at
254 and 320 nm and scanning from 200 to 600 nm). This was interfaced with an HCT Ultra
ionꢀtrap mass spectrometer fitted with an ESI source (Bruker Daltonics HCT Ultra, Bremen,
Germany) operating in full scan, auto MSn mode to obtain fragment ion m/z. As necessary,
MS2, MS3, and MS4 fragmentꢀtargeted experiments were performed to focus only on
compounds producing a parent ion at m/z 191, 173. Tandem mass spectra were acquired in
ManualꢀMSn mode using fixed collision energy. The fragmentation amplitude was set to 0.75
V. Also, direct injection experiments targeting the fragments in MS2, MS3, and MS4 were
performed on all diastereomers of quinic acid keeping the fragmentation amplitude constant at
1.0 volts. MS operating conditions (negative mode) was optimized using (ꢀ)ꢀquinic acid with a
o
capillary temperature of 365 C, a dry gas flow rate of 10 L/min, and a nebulizer pressure of
10 psi.
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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
5mm × 4.6 mm i.d. guard column of the same material (Varian, Darmstadt, Germany). The
elution solvent was water: formic acid (1000:0.05 v/v) delivered at a total flow rate of 800
µL/min by 30 min isocratically.
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NMR
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1H NMR and 13C NMR spectra were acquired on a JEOL ECXꢀ400 spectrometer operating at
400 MHz for 1H NMR and 100 MHz for 13C NMR at room temperature using a 5 mm probe.
The chemical shifts (δ) are reported in parts per million and were referenced to
Tetramethylsilane (TMS at 0 ppm). The coupling constants (J) are quoted in hertz. The
following abbreviations are used: s, singlet; d, doublet; t, triplet; m, multiplet; bs, broad
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 nonꢀselectively by the process
previously described by Maier and coꢀworkers.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
mL) and Amberlite IR120 acidic resin (5g) for 12 h. The reaction mixture was then filtered
and concentrated in vacuo. The product was obtained in more than 95% yield and the purity
of the product was confirmed by NMR. It was then subjected to selective silyl protection on
C3 and C5 of the methyl quinate by tertꢀbutyldimethylsilyl chloride (TBDMSCl).18
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Methyl quinate (2650 mg, 12.86 mmol) was stirred with triethylamine (5 mL) in anhydrous
dimethylformamide (26 mL) in an inert atmosphere and to this solution, tertꢀ
butyldimethylsilyl chloride (5034 mg, 33.436 mmol) was added and the mixture was stirred
for 2 h at 0 0C and 16 h at room temperature. EtOAc was added and the residue was filtered.
Concentrated filtrate was purified using flash chromatography (gradient eluent: 20% EtOAc
in
petroleum
ether)
to
afford
white
crystalline
methyl
3,5ꢀDiꢀOꢀ(tertꢀ
butyldimethylsilyl)quinate 7 in 75% yield, which was confirmed by single crystal Xꢀray
diffraction have free Cꢀ4 on quinic acid skeleton. Compound 7 was used as a precursor for
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the synthesis of epiꢀquinic acid 2 and scylloꢀquinic acid 5 as shown in the supplementary
information.
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DessꢀMartin periodinane (390 mg, 0.92 mmol) was added to the solution of compound 7 (200
mg, 0.46 mmol) dissolved in dichloromethane (15 mL) at room temperature. Reaction was
stirred overnight and diluted with Et2O. Then to this 20 ml of a 1:1 saturated mixture of
Na2S2O3 and NaHCO3 solution was added until the reaction became clear. Organic layer was
collected and the aqueous layer was extracted with EtOAc (3× 20 mL). Combined organic
layers were collected, dried and concentrated in vacuo. Crude product was subjected to flash
chromatography (eluent: 24% EtOAc in petroleum ether), which afforded methyl 3,5ꢀDiꢀOꢀ
(tertꢀbutyldimethylsilyl)ꢀ4ꢀoxoquinate 12 in the form of sticky colorless solid in 87%. 13C
NMR (100 MHz, CDCl3): 206.51, 173.15, 75.98, 75.33, 69.81, 52.94, 46.71, 41.22, 25.79,
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 Cꢀ4 by the action of NaBH4 and Lꢀselectride. Out
of these two reduction procedures, reduction with NaBH4 showed higher selectivity towards
the formation of epiꢀ derivative as compared to Lꢀselectride. 177 mg of compound 12 (0.4074
mmol) was dissolved in ethanol and the reaction flask was immersed in an acetone bath.
Liquid nitrogen was slowly added until the bath temperature reached ꢀ30 0C. NaBH4 (23 mg,
0.6111 mmol) was added and the mixture was stirred for 40 min. Solvent was removed
0
immediately under reduced pressure at 30 C in a rotary evaporator. The residue was then
extracted with water and EtOAc mixture three times. Organic layers were collected and dried
over Na2SO4 and concentrated. 13C NMR of the crude product confirmed reduction at C4
indicated by the loss of a peak at 206 ppm. The crude product was directly subjected to
hydrolysis by 2M HCl and water without further purification. Product of the hydrolysis was
diluted with water and extracted with water and EtOAc mixture thrice. Aqueous layers were
collected and concentrated in vacuo. Resulting white product was used in HPLCꢀMS analysis.
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Synthesis of the muco-quinic acid (3)
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mucoꢀQuinic acid 3 was obtained from the methyl TMBꢀmucoꢀquinate (TMB Ley’s
tetramethoxybutane), which was synthesized by the procedure previously reported by Jaiswal
et al..8, Methyl TMBꢀmucoꢀquinate (500 mg, 1.56 mmol) was subjected to hydrolysis by 70%
aqueous trifluoroacetic acid for 1 hour. Resulting methyl mucoꢀquinate was stirred with 2M
KOH for 20 min followed by neutralization by Amberlite IR 120 acidic resin for 10 min. The
mixture was filtered and concentrated under reduced pressure. The resulting yellowish solid
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was analyzed by NMR. H NMR (400 MHz, D2O): 1.73 (dd, 1H, H6ax, JHH 12.4), 1.75 (dd,
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, JHH 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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3,4ꢀOꢀCyclohexylideneꢀ1,5ꢀquinide 13 was synthesized by adding quantities of 10.00 g
(52.04 mmol) of quinic acid and 200 mg (1.05 mmol) of pꢀtoluenesulfonic acid monohydrate
(PTSA·H2O) to 100 mL of cyclohexanone to give a white suspension. The reaction was then
refluxed for 24 h to give a yellow solution which was cooled to 50 ºC and neutralized with a
solution of NaOEt (71.5 mg) in EtOH (5 mL) to give a yellow clear solution. The solvents
were removed under reduced pressure and to the resulting yellow viscous liquid a volume of
100 mL of EtOAc was added. The organic phase was washed with 50 mL of H2O and the
aqueous phase was backꢀextracted with 30 mL EtOAc. The combined organic layers were
washed with a halfꢀsaturated NaHCO3 solution, dried on Na2SO4, filtered and evaporated. The
resulting yellow viscous liquid was recrystallized from a 1:1 nꢀheptane:EtOAc solution to
afford white crystals of compound 13 (9.26 g, 36.43 mmol, 70%, mp. 172ꢀ174 °C); 1HꢀNMR
(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 =
6.4, 2.8, 1.4 Hz), 2.66 (d, 1H, J = 11.9 Hz), 2.38ꢀ2.31 (ddd, 1H, J = 14.7, 7.8, 2.3 Hz), 2.31ꢀ
2.25 (ddt, 1H, J = 11.9, 6.4, 2.3 Hz), 2.17 (dd, 1H, J = 14.7, 3.2 Hz), 1.73ꢀ1.68 (m, 2H), 1.67ꢀ
1.60 (m, 2H), 1.57ꢀ1.51 (m, 4H), 1.43ꢀ1.36 (m, 2H); 13CꢀNMR (CDCl3): δC 178.9 (ꢀCOOR),
110.7 (Cꢀ1'), 76.1 (Cꢀ4), 71.8 (Cꢀ1), 71.6 (Cꢀ3), 71.2 (Cꢀ5), 38.6 (Cꢀ6), 37.0 (Cꢀ2), 34.5 (Cꢀ
6'), 33.7 (Cꢀ2'), 25.1 (Cꢀ5'), 24.0 (Cꢀ3'), 23.6 (Cꢀ4').
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3,4ꢀOꢀCyclohexylideneꢀ1,5ꢀquinide 13 (8.75 g, 34.41 mmol) was dissolved in 100 mL MeOH
and a 21% solution NaOMe/MeOH was added (187 mg NaOMe). The clear solution was
stirred overnight, the mixture was then quenched with glacial acetic acid (232 µL) and the
volatile components were removed under vacuum. The resulting mixture was dissolved in
EtOAc and washed 3 times (3x40 mL). The organic layer was dried over Na2SO4, filtered and
the solvent was removed under low pressure. The crude product was purified by column
chromatography on silica gel (20ꢀ50% EtOAc/petroleum ether) to give methyl 3,4ꢀOꢀ
1
cyclohexylideneꢀquinate 14 as a white solid (5.87 g, 20.51 mmol, 60%). HꢀNMR (CDCl3):
δH 4.46 (dt, 1H, J = 5.9, 3.7 Hz), 4.14ꢀ4.07 (m, 1H), 3.97 (t, 1H, J = 5.9 Hz), 3.79 (s, 3H),
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,
13.7, 11.0 Hz), 1.74ꢀ1.68 (m, 2H), 1.68ꢀ1.52 (m, 6H), 1.44ꢀ1.33 (m, 2H); 13CꢀNMR (CDCl3):
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δC 175.6 (ꢀCOOCH3), 110.1 (Cꢀ1'), 79.4 (Cꢀ4), 74.1 (Cꢀ1), 73.1 (Cꢀ3), 68.7 (Cꢀ5), 53.0 (ꢀ
CH3), 39.0 (Cꢀ6), 38.0 (Cꢀ2), 34.9 (Cꢀ6'), 34.8 (Cꢀ2'), 25.0 (Cꢀ5'), 24.1 (Cꢀ3'), 23.7 (Cꢀ4').
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To a suspension of DessꢀMartin periodinane (2.96 g, 6.99 mmol) in anhydrous CH2Cl2 (65
mL) compound 14 (1.82 g, 6.36 mmol) was added. The reaction mixture was stirred at room
temperature for 18 h, was then diluted with Et2O (100 mL) and 50 mL of a saturated aqueous
Na2S2O3 and 50 mL of a saturated NaHCO3 solution (100 mL) were added. The mixture was
stirred until the solids were dissolved (20 min). The aqueous layer was extracted with Et2O
(2× 50 mL) and the combined organic layers were dried over Na2SO4, filtered and
concentrated in vacuum. Product methyl 3,4ꢀOꢀcyclohexylideneꢀ5ꢀoxoquinate 15 (1.81 g,
6.36 mmol, 100%) was used for the next step without further purification. 1HꢀNMR (CDCl3):
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,
1H, J = 14.7, 2.3 Hz), 2.56 (t, 1H, J = 2.3 Hz), 2.55 (d, 1H, J = 4.1 Hz), 1.74ꢀ1.64 (m, 2H),
1.64ꢀ1.56 (m, 4H), 1.56ꢀ1.47 (m, 2H), 1.43ꢀ1.32 (m, 2H); 13CꢀNMR (CDCl3): δC 204.4 (Cꢀ5),
173.0 (ꢀCOOCH3), 111.7 (Cꢀ1'), 78.2 (Cꢀ4), 76.9 (Cꢀ1), 75.9 (Cꢀ3), 53.5 (ꢀCH3), 49.1 (Cꢀ6),
37.0 (Cꢀ2), 35.3 (Cꢀ6'), 34.7 (Cꢀ2'), 24.9 (Cꢀ5'), 23.9 (Cꢀ3'), 23.8 (Cꢀ4').
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To obtain methyl 3,4ꢀOꢀcyclohexylideneꢀepiꢀquinate 16, compound 15 (1.53 g, 5.33 mmol)
was dissolved in a 1:1 mixture (v/v) MeOH/THF (100 mL) and was cooled to ꢀ30 ˚C with an
acetone/liquid nitrogen bath. NaBH4 (222 mg, 5.86 mmol) was added and the mixture was
stirred at ꢀ30 ˚C for 1 h. The solvents were removed in vacuum and the residue was extracted
three times with a water/EtOAc mixture. The organic layers were dried over Na2SO4, filtered
and the solvent was removed under reduced pressure. The product was purified by column
1
chromatography (40% EtOAc/petroleum ether). HꢀNMR (CDCl3): δH 4.52 (dt, 1H, J = 7.6,
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), 2.18ꢀ2.06
(m, 3H), 2.02 (dd, 1H, J = 14.2, 10.1 Hz), 1.75 (m, 2H), 1.69ꢀ1.59 (m, 4H), 1.59ꢀ1.51 (m,
2H), 1.44ꢀ1.35 (m, 2H); 13CꢀNMR (CDCl3): δC 175.4 (ꢀCOOCH3), 110.0 (Cꢀ1'), 73.8 (Cꢀ4),
73.4 (Cꢀ1), 72.5 (Cꢀ3), 66.1 (Cꢀ5), 53.0 (ꢀCH3), 38.1 (Cꢀ6), 36.4 (Cꢀ2), 35.6 (Cꢀ6'), 34.0 (Cꢀ
2'), 25.2 (Cꢀ5'), 24.1 (Cꢀ3'), 23.7 (Cꢀ4').
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Crystals of 4 suitable for single crystal Xꢀray diffraction were obtained in an NMR tube
containing demethylated 16 dissolved in CDCl3 by removal of the acidꢀlabile cyclohexylidene
1
protection promoted by the trace amounts of HCl present in the deuterated solvent. HꢀNMR
(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 =
12.4, 4.1 Hz), 1.62 (t, 2H, J = 12.4 Hz); 13CꢀNMR (D2O): δC 177.1 (ꢀCOOCH3), 72.4 (Cꢀ4),
71.1 (Cꢀ1), 66.9 (Cꢀ3, Cꢀ5), 35.9 (Cꢀ2, Cꢀ6).
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Synthesis of the scyllo-quinic acid (5)
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Methyl 3,5ꢀDiꢀOꢀ(tertꢀbutyldimethylsilyl)quinate (7) (500 mg, 1.15 mmol) was mixed and
0
stirred with 12 mL of anhydrous pyridine and cooled to 0 C. Methanesulfonyl chloride was
added drop wise to the mixture. Reaction was stirred at room temperature for overnight. 10
mL NaHCO3 added and after stirring for 10 min, aqueous phase was washed two times with
Et2O. Organic layers were collected, dried over Na2SO4 and concentrated. Crude product was
subjected to flash chromatography (eluent: 22% EtOAc in petroleum ether), which afforded
1
methyl 3,5ꢀdiꢀOꢀ(tertꢀbutyldimethylsilyl)ꢀ4ꢀOꢀmethanesulfonylquinate 8 in 95% yield. H
NMR (400 MHz, CDCl3): 0.06 (s, 3H, MeSi), 0.08 (s, 3H, MeSi), 0.11 (s, 3H, MeSi), 0.12 (s,
2
3
3H, MeSi), 0.86 (s, 9H, Me3CSi), 0.88 (s, 9H, Me3CSi), 1.94 (dd, 1H, H6ax, JHH 13.74, JHH
10.07, Hz), 2.00ꢀ2.10 (m, 1H, H2ax), 2.12ꢀ2.31 (m, 2H, H6eq, H2eq), 3.04 (s, 3H, MeS), 3.77 (s,
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
and to this was added cesium fluoride (790 mg, 5.2 mmol). The mixture was stirred for 30
min and cinnamic acid (770 mg, 5.2 mmol) was added. The reaction was heated to 90 0C and
stirred for 36 h. DMF was removed in vacuum and the obtained crude product was subjected
to column chromatography (eluent: 45% EtOAc, 55% petroleum ether). One fraction
collected from the column chromatography confirmed the formation of the desired product in
the NMR analysis and formed a crystal in the NMR tube suitable for single crystal Xꢀray
diffraction.
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The mixture of compound 9 and compound 10 was subjected to hydrolysis through the same
procedure as described earlier. Product of the hydrolysis was diluted with water and extracted
with water and EtOAc mixture three times. Aqueous layers were collected and concentrated
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
water for 1 hour. The mixture was then filtered and the water in the filtrate was removed
under reduced pressure. Resulting residue was subjected to base hydrolysis by treatment of 25
mL 2M NaOH for 2 h. The reaction was neutralized by addition of 2M HCl. The mixture was
diluted with 25 mL water and the aqueous layer was extracted with EtOAc (3 × 20 mL). The
aqueous layers were collected and concentrated in vacuo. Resulting yellowish product was
used directly in HPLCꢀMS analysis.
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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
cisꢀquinate 17 were prepared by refluxing respective diastereomers in MeOH with equal
amount of Amberlite IR120 acidic resin for 12 h. The reaction mixture was then filtered and
concentrated in vacuo. The resulting product was used directly in HPLC/MS analysis in case
of methyl epiꢀquinate, methyl scylloꢀquinate and methyl mucoꢀquinate. Crystals of methyl cisꢀ
quinate 17 suitable for single crystal Xꢀray diffreaction (Figure 4) were obtained in an NMR
tube containing methyl 3,4ꢀOꢀcyclohexylideneꢀepiꢀquinate 16 dissolved in CDCl3 by removal
of the acidꢀlabile cyclohexylidene protection promoted by the trace amounts of HCl present in
the deuterated solvent. The methyl quinate of (ꢀ)ꢀquinic acid was confirmed by the NMR and
then utilized in HPLC/MS.
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X-ray crystallography
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Crystals were mounted on a Hampton cryoloop in light oil for data collection at 100 K.
Indexing and data collection were performed on a Bruker D8 SMART APEX II CCD
diffractoꢀmeter with κ geometry and Mo Kα radiation (graphite monoꢀ chromator, λ =
0.71073 Å; Karlsruhe, Germany). Data integration was performed using SAINT. Routine
Lorentz and polarization corrections were applied. The SHELX package was used for
structure solution and refinement. Refinements were fullꢀmatrix leastꢀsquares against F2 using
all data. In the final refinement, all nonꢀhydrogen atoms were refined anisotropically and
hydrogen atoms were either found directly and refined isotropically or placed in calculated
positions. Crystallographic data (CIF files) are available at www.ccdc.cam.ac.uk.
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RESULTS AND DISCUSSIONS
Synthesis of quinic acid diastereomers
18
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Several methods for the synthesis of the epiꢀquinic acid 2 have been reported, exploiting
formation of a 1, 5ꢀquinide followed by selective protection of Cꢀ3ꢀOH by silyl protecting
group and subsequent inversion of configuration at C4 by oxidationꢀreduction sequences. This
approach was found to be unreliable in our hands, since silylꢀprotection was observed to be
nonꢀselective and separation low yielding. In the present work, once the methodology to
obtain 7 in pure form was developed, the ambiguity in the selective protection was eliminated
in order to leave Cꢀ4ꢀOH free for further treatment (Figure 2). Methyl 3,5ꢀdiꢀOꢀ(tertꢀ
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butyldimethylsilyl) quinate 7 served as a precursor for both of the diastereomers, epiꢀquinic
acid 2 and scylloꢀquinic acid 5. Of these, diastereomer 2 was obtained by oxidationꢀreduction
pathway and 5 was synthesized by incorporating SN2 reaction strategy, using cinnamic acid as
a nucleophile leading to inversion of configuration at C4. However, in fact the actual product
isolated, was found to have rearranged to yield 3ꢀOꢀcinnamoylꢀ1,4ꢀscylloꢀquinide 9 possibly
due to the traces of acid present in the workꢀup procedure. 3ꢀOꢀCinnamoylꢀ1, 4ꢀscylloꢀquinide
9 and 3ꢀOꢀcinnamoylꢀ1,5ꢀquinide 10 were coꢀcrystallized and were obtained in very low
yield. Hence, attempts towards the isolation of compound 9 were avoided and the crystals
were directly hydrolyzed to obtain a 9: 1 mixture of scylloꢀquinic acid 5 and quinic acid as
judged by NMR and LCꢀMS data.
19
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CisꢀQuinic acid 4 was obtained starting from 1,3ꢀcyclohexylidene protected quinide
13
(Figure 3). Opening of the lactone ring using methoxide furnished methylester 14, which was
subsequently oxidized to ketone 15 using the DessꢀMartin periodinane reagent.
Diastereoselective reduction using NaBH4 yielded a 4:1 mixture of diastereomers, from,
which methyl ester 17 could be obtained in pure form by crystallization. Hydrolysis yielded
the target compound 4 in poor yield, however in crystalline form suitable for single crystal Xꢀ
ray diffraction studies, hence confirming its structure.
320
Discussion of single crystal X-ray structures
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For five synthetic intermediates crystals suitable for single crystal Xꢀray diffraction could be
obtained. The resulting Xꢀray structures confirm the relative stereochemistry of the quinic
acid scaffold as diastereomers of quinic acid and therefore support the relative
stereochemistry of the final quinic acid diastereomer products. In the crystal of cisꢀquinic acid
4 (Figure 4), we observed two molecules in the asymmetric unit representing the same
structure. Compounds 3ꢀOꢀcinnamoylꢀ1,4ꢀscylloꢀquinide 9 and 3ꢀOꢀcinnamoylꢀ1,5ꢀquinide 10
coꢀcrystallized along with one molecule of chloroform in one asymmetric unit. The crystal
structure of 3,5ꢀdiꢀOꢀ(tertꢀbutyldimethylsilyl)quinate 7 showed conformational disorder in
one of the silyl groups (Si1A and Si1B), both conformations are shown in Figure 4. The
disorder was modeled and showed a value of 50% for each conformation. Table 2 illustrates
the crystal data and structure refinement for compounds methyl cisꢀquinate 17, cisꢀquinic acid
4,
3ꢀOꢀcinnamoylꢀ1,4ꢀscylloꢀquinide(9),
3ꢀOꢀcinnamoylꢀ1,5ꢀquinide
10,
3,4ꢀOꢀ
Cyclohexylideneꢀ1,5ꢀquinide (13) and methyl 3,5ꢀDiꢀOꢀ(tertꢀbutyldimethylsilyl)quinate 7.
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LC-MS analysis of quinic acid diastereomers
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With authentic standards in hand for four quinic acid diastereoismers from this work and
muco quinic acid from previous work,8 a HPLC method was developed allowing separation
of quinic acid diastereomers for future food analysis and tandem MS investigations. All
quinic acid diastereomers can be readily detected in negative ion mode as pseudomolecular
ions ([MꢀH], C7H11O6) at m/z 191. 0550 +/ꢀ 0.0003 using high resolution MS or at 191.1 using
an ESI ion trap mass spectrometer. Using Maier’s protocol for unselective isomerization using
6
concentrated acid the missing isomer neoꢀquinic acid 6 could be tentatively assigned by
arguments of exclusion. Observation the retention times of all the diastereomers, shows that
they elute very close to each other . The elution order can be given as, 1>5>2>3=4. Where 1
(quinic acid) elutes at 3.9 min and 3/4 mucoꢀ and/or cisꢀquinic acid elute at 3.4 minutes (Table
1). Although diastereomers 3 and 4 partially coꢀelute at the same retention time, their tandem
MS shows clear distinction as can be seen in Figure 7 and 8. All tandem MS data for quinic
acid diastereomers 1ꢀ5 with the precursor ion at m/z 191.0 are shown in Figures 5ꢀ9.
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An analysis of a roasted and subsequently hydrolyzed Arabica coffee extract shows that in an
extracted ion chromatogram at m/z 191 four diastereomers of quinic acid can be observed and
assigned (Figure 10) The identified compounds are epiꢀquinic acid 2, mucoꢀquinic acid 3 and
cisꢀquinic acid 4 along with quinic acid 1. Following an earlier report we assume that these
compounds are formed at elevated temperature during coffee roasting, since they are absent
6, 20ꢀ23
from the corresponding green coffee bean material.
It is surprising to note that quinic
acid secondary alcohols are prone to epimerization under thermal conditions, whereas
structurally related carbohydrates are not. 24ꢀ26
357
Tandem mass spectrometry of quinic acid diastereomers
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Recent advances in tandem mass spectrometry have shown that certain classes of isomeric
compounds can be reliably distinguished by tandem mass spectrometry based on significant
differences in their tandem mass spectra. In particular regioisomers have been shown to
display characteristic fragmentation mechanisms allowing a reliable assignment of structure.
27ꢀ29
Much less is known about differences in fragment spectra of sets of diastereomers with
the compounds available here allowing a proof of principle study that diastereomeric
structures can be distinguished and assigned based on their tandem MS spectra.
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Fragment spectra were obtained in negative ion mode using collision induced dissociation
(CID) using an ion trap mass spectrometer. MS2, MS3 and in some cases MS4 spectra were
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recorded. The MS2 fragmentation all of the diastereomers was investigated under constant
collision energy in the LCꢀMS runs at 0.75 volts (see Table 1). Optimum value for the
collision energy was obtained by taking (ꢀ)ꢀquinic acid 1 as a standard. All diastereomers
show characteristic fragmentations including neutral loss of 46 Da (ꢀCH2O2) and sequential
neutral loss of 18 Da (ꢀH2O). Comparison of MSn spctra of fragment ions with MS2 spectra of
reference compounds the structure of the fragment ions resulting from water loss can differ
leading either to olefinic fragment ions or to epoxide fragment ions. The diastereomers differ
however significantly in the intensity of fragment ions and the order of fragmentation events
in MSn.
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Diastereomers 2 and 5 elute very close to each other and they fragment almost identically in
MS2, however, in MS3, scylloꢀquinic acid 5 produces a base peak at m/z 93 (C6H5O) whereas
epiꢀquinic acid 2 gives a base peak at m/z 111 (C6H7O2) in MS3. In another case, (ꢀ)ꢀquinic
acid 1 and mucoꢀquinic acid 3 show similar fragmentation patterns giving base peaks at m/z
127 (C6H7O3) and m/z 109 (C6H5O2) in MS2 and MS3 respectively but, they differ in the
retention times with compound 3 eluting 0.5 min earlier than compound 1. In another case
distinction between diastereomers 1 and 3 on the basis of their tandem MS, fragmentation had
to be carried out in MS4.
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As shown in scheme A in Figure 11, (ꢀ)ꢀquinic acid (1) with a pseudomolecular ion at m/z
191.1 (C7H11O6) as precursor ion undergoes concomitant dehydration and deformylation to
produce a base peak at m/z 127 (C6H7O3) corresponding to fragment Q1 in MS2. Loss of 46
Da and 18 Da takes place simultaneously as no secondary peak at m/z 145, corresponding to
deformylated quinic acid can be detected. Interestingly the carboxyl moiety on Cꢀ1 is
formally fragmented as formic acid reminiscent of an ester elimination or Tchugaev
elimination in mechanistic organic chemistry. Presumably dehydration takes place in between
Cꢀ2 and Cꢀ3 due to the fact that the proton on Cꢀ2 and hydroxyl group on Cꢀ3 share an antiꢀ
periplanar arrangement. The precursor ion at m/z 127 further fragments in MS3 to give base
peak at m/z 109, which could suggest the presence of dihydroxybenzeneꢀtype structure.
However, since Cꢀ4ꢀOH and Cꢀ5ꢀOH do not possess the antiꢀperiplanar arrangement between
either protons and hydroxyl groups required for stereoelectronic reasons for an E2 type
elimination a different pathway is taken. Comparison of MS2 spectra of all three
dihydroxybenzenes (1,3ꢀ, 1,4ꢀ and 1,2ꢀC6H6O2) shows that none of them is identical to the
MS4 spectrum of 1 with m/z 127 as precursor ion (see Figure S5 in supplementary
information). The fragmentation of an epoxide such as cyclohexene oxide in MS2 gives only a
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peak at m/z 81 with a neutral loss of 16 Da reminiscent of the MS4 of (ꢀ)ꢀquinic acid 1. Hence
an epoxide fragment ion Q3 is proposed as product of fragmentation of Q1.
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Scheme B in Figure 11 shows the proposed fragmentation pathway for epiꢀquinic acid 2. The
fragmentation can be explained if elimination of water through an E2 route takes place
between Cꢀ1 and Cꢀ2 to give epiꢀshikimic acid with a base peak at m/z 173 (C7H9O5) (Q3).
Indeed the MS3 with m/z 173 as precursor ion is almost identical to the MS2 spectrum of
shikimic acid.29 A direct infusion experiment shows that m/z 111 (C6H7O2) is a primary peak
in the MS3 of epiꢀquinic acid (2) (Figure 11). It can be explained through the assumption of
the presence of Q4 arising through the dehydration in between Cꢀ4 and Cꢀ5, which possess
the required antiꢀperiplanar arrangement of a proton and –OH group as can be seen in the
Figure 11.
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The suggested fragmentation of mucoꢀquinic acid 3 is shown in scheme C. The carboxyl
group on Cꢀ1 is cleaved as a formic acid and the dehydration takes place in between Cꢀ2 and
Cꢀ3 due to antiꢀperiplanar arrangement similar to the fragmentation in 1 to give base peak in
MS3 at m/z 127 (Q5). As shown in the scheme, when the cyclohexane ring is inverted, an
antiꢀperiplanar geometry between Cꢀ2ꢀH and Cꢀ3ꢀOH arises suitable for dehydration between
Cꢀ2 and Cꢀ3. As can be observed in the direct infusion experiment in Figure 13, m/z 109
fragments further to give m/z 81.2 exactly similar to the MS4 of (ꢀ)ꢀquinic acid (1). This
means that the fragments Q2 and Q6 are fragment ions of identical structure. Diastereomers 1
and 3 can be distinguished in MS2 of 3, as a secondary peak at m/z 145 was observed, which
signifies the decarboxylated molecule (Figure 10). In the structural arrangement of the
diastereomer 3, absence of the hydrogen bonding between 1,3ꢀdiaxially positioned oxygen
anion on C1 and hydrogen on Cꢀ3ꢀOH does not facilitate the elimination of Cꢀ3ꢀOH as
observed in case of (ꢀ)ꢀquinic acid 1.
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MS2 of the cisꢀquinic acid 4 gives base peak at m/z 93 along with the secondary peaks at m/z
173 and m/z 111. Base peak at m/z 93 suggests that decarboxylation and three dehydration
processes had taken place simultaneously to obtain the stable aromatic phenolate fragment ion
Q9. Unfortunately, phenol as an external standard and m/z 93 in compound 4 do not fragment
further as observed in direct infusion experiments, even at high collision energies. However
mechanistic arguments show that the phenolate structure of Q9 is reasonable if not expected,
since in intermediate Q7 all CꢀOH moieties show an antiꢀperiplanar arrangement with Q8 at
m/z 111 as a secondary peak and reaction intermediate in MS2.
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scylloꢀQuinic acid 5 shows unique fragmentation pattern if compared to the other
diastereomers in present work. In MS2 it shows the pattern similar to the MS2 of the epiꢀ
quinic acid 2 and in MS3, m/z 173 directly fragments into m/z 93 similar to the MS2 of the cisꢀ
quinic acid 4 as can be seen in Figure 10. It is unclear at this point of time how the
fragmentation scheme in case of 5 can be explained mechanistically.
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In conclusion, we have selectively synthesized all the diastereomers of the quinic acid except
for the neoꢀquinic acid 6 to prove that they can be chromatographically resolved and can be
identified by their characteristic fragmentation behavior in tandem MS spectra. This study
also illustrates the importance of the tandem mass spectrometry in the area of identification
and confirmation of the stereochemistry of comparatively small molecules like the
diastereomers of the quinic acid. It is also worth mentioning that it is the first time the crystal
structures of 3ꢀOꢀcinnamoylꢀ1,4ꢀscylloꢀquinide 9 , 3ꢀOꢀcinnamoylꢀ1,5ꢀquinide 10, cisꢀquinic
acid 4, methyl cisꢀquinate 17 and 3,4ꢀOꢀCyclohexylideneꢀ1,5ꢀquinide 13 have been reported
(Figure 4). The fragmentation mechanisms are explained on the basis of the conformational
differences between the diastereomers of the quinic acid. Presence of the diastereomers like 3,
5 and 2 in hydrolyzed roasted coffee sample proves the existence of selected diastereomers of
chlorogenic acids in roasted coffee. However, it still remains unclear if the epimerization
occurs after the esterification with cinnamoyl functionality or the cinnamoyl group esterifies
with epimerized quinic acid during the food processing.
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Supplementary informations are available containing crystallographic data, further synthetic
details and chromatograms and spectral data for all quinic acid diasteromers discussed.
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455
ACKNOWLEDGEMENTS
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The authors gratefully acknowledge the financial support from Kraft Food (now Mondelez)
and Jacobs University Bremen gGmbH. Furthermore, excellent technical support from Ms.
Anja Mueller and use of the Xꢀray facility of Prof. Dr. Kortz laboratory is acknowledged.
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chlorogenic acids containing aliphatic side chains in Burdock (Arcticum lappa L.)
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530
531
532
533
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roots by liquid chromatography tandem mass spectrometry. Food and Function 2011,
2, 63ꢀ71.
(24) Moreira, A. S. P.; Coimbra, M. A.; Nunes, F. M.; Simoes, J.; Domingues, M. R. M.,
Evaluation of the Effect of Roasting on the Structure of Coffee Galactomannans Using
Model Oligosaccharides. Journal of Agricultural and Food Chemistry 2011, 59,
10078ꢀ10087.
(25) Nunes, F. M.; Reis, A.; Domingues, M. R. M.; Coimbra, M. A., Characterization of
galactomannan derivatives in roasted coffee beverages. Journal of Agricultural and
Food Chemistry 2006, 54, 3428ꢀ3439.
(26) Golon, A.; Kuhnert, N., Unraveling the Chemical Composition of Caramel. Journal of
Agricultural and Food Chemistry 2012, 60, 3266ꢀ3274.
(27) Clifford, M. N.; Knight, S.; Kuhnert, N. Discriminating between the six isomers of
dicaffeoylquinic acid by LCꢀMSn. J. Agr. Food Chem. 2005, 53, 3821ꢀ3832.
(28) Jaiswal, R.; Matei, M. F.; Glembockyte, V.; Patras, M. A.; Kuhnert, N., Hierarchical
Key for the LCꢀMSn Identification of All Ten Regioꢀ and Stereoisomers of
Caffeoylglucose. Journal of Agricultural and Food Chemistry 2014, 62, 9252ꢀ9265.
(29) Jaiswal, R.; Sovdat, T.; Vivan, F.; Kuhnert, N., Profiling and Characterization by LCꢀ
MSn of the Chlorogenic Acids and Hydroxycinnamoylshikimate Esters in Mate (Ilex
paraguariensis). J. Agric. Food Chem. 2010, 58, 5471ꢀ5484.
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551
552
553
554
555
556
557
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558
559
560
561
562
563
564
TABLE LEGEND
Table 1. MS2 data of quinic acid diastereomers in negative ion mode at 75% collision energy
FIGURE LEGEND
Figure 1. Stereoisomers of quinic acid
Figure 2. Reaction scheme for obtaining scylloꢀquinic acid 5 and epiꢀquinic acid 2
Figure 3: Reaction scheme for the synthesis of cisꢀquinic acid 4
565
566
567
568
Figure 4: Single crystal Xꢀray structures of cisꢀquinic acid (4) , 3,4ꢀOꢀCyclohexylideneꢀ1,5ꢀ
quinide (13), 3ꢀOꢀcinnamoylꢀ1,4ꢀscylloꢀquinide (9), 3ꢀOꢀcinnamoylꢀ1,5ꢀquinide (10), methyl
cisꢀquinate (17), methyl 3,5ꢀDiꢀOꢀ(tertꢀbutyldimethylsilyl)quinate (7) Conformer A and of
methyl 3,5ꢀDiꢀOꢀ(tertꢀbutyldimethylsilyl)quinate (7) Conformer B.
569
570
571
572
573
Figure 5: MS2 and MS3 spectra of (ꢀ) quinic acid 1 in negative ion mode
Figure 6: MS2 and MS3 spectra of epiꢀ quinic acid 2 in negative ion mode
Figure 7: MS2 and MS3 spectra of mucoꢀquinic acid 3 in negative ion mode
Figure 8: MS2 spectrum of cisꢀ quinic acid 4 in negative ion mode
Figure 9: MS2 and MS3 spectra of scylloꢀ 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
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Tables
Table 1. Chromatographic and MS2 data of quinic acid diastereomers 1-5 in negative ion
mode at 75% collision energy
Compound
No.
MS2 (m/z 191.0 as precursor ion)
Secondary peak
Retention
Time
[min]
Base peak
m/z
m/z
int%
m/z
int%
m/z
int%
m/z
int%
1
2
3
4
5
3.9
3.6
3.4
3.4
4.2
127
173
127
93
173
127
173
173
127
82
86
64
37
91
85
145
85
56
30
45
34
50
93
85
51
24
20
7
111
111
145
61
23
17
19
5
109
155
111
111
85
173
48
93
46
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Journal of Agricultural and Food Chemistry
Figures
HO
COOH
OH
HO
OH
2
epi-quinic acid
C-1
HO
COOH
HO
HO
COOH
OH
HO
COOH
OH
C-3
1
C-5
3
5
HO
OH
HO
OH
OH
OH
cis-quinic acid
(-) quinic acid 1
muco-quinic acid
parent compound
4
3
C-4
HO
COOH
OH
HO
COOH
OH
HO
COOH
C-3
C-5
HO
HO
HO
OH
OH
OH
scyllo-quinic acid
OH
neo-quinic acid
(+) quinic-acid
5
6
OH
OH
OH
OH
OH
O
O
O
OH
OH
OH
OH
HO
OH
HO
HO
HO
OH
OH
epi-quinic acid 2
OH
OH
(-) quinic acid 1
muco-quinic acid 3
OH
O
OH
O
O
HO
OH
HO
HO
HO
OH
OH
OH
OH
OH
cis-quinic acid 4
scyllo-quinic acid 5
neo-quinic acid 6
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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
O
O
HO
HO
HO
OH
OH
O
O
O
a, b
c
Si
Si
Si
Si
HO
O
O
O
OH
O
OH
S
1
7
8
O
O
e
O
HO
O
O
HO
HO
O
O
O
f
d
11
Si
Si
Si
O
O
Si
O
O
OH
O
O
O
HO
HO
g
12
O
O
O
+
HO
HO
g
O
OH
OH
OH
OH
O
5
1
+
OH
+
O
O
HO
OH
HO
OH
OH
2
1
9
10
Reagents and conditions: (a) MeOH, reflux, 12 h, Amberlite IR 120, 100%; (b) 2.6 equiv
0
TBDMSCl, 2.6 equiv Et3N, DMF 0 C, 2 h and then to RT, 16 h 75%; (c) 1.5 equiv MsCl,
0
Py, RT, overnight, 95%; (d) 11, CsF, DMF, 90 C, 24 h 2% 9 and 10; (e) 2 equiv Dessꢀ
0
Martin periodinane, CH2Cl2 , overnight, RT, 87%; (f) 1.5 equiv NaBH4, Ethanol, ꢀ30 C, 40
min.; (g) 2M HCl, H2O, 1 h.
Figure 2. Reaction scheme for obtaining scylloꢀquinic acid (5) and epiꢀquinic acid (2)
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O
O
O
O
HO
HO
HO
HO
OH
OH
O
O
O
a
c
b
O
O
O
OH
O
HO
O
O
O
OH
1
13
14
15
d
O
O
HO
HO
O
OH
OH
f
O
OH
HO
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) DessꢀMartin 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 cisꢀquinic acid 4
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4
10
17
7 conformer A
9
7 conformer B
Figure 4: Single crystal Xꢀray structures of cisꢀquinic acid (4) , 3,4ꢀOꢀCyclohexylideneꢀ1,5ꢀ
quinide (13), 3ꢀOꢀcinnamoylꢀ1,4ꢀscylloꢀquinide (9), 3ꢀOꢀcinnamoylꢀ1,5ꢀquinide (10), methyl
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Journal of Agricultural and Food Chemistry
cisꢀquinate (17), methyl 3,5ꢀDiꢀOꢀ(tertꢀbutyldimethylsilyl)quinate (7) Conformer A and of
methyl 3,5ꢀDiꢀOꢀ(tertꢀbutyldimethylsilyl)quinate (7) Conformer B.
-MS2(190.7), 3.9min
[%]
(1)
126.8
100
85.2
93.0
172.7
188.6
110.9
108.9
61.5
154.9
0
-MS3(190.8>126.9), 4.0min
100
85.5
80
0
40
60
100 120 140 160 180 200 m/z
Figure 5: MS2 and MS3 spectra of (ꢀ) quinic acid 1 in negative ion mode
[%]
100
-MS2(190.7), 3.6min
126.8
144.8
(2)
172.7
85.1
108.8
59.5
0
-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 epiꢀ quinic acid 2 in negative ion mode
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-MS2(190.7), 3.4min
126.8
[%]
100
(3)
172.7
144.8
154.7
-MS3(190.8->126.8), 3.5min
85.1
108.9
108.8
0
100
85.1
80
0
40
60
100 120 140 160 180 200 m/z
Figure 7: MS2 and MS3 spectra of mucoꢀquinic acid 3 in negative ion mode
[%]
100
-MS2(190.7), 3.4min
93.1
(4)
80
60
40
20
0
172.7
110.9
136.8
59.1
60
154.7
86.1
80
40
100 120 140 160 180 200 m/z
Figure 8: MS2 spectrum of cisꢀ quinic acid 4 in negative ion mode
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Journal of Agricultural and Food Chemistry
-MS2(190.7), 3.7min
[%]
100
(5)
172.6
126.8
85.1
110.9
188.7
154.7
71.3
0
-MS3(190.8->173.3), 3.8min
93.0
100
127.8
152.7
110.9
0
50
100
150
200
250
300 m/z
Figure 9: MS2 and MS3 spectra of scylloꢀ quinic acid 5 in negative ion mode
Intens.
EIC 191.0
6
x10
Hydrolysed roasted coffee
1.0
0.8
0.6
0.4
0.2
0.0
1
3 5 2
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)
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O
OH
OH
OH
OH
O
O
H
O
H
A
-HCO2H
-H2O
-H2O
O
OH
OH
OH
OH
H
O
O
O
O
O
OH
H
m/z 127
m/z 109
Q1
Q2
OH
OH
OH
OH
OH
5 OH
3
O
O
H
OH
4
-H2O
-H2O
HO
1
-HCO2
6
5
OH
4
O
O
B
C
3
2
6
OH
1
2
OH OH
OH
OH
OH
H
m/z 111
m/z 173
Q4
Q3
O
O
OH
OH
OH
OH
H
O
OH
H
O
OH
-H2O
-HCO2H
-H2O
OH
OH
OH
OH
2
H
HO
OH
3
H
O
O
O
O
OH
H
OH
m/z 109
m/z 127
Q5
Q6
O
H
H
O
O
H
O
OH
O
OH
D
OH
O
OH
OH
OH
H
OH
OH
O
OH
OH
H
m/z 173
m/z 93
m/z 111
Q7
Q9
Q8
Figure 11: Proposed fragmentation scheme of quinic acids diastereomers in negative ion
mode
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TOC Graphic
OH
OH
OH
OH
O
O
OH
HO
HO
OH
OH
OH
OH
O
O
OH
HO
HO
HO
HO
OH
OH
OH
OH
OH
OH
OH
O
OH
O
OH
OH
OH
HO
HO
OH
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