Investigation of Acyl Migration in Mono- and Dicaffeoylquinic Acids

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Investigation of acyl migration in mono- and di-caffeoylquinic acids under aqueous basic, aqueous acidic and dry roasting conditions Sagar Deshpande, Rakesh Jaiswal, Marius Febi Matei, and Nikolai Kuhnert J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5017384 • Publication Date (Web): 12 Aug 2014 Downloaded from http://pubs.acs.org on August 21, 2014

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Journal of Agricultural and Food Chemistry

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Investigation of acyl migration in mono- and di-caffeoylquinic acids under aqueous

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basic, aqueous acidic and dry roasting conditions

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Sagar Deshpande, Rakesh Jaiswal, Marius Febi Matei and Nikolai Kuhnert*

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School of Engineering and Science, Chemistry, Jacobs University Bremen, 28759 Bremen,

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Germany

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*Author to whom correspondence should be addressed Tel: 49 421 200 3120; Fax: 49 421

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200 3229; E-mail: [email protected]

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Abstract

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Acyl migration in chlorogenic acids describes the process of migration of cinnamoyl moieties

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from one quinic acid alcohol group to another thus interconverting chlorogenic acid

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regioisomers. It therefore constitutes a special case of a transesterification reaction. Acyl

25

migration constitutes an important reaction pathway in both coffee roasting and brewing

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altering the structure of chlorogenic acid initially present in the green coffee bean. In this

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contribution we describe detailed and comprehensive mechanistic studies comparing inter-

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and intra-molecular acyl migration involving the seven most common chlorogenic acids in

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coffee. We employ aqueous acidic and basic conditions mimicking the brewing of coffee

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along with dry roasting conditions. We show that under aqueous basic conditions

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intramolecular acyl migration is fully reversible with basic hydrolysis competing with acyl

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migration. 3-caffeoylquinic acid was shown to be most labile to basic hydrolysis. We

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additionally show that the acyl migration process is strongly pH dependant with increased

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transesterification taking place at basic pH. Under dry roasting conditions acyl migration

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competes with dehydration to form lactones. We argue that acyl migration precedes

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lactonisation with 3-caffeoylquinic acid lactone being the predominant product.

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Introduction

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Coffee is one of the most valued agricultural commodities in terms of the economic aspects of

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the exports from the developing coffee producing countries, accounting to ca. 8 million metric

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tonnes per year. Approximately, 2.3 billion cups of coffee are consumed worldwide per day.1,

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2

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coffee) are the two types of coffee holding 70% and 30%, respectively of the total coffee

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market in the world.3 Chlorogenic acids are present in the range of 6-12% of the dry weight of

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the green coffee bean and constitute the most abundant class of secondary metabolites.4

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Chlorogenic acids (CGAs) are a large group of esters formed between one or more cinnamic

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acid derivatives and D-(-)-quinic acid. CGAs are classified on the basis of the number of the

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cinnamoyl residues esterified with the quinic acid as well as the functional groups present on

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the aromatic moiety of the cinnamoyl residues. Out of the total content of the CGAs in green

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coffee, 5-O-caffeoylquinic acid (3) comprises about 50%. Other subclasses like

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caffeoylquinic acids, dicaffeoylquinic acids, feruloylquinic acids and p-coumaroylquinic acids

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contribute to a large extent to the other 50% of the total CGAs present in coffee. In total 45

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CGAs have been reported in Arabica and 85 in Robusta green coffee beans.1, 2 CGAs are very

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important plant secondary metabolites due to their pharmacological properties, such as

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antioxidant property5, anti-hepatitis B virus activity6, antispasmodic activity4, anti-diabetic

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activity7, inhibition of the HIV-1 integrase8,

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carcinogenic compounds.4

Coffea arabica (known as Arabica coffee) and Coffea canephora (known as Robusta

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and inhibition of the mutagenicity of

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The various roasting conditions affect drastically the concentration and composition of CGAs

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in coffee. The chemistry at elevated temperatures of CGAs has been described in detail with

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epimerisation, dehydration and transesterifications (acyl migration a special case) dominating

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the mechanistic spectrum.2 The evidence for transesterification phenomena was demonstrated

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indirectly by the presence of a series of 1-substitited CGA derivatives in roasted coffee, which

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are absent in the green bean. For every 1% of the dry matter of the total CGA content in the

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green coffee beans, 8-10% of the original CGAs are transformed or decomposed into

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respective cinnamic acid derivatives and quinic acid.1,2 The lightest drinkable roast (the so-

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called ‘Cinnamon’ roast) involves roasting of green coffee beans at around 180 °C until the

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coffee beans just encounter the ‘first crack’. It was reported by Clifford et al. 1 that during the

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early stage of the roasting process, transformations such as isomerization (acyl migration) or

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hydrolysis of the ester bond, take place in the CGAs. Later, chemical transformations like

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decarboxylation of cinnamoyl moieties to produce a number of phenylindans, epimerization at

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the quinic acid and dehydration to produce cyclohexenes and lactones take place.2, 3

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Clifford et al.

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and transesterification in 5-O-caffeoylquinic acid (3), 3-O-caffeoylquinic acid (2), 4-O-

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caffeoylquinic acid (4), 3,4-di-O-caffeoylqunic acid (8), 3,5-di-O-caffeoylqunic acid (9), 4,5-

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di-O-caffeoylqunic acid (10), 5-O-p-coumaroylquinic acid (13) and 5-O-feruloylqunic acid

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(23) in which, the identification of some of the transformed products was based on the

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putative conclusions acquired by analytical HPLC. Dawidowicz et al.12

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transformation products of 5-O-caffeoylquinic acid (3) after five hours of reflux in an acid-

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water solution including two water addition products. This study only incorporated 5-CQA

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and the brewing time was not in line with common consumer practice. No comprehensive

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mechanistic study has been previously reported which investigated the intra- versus inter-

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molecular acyl migration under different conditions incorporating all major commercially

10, 11

also reported the basic hydrolysis induced intramolecular isomerization


found nine

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available regio-isomers of mono- and di-caffeoyl chlorogenic acids. Most importantly data

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on acyl migration under roasting conditions are absent from the literature.

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The complexity in the data interpretation for the structural analyses of the CGAs present in

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the roasted coffee melanoidins arises from the regio- and stereoisomeric compounds in the

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natural sources. For this reason, model roasting experiments on the commercially available

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mono- and dicaffeoylquinic acids were attempted in the present work in order to study the

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transformations taking place in CGAs during the early roasting stages. Also, isomerization

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(acyl migration) was induced by both basic hydrolysis and simple hydrolysis (brewing) in

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these reference standards to observe the isomeric transformations on the basis of relative

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quantitation. Recently, tandem mass spectrometry has allowed accurate structural assignment

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and identification of the CGA regioisomers. The advantage of multi-dimensional specificity

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of LC-MSn enabled isomeric resolution and relative quantitation of the early roasting

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transformations in the CGAs.13

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Materials and Methods

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Chemicals and materials

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All the chemicals (analytical grade) were purchased from Sigma-Aldrich (Bremen, Germany).

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Commercially available mono- and di-caffeoylquinic acids such as 5-O-caffeoylquinic acid

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(3), 3-O-caffeoylquinic acid (neo-chlorogenic acid) (2), 4-O-caffeoylquinic acid (crypto-

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chlorogenic acid) (4), 1,3-di-O-caffeoylquinic acid (cynarin) (5), 3,4-di-O-caffeoylquinic acid

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(8), 3,5-di-O-caffeoylquinic acid (9), 4,5-di-O-caffeoylquinic acid (10) were purchased from

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PhytoLab GmbH & Co. KG (Vestenbergsgreuth, Germany).

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Hydrolysis by tetramethylammonium hydroxide (TMAH)



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All the seven CGAs reference standards were treated with aqueous TMAH (25 g/L). Each

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sample was diluted by 5 mL of aqueous TMAH and stirred at room temperature. pH of the

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mixture was observed to be around 12. 1 mL solution from each sample was taken out at 2, 5,

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10, 30 and 60 min time intervals. Each sample was saturated with brine and extracted twice

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with ethyl acetate. Combined organic layers were concentrated in vacuo and each sample was

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prepared in 1 mL of methanol for analysis by LC-MSn for intramolecular acyl migration.

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To study the intermolecular acyl migration (cross-over experiment), 5-CQA (25 mg, 0.07062

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mmol) was added to a round bottom flask containing ferulic acid (13.7 mg, 0.07062 mmol). 5

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mL of 10 times diluted (25 g/litre) TMAH was added to the flask and the mixture was stirred

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at room temperature. 1 mL samples were taken out from the flask at 2, 5, 10, 15 and 30 min

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time intervals. Each sample was saturated with brine and extracted twice with ethyl acetate.

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The combined organic layers were concentrated in vacuo and each sample was prepared in

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methanol to be analysed by LC-MSn. The same procedure was repeated with p-coumaric acid

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(11.57 mg, 0.07062 mmol) and 5-CQA (25 mg, 0.07062 mmol).

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Model roasting

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All the seven CGAs reference standards were heated at 180 °C for 12 min separately to study

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the intramolecular acyl migration. Equimolar quantities of 5-CQA with p-coumaric acid and

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5-CQA with ferulic acid were heated together at 180 °C for 12 min to study the intermolecular

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acyl migration (cross-over experiment). All the samples were heated in a Buechi Glass Oven

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B-585 and prepared in 1 mL methanol for LC-MSn analysis.

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Brewing of CGAs (2-5 and 8-10)

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Commercially available chlorogenic acids standards (each sample 500 µg) were infused in 3

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mL of hot water each and stirred for 5 h under reflux. pH of each sample was determined to


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be 5 with pH meter. The solvent was removed under low pressure and the samples were

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dissolved in 1mL MeOH and used for LC-MSn.

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LC-MSn

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The 1100 series LC equipment (Agilent, Bremen, 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 ion-trap mass

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spectrometer fitted with an HCT Ultra ESI source (Bruker Daltonics, Bremen, Germany)

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operating in full scan, auto MSn mode to obtain fragment ion m/z. Tandem mass spectra were

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acquired in Auto-MSn mode (smart fragmentation) using a ramping of the collision energy.

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Maximum fragmentation amplitude was set to 1 V, starting at 30% and ending at 200%. The

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MS operating conditions (negative mode) had been optimized using 5-caffeoylquinic acid

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

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pressure of 10 psi.

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HPLC

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Separation was achieved on a 150 x 3 mm i.d., 5 µm diphenyl column, with a 5 mm x 3 mm

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i.d. guard column (Varian, Darmstadt, Germany). Alternatively, separation was also achieved

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on a 250 mm x 3 mm i.d., 5 µm C18-amide column, with a 5 mm x 3 mm i.d. guard column

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of the same material (Varian, Darmstadt, Germany) for the cases of hydrolysis (brewing) of

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reference standards experiments. Solvent A was water/formic acid (1000:0.05, v/v) and

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solvent B was methanol. Solvents were delivered at a total flow rate of 500 µL/min. The

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gradient profile was from 10 % B to 70 % B linearly in 60 min followed by 10 min isocratic,

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and a return to 10 % B at 90 min and 10 min isocratic to re-equilibrate.

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Preliminary assessment of data



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All the data for the chlorogenic acids use the recommended IUPAC numbering system;14 the

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same numbering system was adopted for chlorogenic acids, their cis-isomers, their acyl-

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migration isomers and water addition products (Figure 1). The relative concentrations of the

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transformed products are expressed here in terms of the peak areas obtained in their UV

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chromatograms assuming the relative response factor in UV close to one, based on identical

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absorptivity of all mono CGA.13,

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accordingly.

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Results and Discussion

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Intramolecular acyl migration: hydrolysis by TMAH of 2-5 and 8-10

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The most common CGAs in the green coffee bean 2-5 and 8-10 were selected and subjected

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to aqueous TMAH, with samples taken at intervals from 2-60 minutes. Samples were directly

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analyzed by LC-MS with compound identification and assignment achieved through

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comparison of retention times and fragment spectra.2,

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was carried out using HPLC-UV traces monitoring cinnamoyl absorption at 320 nm. The

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concentration of the samples was chosen to be sufficiently low (1-1.5 mg/ mL) to prevent

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intermolecular acyl migration or transesterification, yet corresponding to typical coffee brew

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CGA levels (around 200 mg/cup = 1 mg/mL) therefore allowing observation of intra

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molecular acyl migration exclusively. In the hydrolysis of 3-CQA (2), 5-CQA (3) and 4-CQA

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(4), all the other mono-acyl derivatives were identified during the hydrolysis except for 1-

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CQA (1). Also, we did not observe the formation of any di-acyl derivatives in the hydrolysis

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of the mono-acyl quinic acids. This observation confirms that the acyl migration we observed

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in this study was in fact an intramolecular process. Figure 2 represents the UV

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chromatograms of the 5-CQA in basic solution at different time intervals. It should be noted

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that the UV response in mono- and di-acylquinic acids has been used in the past as a reliable

15

In tables and figures, the peak area values are stated

18-23

Relative or absolute quantitation


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relative response factor.1 Hanson et al.16 used radiolabelled quinic acid to investigate the acyl

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migration pathway in cinnamoylqunic acids; in accordance with these findings, we assumed

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that the mechanism of the acyl migration follows the 1,2-ortho ester intermediate formation.

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This assumption is also supported by the study of Xie et al.17 The transformations of 5-CQA

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(3), 4-CQA (4) and 3-CQA (2) with time are presented in Figure 3. From the results, we can

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conclude that 5-CQA is much more stable than 4-CQA and 3-CQA and the order of the

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stability is 5>4>3 in terms of the hydrolysis of the caffeoyl ester. This stability was observed

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to provide the resistance to decomposition thus allowing 5-CQA to form the acyl migrated

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products over longer hydrolysis durations. On the other hand, with 3-CQA being the least

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stable of the three mono-acylquinic acids, it decomposes to form caffeic acid and quinic acid

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even before acyl migration takes place.

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Considering the mechanism of the acyl migration proceeding through an 1,2-ortho-ester

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intermediate formation as 5-CQA ⇋ 4-CQA ⇋ 3-CQA, the reverse equilibrium, through

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which the migrated acyl group reverts back to its original position appears to be slower. It is

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difficult to comment on the thermodynamic equilibrium of the acyl migration because of the

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ongoing simultaneous ester hydrolysis competing with the acyl migration process.

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The equilibrium between 3-CQA (2) and 4-CQA (4) is readily achieved because the ortho-

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ester intermediate is more stable due to the cis geometry (Figure 4). Compound 2 shows a

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tendency to hydrolyse to generate caffeic acid and quinic acid rather than to undergo acyl

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migration presumably due to the 1,3-syn-diaxial arrangement between the C1 hydroxyl group

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and the C3 ester. The hydrogen bonding between C1-OH and the carbonyl oxygen on the ester

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on C3 results in steric hindrance preventing the nucleophilic attack on the carbonyl carbon by

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the C4 hydroxyl group and facilitates the hydrolysis of the ester bond thus dissociating the

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caffeoyl moiety in basic conditions. At the same time this hydrogen bonding presumably

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activates the ester at C3 for hydrolytic cleavage. 9 ACS Paragon Plus Environment


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Similar to monocaffeoyl derivatives, dicaffeoyl quinic acids were subjected to aqueous

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TMAH treatment and analysed in the same manner. Previous studies have shown that 1,5-

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diCQA (7) was converted into 1,3-diCQA (5) and 5-CQA (3) rapidly and extensively by

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TMAH treatment within one minute of hydrolysis.18, 19 In the present study however, basic

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hydrolysis of 1,3-diCQA (5) did not show any presence of 1,5-diCQA (7) or 1,4-diCQA (6).

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1,3-diCQA (5) decomposed largely into 1-CQA (1) rather than 3-CQA (2) in the ratio 2.2:1

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after two min of base treatment. 1,3-diCQA (5) did not transform preferably into 3,5-diCQA

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(9) whereas 3,4-diCQA (8) and 4,5-diCQA (10) were formed in very small quantities from (5)

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(Figure 5). 1-CQA (1) was found to be present entirely as a decomposition product of 1,5-

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diCQA (7) or 1,4-diCQA or (6) 1,3-diCQA (5) rather than a product of acyl migration as it

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was not observed as an acyl migration product of any other mono- or di-acylated substrates;

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this fact also supports the ortho-ester propagation of acyl migration process. Moreover, this

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observation confirms the hydrolytic lability of the esters in the 3-acylated position.

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In the case of hydrolysis of 4,5-diCQA (10), we observed that 4,5-diCQA transformed mainly

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into 3,4-diCQA (8) after two min of base treatment. Additionally CQAs, in particular 5-CQA

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(3), 4-CQA (4) and 3-CQA (2) were observed after two min reaction time. This trend

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continued for five minutes during the base treatment, after which all of the CGA derivatives

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were completely decomposed. 3,4-diCQA (8) was observed to be the least stable isomer

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during the base treatment study. At two minutes, 8 was found to be in equilibrium with 3,5-

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diCQA (9) however after five minutes of treatment, 3,5-diCQA (9) was observed to display a

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slightly larger peak area than 3,4-diCQA (8). 3-CQA (2) was not observed in any sample

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throughout the duration of the base treatment of 3,4-diCQA due to its tendency to decompose

231

rapidly by hydrolysis.

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Observations based on the results from the base treatment on 3,5-diCQA (9) were quite

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distinctive. After five minutes 3,5-diCQA (9) was observed to possess surprising stability to 10 ACS Paragon Plus Environment

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the hydrolysis of the ester as we did not detect any of the mono-CQA derivatives even after

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60 min of base treatment (Figure 5). 3,5-diCQA (9) transformed mainly into 3,4-diCQA (8)

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followed by 4,5-diCQA (10). The ratio of 9:8:10 remained approximately constant throughout

237

the 60 min of base treatment.

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Intermolecular acyl migration (Transesterification): hydrolysis by TMAH (cross-over

239

experiment)

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In these experiments, we investigated intermolecular acyl migration by carrying out cross-

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over experiments, in which 5-CQA was reacted with the free acids like ferulic and p-coumaric

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acids at 1:1 stoichiometry in presence of a base. The change of substituents hereby allows

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easy identification of products by LC-MS based on the m/z value of the pseudomolecular ions

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of the products. The intermolecular acyl migration was found to be simultaneously competing

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with intramolecular acyl migration as well as hydrolysis of the CQA. The products identified

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in the reaction of 5-CQA with ferulic acid and p-coumaric acid at 2, 5, 10, 15 and 30 min of

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cross-over experiment are summarized in Table 1. Along with the transesterification products

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of 5-CQA and respective free acid, formation of the cis-cinnamoyl isomers was also observed

249

in ferulic, caffeic and p-coumaric acids. All products were identified according to the

250

fragmentation schemes reported previously.18-21

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In the case of the base treatment of an equimolar mixture of p-coumaric acid and 5-CQA (3),

252

the intramolecular acyl migration within 5-CQA seemed to be dominating the hydrolysis and

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the intermolecular acyl migration. According to the peak areas observed, the formation of the

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intermolecular acyl migrated species (transesters) was found to be least favoured (Figure 6).

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For example, 3-CQA (2), 5-CQA (3) and 4-CQA (4) formed predominantly over caffeic acid

256

(32) during two minutes of TMAH treatment of 5-CQA. p-Coumaric acid (33) did not esterify

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with the quinic acid generated from the hydrolysis of the 5-CQA (3) after two minutes hence,

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no p-coumaroylquinic acids were observed. However, after 5 minutes of base treatment the 11 ACS Paragon Plus Environment


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first transesterification product appeared in the form of 4-pCoQA (14) and after ten minutes

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both 5-pCoQA (13) and 4-pCoQA (14) were observed. Unfortunately, due to very low

261

concentration we could not compare the peak areas of compounds 13 and 14 in the UV

262

chromatogram and hence cannot comment on the kinetics of the acyl migration. Although it

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was clear that 4-pCoQA (14) was formed earlier than 5-pCoQA (13), it was not obvious

264

whether 13 was an acyl migration product of 14. Formation of intramolecular acyl migration

265

products takes place according to the conclusions established earlier in this study. After two

266

minutes the rate of hydrolysis of 5-CQA was very low and the amounts of 3-CQA (2), 5-CQA

267

(3) and 4-CQA (4) were the highest. Between five to ten minutes of basic hydrolysis,

268

equilibrium was reached where 5-CQA was found to be the predominant isomer. Formation

269

of the cis-cinnamoyl derivatives supposedly follow a water addition-elimination pathway to

270

the double bond in the cinnamoyl moiety during the basic hydrolysis as described earlier.25

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When an equimolar mixture of ferulic acid (31) and 5-CQA (3) was treated with base we

272

observed three regio-isomers of feruloylquinic acid resulting from intermolecular

273

transesterification: 3-FQA (22), 5-FQA (23) and 4-FQA (24).22 The peak area of the

274

compounds 22-24 is very low compared to the intramolecular acyl migration products formed

275

in this experiment hence, they do not appear in the plot shown in Figure 6. The same was not

276

observed in the case of the p-coumaric acid experiment. After two minutes, 3-FQA (peak area

277

= 3215) was formed predominantly over 5-FQA and 4-FQA. 5-FQA (23) and 4-FQA (24)

278

showed negligible peak areas. Only 5-FQA remained stable enough to be detected after 10

279

and 15 min of basic hydrolysis. After 30 min all the transesters and the substrate 5-CQA was

280

found to be decomposed completely as we could identify caffeic acid only. Formation of 3-

281

FQA was found to be kinetically favoured. This was found to be consistent with the fact that

282

during the first few minutes of the base treatment of 5-CQA to study intramolecular acyl

283

migration, 3-CQA dominates the acyl migration product spectrum. We also identified cis-112 ACS Paragon Plus Environment

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O-caffeoylquinic acid (47) in the EIC (extracted ion chromatogram) of m/z 353 and the UV

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chromatogram on the basis of its early elution and fragmentation (Figure 7).23 Similar to 1-

286

cis-CQA (47), 4-cis-CQA (48) was also identified as another isomerised caffeoylquinic acid

287

derivative, both of which are assumed to be formed by an addition elimination of the water

288

molecule across the double bond in caffeic acid. 4-cis-CQA (48) was observed to be in

289

equilibrium with 4- trans-CQA (4) after two minutes but with an increase in the reaction time

290

only 4-CQA was observed to survive the base treatment. Unexpectedly, we identified two

291

chlorogenic acid lactones as minor by products, namely 3-CQL (37) and 4-CQL (38) in this

292

sample. Under basic conditions the dehydrated product of caffeoylquinic acid must be in

293

continuous equilibrium with caffeoylquinic acid. A single hetero-diacyl chlorogenic acid was

294

observed in the chromatogram in the form of caffeoyl-feruloylquinic acid (49) after 5 to 15

295

minutes of basic hydrolysis (Figure 6). Figure 7 shows the fragmentation pathway for

296

compound (49), in which it loses the ferulic acid moiety and undergoes simultaneous

297

dehydration to give m/z 335 as a base peak in MS2. Furthermore, in MS3 the dehydrated

298

caffeoylquinic acid entity undergoes decarboxylation to give a base peak at m/z 291 and also

299

shows the presence of ferulic acid as a secondary peak at m/z 193. This fragmentation

300

pathway for a caffeoyl-feruloylquinic acid was found to be inconsistent with the

301

fragmentation pathways of 1-caffeoyl-3-feruloylquinic acid, 3-feruloyl-5-caffeoylquinic acid,

302

cis-4-feruloyl-5-caffeoylquinic

303

feruloylquinic acid and cis-3-feruloyl-5-caffeoylquinic acid previously reported by Jaiswal et

304

al..21 Hence, the regio-chemistry of the acyl groups in caffeoyl-feruloylquinic acid (49)

305

remains unknown.

306

Intramolecular acyl migration: model roasting of 2-5 and 8-10

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Compounds 3-CQA (2), 5-CQA (3), 4-CQA (4), 1,3-diCQA (5), 3,4-diCQA (8), 3,5-diCQA

308

(9) and 4,5-diCQA (10) were heated at 180 °C for 12 min separately to study the

acid,

4-feruloyl-5-caffeoylquinic


acid,

4-caffeoyl-5-


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intramolecular acyl migration in the absence of solvents in conditions closely mimicking

310

coffee roasting. From the data summarized in Table 2, we clearly see that only 4-CQA (4),

311

3,4-diCQA (8) and 3,5-diCQA (9) undergo transformations to generate various dehydrated

312

products mainly in the form of caffeoyl quinic acid lactones. In the case of mono-acylated

313

chlorogenic acid reference standards, only 4-CQA undergoes acyl migration with

314

simultaneous dehydration. 3-CQL (37) was found to be the predominant transformation

315

product in the heat treatment of 4-CQA. 5-caffeoyshikimic acid was also identified but was

316

found to be the least favoured dehydration product after 4-CQL. In this experiment, we

317

observed that 3-CQL (37) was forming predominantly over 4-CQL (38) irrespective of the

318

substrate, possibly because of the additional stability gained by the equatorial position that 3-

319

CQL assumes in the inverted chair conformation required for stereoelectronic reasons for

320

lactonization. Therefore we suggest that the dehydration processes such as lactone and

321

shikimic acid formation at the quinic acid moiety follows acyl migration. i.e. in the simulated

322

roasting environment, acyl migration takes place before dehydration at the quinic acid moiety.

323

Additionally, lactonization dominates over the alternative shikimate formation in the model

324

roasting of all the substrates (Table 2). 1,3-diCQA (5) did not undergo noticeable

325

transformation by the heat treatment whereas 3,4-diCQA (8) generated most of the

326

transformation products among all four di-CQAs. It is likely that the observed products 3-

327

CQL (37) and 4-CQL (38) resulting from 3,4-diCQA (8) were formed by lactonization and

328

loss of one of the two acyl moieties following the order of the processes as dissociation first

329

and dehydration at the quinic acid moiety later; attributed to the fact that both of the caffeoyl

330

lactones possess similar peak areas. 3,4-di-O-caffeoyl-1,5-quinide (46)

331

formed in the heat treatment of 3,4-diCQA. The presence of the two different peaks eluting at

332

51.2 and 52.4 min having the same fragmentation pattern as 3,4-diCQL (46) suggested the

333

presence of a cis isomer of compound 46 (Figure 8). Both isomers of 3,4-diCQL (46)


was preferably

Page 15 of 43


334

generate a base peak at m/z 335 with virtually non-existent secondary peaks confirming the

335

regio-chemistry of 3,4-diCQL.

336

Among the unchanged substrates throughout the heat treatment at 180 °C for 12 min such as,

337

5-CQA, 3-CQA, 1,3-diCQA and 4,5-diCQA the stability of 5-CQA (3) and 1,3-diCQA (5) to

338

high temperatures was confirmed by heating them at 200 °C for 10 min.

339

Transesterification (Intermolecular acyl migration): model roasting (cross-over

340

experiment)

341

In these sets of experiments, we explored the possibilities of transesterification or

342

intermolecular acyl migration by carrying out cross-over experiments, in which 5-CQA was

343

subjected to heat treatment in the presence of free acids like ferulic acid and p-coumaric acid

344

at 1:1 stoichiometry. In the first case an equimolar mixture of pCoA and 5-CQA (3) was

345

heated together. It was observed that 5-CQA (3) mostly remained unchanged by the heat

346

treatment with the peak area corresponding to 5-CQA accounting for five times the peak area

347

of all the transformation products combined (Table 3). Again caffeoyl quinic acid lactones

348

dominated as the main transformation products. Since it was established earlier by Clifford et

349

al.3 that acyl migration takes place before dehydration in chlorogenic acid when there is still

350

some water present in the sample, it can be assumed that the 5-acyl group migrates to

351

positions C3 and C4 first and then both products undergo dehydration to yield the

352

corresponding lactones in the form of 3-CQL and 4-CQL. 4-CQL (38) was formed in larger

353

quantities if compared to 3-CQL (37). Only a single transesterification product was identified

354

in the form of 4-pCoQA (14), which formed during model roasting (Table 3 and Figure 8). It

355

is speculated that the formation of the 1,5-quinide takes place earlier making C5 on quinic acid

356

unavailable for the possible condensation with p-coumaric acid hence, we do not observe 5-

357

pCoQA (13) but 4-pCoQA (14).



Page 16 of 43

358

Model roasting experiment between 5-CQA (3) and ferulic acid (31) generated a larger

359

number of transformation products compared to the number of transformation products

360

detected from the same experiment with pCoA (33) and 5-CQA (3). Similar trend in terms of

361

number of the transformed products was observed in the cross-over experiment by TMAH

362

treatment. The list of the transformation products in this experiment looks very similar to the

363

list in Table 3. An additional two products are found to be the acyl migration products of 5-

364

CQA. The acyl group in 5-CQA (3) was found to presumably migrate intramolecularly to

365

produce 3-CQA (2) and 4-CQA (4) but the corresponding caffeoyllactones 3-CQL (37) and 4-

366

CQL (38) were formed more in quantity. The ratio of 3-CQL (37) to 3-CQA (2) was 11:1

367

whereas, the ratio of 4-CQL (38) to 4-CQA (4) was found to be 1:1.3 since, 4-CQL formed in

368

higher quantity if compared to 3-CQL judged by comparison of the corresponding peak areas

369

(Table 3). In this experiment it was observed that 5-CQA (3) remained unchanged to even

370

greater extent than in previous experiments by the heat treatment since the peak area under 5-

371

CQA alone is more than 16 times the sum of peak areas of all the transformation products

372

combined (Table 3). 5-FQA (23) was formed in considerable quantity confirming

373

intermolecular acyl migration between ferulic acid and 5-CQA (3) (Figure 9).

374

Intramolecular acyl migration: brewing of CGAs

375

In these experiments we investigated acyl migration products of mono- and di-acyl CGAs (2-

376

5 and 8-10) formed during the brewing process (Table 4). Typically, the pH of a coffee brew

377

is between 4.7-5.1. In our case the pH of the model brew was measured at 5.0. We observed

378

that hot water also serves as a reactive reagent other than just as a simple solvent in coffee

379

brewing similar to previous work on tea fermentation, where water was shown to be the key

380

reagent in thearubigin formation.24 Apart from the acyl migration products and trans-cis

381

isomerization (cis-caffeoylquinic acids) products; the resulting chromatograms showed


Page 17 of 43


382

transformation products referred here as hydroxy-dihydrocaffeoylquinic acids arising through

383

conjugate addition of water molecule to the olefinic cinnamoyl moiety.25

384

Similar to the model roasting experiment, 5-CQA (3) did not show any acyl migrated

385

products in hydrolysis (brewing). The acyl moiety in 4-CQA (4) and 3-CQA (2) did not

386

migrate to C5 of the quinic acid but acyl moiety interchange between C3 and C4 was observed

387

due to the stability of the ortho-ester intermediate arising from the cis geometry (Figure 4).

388

Acyl migration to C5 from C3 and C4 was found to be highly pH dependent as we only

389

observed it in case of basic hydrolysis.

390

Cynarin (1,3-diCQA) did not show any transformation products under acidic brewing

391

conditions after 5 h. of reflux. Among the remaining di-acylated reference standards 3,4-di-

392

acylated esters were preferably formed irrespective of the substrate. This observation can

393

possibly be attributed to the fact that the parallel aromatic rings allow for

394

interactions providing added stability to the 3,4-diCQA in the minimum energy chair

395

conformation of qunic acid moiety. By comparing the peak areas in the UV chromatogram it

396

was observed that the decomposition of the di-CQAs to produce mono-CQAs was taking

397

place in minute quantity as compared to the basic hydrolysis experiment to study

398

intermolecular acyl migration. In the case of mono-acylated chlorogenic acids, up to 1.5-2.0%

399

of the chlorogenic acids were transformed into their hydroxylated derivatives and the

400

diacylated chlorogenic acids up to 4-4.5% if the relative peak areas in EIC are considered.25

401

but their peak areas in UV chromatograms were found to be negligible. The structures for the

402

water addition compounds can be found in in our previous publication25 and are not further

403

commented on here.

404

In this work we investigated acyl migration under three conditions relevant to the processing

405

of coffee, aqueous basic and acidic and dry roasting conditions. Under all three conditions

406

acyl migration was observed, however only dominating as a pathway under aqueous basic 17 ACS Paragon Plus Environment

-

stacking


Page 18 of 43

407

conditions. Under aqueous acidic and dry roasting conditions acyl migration products were in

408

contrast observed as minor products. Acyl migration in aqueous solution is under typical

409

chlorogenic acids concentrations in a coffee brew predominantly a reversible intramolecular

410

process, showing a strong pH dependency. We could furthermore show that the acyl

411

migration phenomena occurs in dry roasting before dehydration takes place in the quinic acid

412

moiety. Acyl migration is facilitated in presence of the liquid media, as the esters present on

413

C3 position of the quinic acid are prone to hydrolysis of the ester bond, rather than undergoing

414

acyl migration in any experimental condition. In cross-over experiments (TMAH and model

415

roasting), with 5-CQA (3) ferulic acid if compared to p-coumaric acid showed increased

416

reactivity generating a large number of transesterification products. A significant proportion

417

of 3-CQA and 4-CQA quantities and their derivatives in a cup of coffee must be assumed to

418

be a result of acyl migration occurring in roasting and brewing processes originating from 5-

419

CQA.

420

Acknowledgements

421

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

422

International) and Jacobs University Bremen gGmbH. We acknowledge support from Dr.

423

Frank Ullrich during the project. Furthermore we acknowledge excellent technical support by

424

Ms. Anja Müller.

425

Supporting Information Available:

426

Table S1: Initial concentrations of the reference standards.

427

Figure S1: Structures identified after brewing of the reference standards.

428

Figure S2: Amount of the transformation products after base hydrolysis for different time

429

intervals of 3,4- and 4,5-diCQA reference standards. 18 ACS Paragon Plus Environment

Page 19 of 43

430


This material is available free of charge via the Internet at http://pubs.acs.org

431 432 433 434 435 436

437

Figure Legends

438

Figure 1. Structure of mono- and di-caffeoylquinic, p-coumaroylquinic and feruloylquinic

439

acids

440

Figure 2. UV Chromatograms (318-322 nm) at 2, 5, 10 and 30 min of basic hydrolysis of 5-

441

CQA

442

Figure 3. Amount of the transformation products after basic hydrolysis for different time

443

intervals of 5-CQA, 4-CQA and 3-CQA

444

Figure 4. Mechanism of the acyl migration through an ortho-ester intermediate formation

445

Figure 5. Amount of the transformation products after basic hydrolysis for different time

446

intervals of di-acylated reference standards

447

Figure 6. Comparison between the peak areas of compounds formed during TMAH treatment

448

of 5-CQA with p-coumaric acid and 5-CQA with ferulic acid

449

Figure 7. EIC and fragmentation patterns for 1-cis-caffeoylquinic acid (47) at m/z 353 and

450

caffeoyl-feruloylquinic acid (49) at m/z 529 in transesterification induced by TMAH 19 ACS Paragon Plus Environment


451

Figure 8. EIC and fragmentation patterns for ion at m/z 497 observed during model roasting

452

Figure 9. MS3 and MS4 of 4-pCoQA (14) and 5-FQA (23) respectively observed during

453

cross-over experiment by model roasting


Page 20 of 43

Page 21 of 43

454


References

455

1. Clifford, M.N. Chlorogenic acids and other cinnamates - nature, occurrence, dietary

456

burden, absorption and metabolism. J. Sci. Food Agric. 2000, 80, 1033-1043.

457

2. Jaiswal, R.; Matei, M.F.; Golon, A.; Witt, M.; Kuhnert, N. Understanding the fate of

458

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

459

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

460

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

461

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

462

4. Farah, A.; De Paulis, T.; Trugo, L.C.; Martin, P.R. Effect of roasting on the formation of

463

chlorogenic acid lactones in coffee. J. Agric. Food Chem. 2005, 53, 1505-1513.

464

5. Kweon, M.H.; Hwang, H.J.; Sung, H.C. Identification and antioxidant activity of novel

465

chlorogenic acid derivatives from bamboo (Phyllostachys edulis). J. Agric. Food Chem. 2001,

466

49, 4646-4655.

467

6. Wang, G.; Shi, L.; Ren, Y.; Liu, Q.; Liu, H.; Zhang, R.; Li, Z.; Zhu, F.; He, P.; Tang, W.;

468

Tao, P.; Li, C.; Zhao, W.; Zuo, J. Anti-hepatitis B virus activity of chlorogenic acid, quinic

469

acid and caffeic acid in vivo and in vitro. Antiviral Res. 2009, 83, 186-190.

470

7. Hemmerle, H.; Burger, H.; Below, P.; Schubert, G.; Rippel, R.; Schindler, P.W.; Paulus, E.;

471

Herling, A.W. Chlorogenic acid and synthetic chlorogenic acid derivatives: novel inhibitors

472

of hepatic glucose-6-phosphate translocase. J. Med. Chem. 1997, 40, 137-145.

473

8. Robinson, W.E.; Reinecke, M.G.; AbdelMalek, S.; Jia, Q.; Chow, S.A. Inhibitors of HIV-1

474

replication that inhibit HPV integrase. Proc. Natl. Acad. Sci. U. S. A. 1996, 93, 6326-6331.



475

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

476

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

477

immunodeficiency virus-1 (HIV-1) integrase. Chem. Pharm. Bull. 2000, 48, 1796-1798.

478

10. Clifford, M.N.; Kellard, B.; Birch, G.G. Characterisation of chlorogenic acids by

479

simultaneous isomerisation and transesterification with tetramethylammonium hydroxide.

480

Food Chem. 1989, 33, 115-123.

481

11. Clifford, M.N.; Kellard, B.; Birch, G.G. Characterization of caffeoylferuloylquinic acids

482

by simultaneous isomerization and transesterification with tetramethylammonium hydroxide.

483

Food Chem. 1989, 34, 81-88.

484

12. Dawidowicz, A.L.; Typek, R. Thermal stability of 5-O-caffeoylquinic acid in aqueous

485

solutions at different heating conditions. J. Agric. Food Chem. 2010, 58, 12578-12584.

486

13. Trugo, L.C.; Macrae, R. A study of the effect of roasting on the chlorogenic acid

487

composition of coffee using HPLC. Food Chem. 1984, 15, 219-227.

488

14. Anonymous IUPAC Commission on the Nomenclature of Organic Chemistry (CNOC)

489

and IUPAC-IUB Commission on Biochemical Nomenclature (CBN). Nomenclature of

490

cyclitols. Recommendations, 1973. Biochem J. 1976, 153, 23-31.

491

15. Clifford, M.N.; Williams, T.; Bridson, D. Chlorogenic acids and caffeine as possible

492

taxonomic criteria in Coffea and Psilanthus. Phytochemistry 1989, 28, 829-838.

493

16. Hanson, K.R. Chlorogenic acid biosynthesis Chemical synthesis and properties of the

494

mono-O-cinnamoylquinic acids. Biochemistry 1965, 4, 2719-2731.

495

17. Xie, C.; Yu, K.; Zhong, D.; Yuan, T.; Ye, F.; Jarrell, J.A.; Millar, A.; Chen, X.

496

Investigation of isomeric transformations of chlorogenic acid in buffers and biological 22 ACS Paragon Plus Environment

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Page 23 of 43


497

matrixes by ultraperformance liquid chromatography coupled with hybrid quadrupole/ion

498

mobility/orthogonal acceleration time-of-flight mass spectrometry. J. Agric. Food Chem.

499

2011, 59, 11078-11087.

500

18. Clifford, M.N.; Johnston, K.L.; Knight, S.; Kuhnert, N. Hierarchical scheme for LC-MSn

501

identification of chlorogenic acids. J. Agric. Food Chem. 2003, 51, 2900-2911.

502

19. Clifford, M.N.; Knight, S.; Kuhnert, N. Discriminating between the six isomers of

503

dicaffeoylquinic acid by LC-MSn. J. Agric. Food Chem. 2005, 53, 3821-3832.

504

20. Jaiswal, R.; Matei, M.F.; Ullrich, F.; Kuhnert, N. How to distinguish between

505

cinnamoylshikimate esters and chlorogenic acid lactones by liquid chromatography-tandem

506

mass spectrometry. J. Mass Spectrom. 2011, 46, 933-942.

507

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

508

of the chlorogenic acids and hydroxycinnamoylshikimate esters in Mate (Ilex

509

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

510

22. Kuhnert, N.; Jaiswal, R.; Matei, M.F.; Sovdat, T.; Deshpande, S. How to distinguish

511

between feruloyl quinic acids and isoferuloyl quinic acids by liquid chromatography/tandem

512

mass spectrometry. Rapid Commun. Mass Spectrom. 2010, 24, 1575-1582.

513

23. Clifford, M.N.; Kirkpatrick, J.; Kuhnert, N.; Roozendaal, H.; Salgado, P.R. LC-MSn

514

analysis of the cis isomers of chlorogenic acids. Food Chem. 2008, 106, 379-385.

515

24. Kuhnert, N.; Drynan, J.W.; Obuchowicz, J.; Clifford, M.N.; Witt, M. Mass spectrometric

516

characterization of black tea thearubigins leading to an oxidative cascade hypothesis for

517

thearubigin formation. Rapid Commun. Mass Spectrom. 2010, 24, 3387-3404.



518

25. Matei, M.F.; Jaiswal, R.; Kuhnert, N. Investigating the chemical changes of chlorogenic

519

acids during coffee brewing: conjugate addition of water to the olefinic moiety of chlorogenic

520

acids and their quinides. J. Agric. Food Chem. 2012, 60, 12105-12115.

521

522


Page 24 of 43

Page 25 of 43


Table 1. Compounds identified after base treatment of CGA with p-coumaric acid and CGA with ferulic acid for various time intervals 5-CQA + p-coumaric acid 5-CQA + ferulic acid Reaction Product RT (min) m/z (M-H) Reaction Product RT (min) m/z (M-H) time(min) number time(min) number 2

33 32 37 38 2 3 4

26.70 19.30 25.70 29.40 11.50 17.10 20.80

162.6 178.6 334.8 334.9 352.8 352.8 352.8

5

37 38 14 1 2 3 4 40 32 33

25.80 29.80 27.60 10.70 12.10 17.30 21.70 30.60 19.50 26.60

335.0 335.1 337.0 353.1 353.0 353.1 353.1 367.1 178.6 162.6

2 37 38

12.20 25.90 29.80

353.1 335.1 335.0

10

2

31 34 22 23 24 47 2 48 3 4 37 38 32 35

27.3 29.6 19.2 24.1 26.4 3.0 11.2 13.1 16.9 20.5 23.6 25.7 7.4 19.2

192.6 192.6 366.7 366.9 366.9 352.8 352.9 352.8 352.8 352.8 334.9 334.9 178.6 178.6

5

23 40 1 37 38 2 3

26.9 28.7 11.10 25.9 29.6 12.5 17.7

367.1 367.1 353.1 335.1 335.1 353.1 353.0



15

30

1 3 4 33 40 13 14 32

10.80 17.50 21.90 26.40 30.60 24.30 28.40 19.70

353.1 353.0 353.1 162.9 367.1 336.8 337.0 178.6

38 2 3 32 4

29.80 12.40 17.70 19.60 22.00

335.1 353.1 353.0 178.9 352.8

3 4 33

18.00 22.60 26.60

353.1 352.8 162.9

4 49

22.2 30.8

353.0 529.0

10

37 1 2 3 4 23 40 49

25.9 10.9 12.2 17.6 22.1 26.8 28.9 31.0

335.1 353.0 353.0 353.0 353.1 367.1 367.1 529.0

15

37 2 3 4 23 40 49

26.2 12.4 17.7 22.3 27.2 28.9 31.2

335.1 353.0 353.0 353.1 367.1 367.1 529.0

30

40 3

28.9 18.0

367.1 353.0


Page 26 of 43

Page 27 of 43


Table 2. Compounds identified after heating (model roasting) reference standards Starting material 3

Compound 5-CQA

Product 3

RT(min) 23.7

Peak Area(UV) 11035

2

3-CQA

2

18.9

6034

4

4-CQA

37 38 41 47 48 52 48

31.8 35.1 32.9 44.9 52.3 46.3 49.5

2418 1543 396 210 197 80 521

5

1,3-diCQA

5

30.2

16245

8

3,4-diCQA

37 38 46 46-cis

31.7 35.2 51.2 52.4

98 74 7099 896

9

3,5-diCQA

46 46-cis

56.2 56.9

3034 119

10

4,5-diCQA

10

46.3

26560



Page 28 of 43

Table 3. Compounds identified after heating (Model roasting) of 5-CQA (3) with p-coumaric acid and 5-CQA (3) with ferulic acid 5-CQA (3) and p-coumaric acid Product RT (min) Peak area number 25.9 1093 37 29.6 1538 38 28.0 330 14 36.9 145 47 39.8 642 52 41.6 2137 52 17.5 30503 3

5-CQA (3) and ferulic acid Product RT (min) Peak area number 26.0 484 37 29.4 535 38 16.8 44224 3 12.2 45 2 21.9 421 4 26.8 484 23 36.8 176 47 39.9 138 52 41.9 500 52


Page 29 of 43


Table 4. Compounds identified after hydrolysis of reference standards (Brewing of CGAs) Starting material Product name

RT (Min) Peak area(UV)

5-CQA(3)

5-CQA cis-5-CQA 5-hCQA I 5-hCQA II

20.1 23.0 7.3 7.9

17610 347 NA NA

4-CQA(4)

3-CQA 4-CQA cis-3-CQA cis-4-CQA 4-hCQA I 4-hCQA II

13.1 20.6 11.9 16.5 6.9 7.9

17576 6098 17 179 NA NA

3-CQA(2)

3-CQA 4-CQA cis-3-CQA 3-hCQA I + II

13.1 20.6 11.9 5.6

639 954 150 17

1, 3-diCQA(5)

1, 3-diCQA

22.9

3422

3, 4-diCQA(8)

3,4-diCQA cis-4,5-diCQA I 4,5-diCQA cis-3,4-diCQA I cis-3,4-diCQA II 3-CQA 4-CQA 5-CQA 3-C-4-hCQA

35.2 38.0 36.9 34.2 35.9 12.3 19.3 15.6 26.4

2558 99 520 393 345 20 27 2 57

3, 5-diCQA(9)

3,4-diCQA 3,5-diCQA 4,5-diCQA 3-C-5-hCQAI 3-hC-5-CQA I 3-hC-cis-5-CQA 3-hC-5-CQA II 3-CQA 5-CQA

36.5 37.3 41.4 23.8 27.8 28.3 31.7 12.9 20.3

1406 1057 1107 14 22 4 4 80 126

4, 5-diCQA(10)

3,4-diCQA

43.7

1825



3,5-diCQA 4,5-diCQA cis-4,5-diCQA I cis-4,5-diCQA II 3-CQA 4-CQA 5-CQA 4-hC-5-CQA 3-hC-5-CQA II 3-C-5-hCQA II

42.6 45.7 47.2 52.0 16.8 27.9 23.1 34.7 35.5 63.1

512 2148 36 7 19 14 18 18 18 23

NA- Insignificant peak area


Page 30 of 43

Page 31 of 43


HO

R'1O

O C

OR5 OR4 R' O 1

O C

OR5 OR4

O C CH HC

HO

O C CH HC

HO

O C CH

HC

OR3

OR1 OR3

OH Q

Number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

S

O

OH

OH

OH

C

pCo

F

HO C CH O CH CH3 HO

Q R1 R3 R4 R5 R'1 1-O-caffeoylquinic acid (1-CQA) C H H H 3-O-caffeoylquinic acid (3-CQA) H C H H 5-O-caffeoylquinic acid (5-CQA) H H H C 4-O-caffeoylquinic acid (4-CQA) H H C H 1,3-di-O-caffeoylqunic acid (1,3-diCQA) C C H H 1,4-di-O-caffeoylqunic acid (1,4-diCQA) C H C H 1,5-di-O-caffeoylqunic acid (1,5-diCQA) C H H C 3,4-di-O-caffeoylqunic acid (3,4-diCQA) H C C H 3,5-di-O-caffeoylqunic acid (3,5-diCQA) H C H C 4,5-di-O-caffeoylqunic acid (4,5-diCQA) H H C C 1-O-p-coumaroylqunic acid (1-pCoQA) pCo H H H 3-O-p-coumaroylqunic acid (3-pCoQA) H pCo H H 5-O-p-coumaroylqunic acid (5-pCoQA) H H H pCo 4-O-p-coumaroylqunic acid (4-pCoQA) H H pCo H 1,3-di-O-p-coumaroylqunic acid (1,3-dipCoQA) pCo pCo H H 1,4-di-O-p-coumaroylqunic acid (1,4-dipCoQA) pCo H pCo H 1,5-di-O-p-coumaroylqunic acid (1,5-dipCoQA) pCo H H pCo -

OH

cis-C

Name and abbreviation

R3 -


S R4 -

R5 -


18 19 20 21 22 23 24 25 26 27 28 29 30 36 37 38 39 40 41 42 43 44 45 46 47 48 Q-

3,4-di-O-p-coumaroylqunic acid (3,4-dipCoQA) H pCo pCo H 3,5-di-O-p-coumaroylqunic acid (3,5-dipCoQA) H pCo H pCo 4,5-di-O-p-coumaroylqunic acid (4,5-dipCoQA) H H pCo pCo 1-O-feruloylquinic acid (1-FQA) F H H H 3-O-feruloylquinic acid (3-FQA) H F H H 5-O-feruloylquinic acid (5-FQA) H H H F 4-O-feruloylquinic acid (4-FQA) H H F H 1,3-di-O-feruloylqunic acid (1,3-diFQA) F F H H 1,4-di-O-feruloylqunic acid (1,4-diFQA) F H F H 1,5-di-O-feruloylqunic acid (1,5-diFQA) F H H F 3,4-di-O-feruloylqunic acid (3,4-diFQA) H F F H 3,5-di-O-feruloylqunic acid (3,5-diFQA) H F H H 4,5-di-O-feruloylqunic acid (4,5-diFQA) H H F F 1-O-caffeoyl-1,5-quinide (1-CQL) C H H L 3-O-caffeoyl-1,5-quinide (3-CQL) H C H L 4-O-caffeoyl-1,5-quinide (4-CQL) H H C L 5-O-p-coumaroyl-methylquinate H H H pCo Me 5-O-caffeoyl-methylquinate H H H C Me 5-O-caffeoylshikimic acid (5-CSA) cis-5-O-caffeoylshikimic acid (5-cis-CSA) 4,5-di-O-caffeoylshikimic acid (4,5-diCSA) 3,5-di-O-caffeoylshikimic acid (4,5-diCQA) 3,4,5-tri-O-caffeoylshikimic acid (3,4,5-triCSA) 3,4-di-O-caffeoyl-1,5-quinide (3,4-diCQL) H C C L cis-1-O-caffeoylquinic acid (1-cis-CQA) cis-C H H H cis-4-O-caffeoylquinic acid (4-cis-CQA) H H cis-C H quinic acid, Ccaffeic acid, pCop-coumaric acid, F-


H H H C C -

H C H cis-C C C H C C C ferulic acid,

Page 32 of 43

L-

lactone,

Me-

methyl

Page 33 of 43


Figure 1. Structure of mono and di caffeoylquinic, p-coumaroylquinic and feruloylquinic acids



Intens. [mAU] 100 75 50 25 0

2 Min

5 Min 100 75 50 25 0

2

3

2

3

Page 34 of 43

4 32

4 32

10 Min

4 2

3

20 10 32 0 3

30 Min 10.0 7.5 5.0

2 4

32

0 10.0

12.5

15.0

17.5

20.0

22.5

25.0

27.5

30.0 Time [min]

Figure 2. UV Chromatograms (318-322 nm) at 2, 5, 10 and 30 min of basic hydrolysis of 5CQA


Page 35 of 43


3000

A Peak area (mAU)

2500 3-CQA 2000

4-CQA 5-CQA

1500 1000 500 0 2 Min.

5 Min. 10 Min. Time (Min)

30 Min.

30

Peak area (mAU)

25

B 3-CQA

20

4-CQA 5-CQA

15 10 5 0 2 Min.


30 Min.

8 Peak area (mAU)

7

C

6 5

3-CQA

4

4-CQA

3

5-CQA

2 1 0 2 Min.


30 Min.

Figure 3. Amount of the transformation products after basic hydrolysis for different time intervals of A. 5-CQA; B. 4-CQA; and C. 3-CQA 35 ACS Paragon Plus Environment


O

O O

O

HO OH

OH

5-CQA (3)

H

O

-H2O

R O

Page 36 of 43

O O

HO OH

OH

O

R

OH

O O

HO OH

OH

O

R

H OH

4-CQA (4)

trans-ortho-ester intermediate

-H2O

O

OH

O OH

HO OH

O

O

R 3-CQA (2)

OH O

HO OH

R

O O

cis-ortho-ester intermediate

Figure 4. Mechanism of the acyl migration through an ortho-ester intermediate formation


Page 37 of 43


900

A

Peak area (mAU)

800

1, 3-diCQA

700

3, 4-diCQA

600

4, 5-diCQA

500

3-CQA

400

5-CQA

300

4-CQA

200

1-CQA

100 0 2 Min.

5 Min. Duration (Min)

300

10 Min.

30 Min.

B

250 Peak area (mAU)

3, 5-diCQA 200

3, 4-diCQA

150

4, 5-diCQA

100 50 0 2 Min.

5 Min.

10 Min.

30 Min.

60 Min.

Duration (Min)

Figure 5. Amount of the transformation products after basic hydrolysis for different time intervals of A. 1,3-diCQA; B. 3,5-diCQA



12000

A

3-CQA

10000

Peak Area (mAU)

Page 38 of 43

5-CQA 4-CQA

8000

Caffeic acid 6000 4000 2000 0 2

5

10

15

30

Duration (Min)

16000 14000

B

3-CQA 5-CQA

10000

4-CQA

Peak Area (mAU)

12000

4-cis-CQA 8000

1-CQA

6000

Caffeic acid

4000 2000 0 2

5

10 Duration (Min)

15

30

Figure 6. Comparison between the peak areas of compounds formed during TMAH treatment of A. 5-CQA (3) with p-coumaric acid (pCoA); B. 5-CQA with ferulic acid (FA)


Page 39 of 43


Intens. 7 x10

3

EIC 353.0

4

3 2

2

48

47 1 0

5

[%]

10

15

20

25

Time [min]

MS2(353)

47

191

100 0 172.5

126.7

100

MS3(353>191)

110.7 0 170.6

100

0

110

154.6

126.6

110.6 120

MS4(353 >191>173)

130

140

150

160

170

180

190

Intens. x107

m/z

EIC 529.0

0.8 0.6 0.4 49

0.2 0.0

22

24

26

28

30

32


34

Time [min]


[%] 49

Page 40 of 43

MS2(529.0)

334.9

100 290.9 0

MS3(529>335)

290.9 100 148.9

192.9

0

MS4(529>335>291) 148.9

100 0

50

100

150

200

250

300

350

400

450

500

m/z

Figure 7. EIC and fragmentation patterns for 1-cis-caffeoylquinic acid (47) at m/z 353 and caffeoyl-feruloylquinic acid (49) at m/z 529 in transesterification induced by TMAH


Page 41 of 43


Intens. x10 6

EIC at m/z 497

46

4

46-cis

2

0

42

44

46

48

50

52

54

56

[%] 100

MS2(497)

335.1

46/ 46-cis

Time [min]

0

MS3(497>335)

160.9 100 135.3

0

MS4(497>335>161) 133

100 0

100

200

300

400

500

m/z

Figure 8. EIC and fragmentation patterns for ion at m/z 497 observed during model roasting



[%] 100

MS, 28.0min

337

4-pCoQA (14)

Page 42 of 43

0

MS2(337), 28.0min

173 100 0

MS3(337>173), 28.0min 110.9 93.1

100 0

100

154.7 150

200

250

300

350

[%]

0 100

127 110.7

0

m/z

MS3(367 >191), 26.8min

173

0 100

450

MS2(366.7), 26.8min

191.1

5-FQA (23) 100

400

MS4(367>191 >173), 26.9min 109

100

125

150

175

200

225

250

275

300

m/z

Figure 9. MS3 and MS4 of 4-pCoQA (14) and 5-FQA (23) respectively observed during cross-over experiment by model roasting


Page 43 of 43


Table of Content


Investigation of Acyl Migration in Mono- and Dicaffeoylquinic Acids

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