Interaction of chlorogenic acids and quinides from coffee with human serum albumin

Article abstract of DOI:10.1016/j.foodchem.2014.07.080

Chlorogenic acids and their derivatives are abundant in coffee and their composition changes between coffee species. Human serum albumin (HSA) interacts with this family of compounds with high affinity. We have studied by fluorescence spectroscopy the spe

Full text of DOI:10.1016/j.foodchem.2014.07.080

Food Chemistry 168 (2015) 332–340
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Interaction of chlorogenic acids and quinides from coffee with human
serum albumin
a
a
b
a,
Valentina Sinisi a, , Cristina Forzato , Nicola Cefarin , Luciano Navarini , Federico Berti
⇑
⇑
a Dipartimento di Scienze Chimiche e Farmaceutiche, Università degli Studi di Trieste, via L. Giorgieri 1, 34127 Trieste, Italy
b illycaffè S.p.A., via Flavia 110, 34147 Trieste, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Chlorogenic acids and their derivatives are abundant in coffee and their composition changes between
coffee species. Human serum albumin (HSA) interacts with this family of compounds with high
affinity. We have studied by fluorescence spectroscopy the specific binding of HSA with eight compounds
that belong to the coffee polyphenols family, four acids (caffeic acid, ferulic acid, 5-O-caffeoyl quinic
Received 10 February 2014
Received in revised form 11 June 2014
Accepted 15 July 2014
Available online 24 July 2014
acid, and 3,4-dimethoxycinnamic acid) and four lactones (3,4-O-dicaffeoyl-1,5-
(dimethoxy)cinnamoyl]-1,5- -quinide, 3,4-O-bis[3,4-(dimethoxy)cinnamoyl]-1,5-
4-O-tris[3,4-(dimethoxy)cinnamoyl]-1,5- -quinide), finding dissociation constants of the albumin–chlor-
ogenic acids and albumin–quinides complexes in the micromolar range, between 2 and 30 M. Such val-
ues are comparable with those of the most powerful binders of albumin, and more favourable than the
values obtained for the majority of drugs. Interestingly in the case of 3,4-O-dicaffeoyl-1,5- -quinide,
c
-quinide, 3-O-[3,4-
c
c-quinide, and 1,3,
Keywords:
c
Human serum albumin
Protein–ligand interaction
Fluorescence spectroscopy
Coffee polyphenols
Chlorogenic acids
l
c
we have observed the entrance of two ligand molecules in the same binding site, leading up to a first dis-
sociation constant even in the hundred nanomolar range, which is to our knowledge the highest affinity
ever observed for HSA and its ligands. The displacement of warfarin, a reference drug binding to HSA, by
the quinide has also been demonstrated.
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
2005). The roasting process causes a partial loss of CGAs, due to
the occurrence of many reactions including isomerization, degra-
Phenolic acids are found as secondary metabolites in leaves,
roots and especially fruits of many plants. Chlorogenic acids (CGAs)
derive from the esterification with D-(–)-quinic acid 1 of certain
cinnamic acids, such as caffeic acid 2, ferulic acid 3 and p-coumaric
acid 4 (Fig. 1), constituting a large family of different molecules in
the form of mono- or multi-esters (Clifford, 2000).
Of all the CGAs present in green coffee beans, which are the best
source of CGAs found in plants with an amount of 5–12 g/100 g
(Farah, Monteiro, Donangelo, & Lafay, 2008), caffeoylquinic acids
(CQAs) represent the main subgroup and 5-O-caffeoylquinic acid
5 (5-CQA, Fig. 1) is the most abundant one, indeed it is usually
called chlorogenic acid. A difference between the two types of cof-
fee was also evidenced since Robusta green coffee turned out to be
richer in CGAs than Arabica (Farah, de Paulis, Trugo, & Martin,
dation, dehydration and lactonization (Fig. 1) (Clifford, 1985;
Scholz & Maier, 1990; Schrader, Kiehne, Engelhardt, & Maier,
1996). The latter reaction leads to chlorogenic acid lactones (CGLs)
which have also shown potential biological activities (de Paulis
et al., 2002).
CGAs and CGLs are extracted during coffee brewing, and their
content in the cup depend on the type of roasted coffee used and
on the extraction method (Gloess et al., 2013); in a traditional
espresso coffee beverage (30 ml) the content of monocaffeoyl qui-
nic acids is on average 70 mg (Navarini et al., 2008). The extraction
efficiency is higher for CGAs than for CGLs, due to their better
water solubility, and in general a lungo (about 120 ml) is more rich
in CGAs than a regular espresso coffee.
Many studies reported that polyphenols are capable to perme-
ate the gastrointestinal barrier and are absorbed in humans, being
found in plasma as both intact molecules and as their hydrolysis
metabolites, in particular as caffeic acid (Farah et al., 2008;
Monteiro, Farah, Perrone, Trugo, & Donangelo, 2007; Nardini,
Cirillo, Natella, & Scaccini, 2002; Olthof, Hollman, & Katan, 2001;
Renouf et al., 2010); CQAs have been detected in plasma even 4 h
after the ingestion.
Abbreviations: CGAs, chlorogenic acids; CQAs, caffeoylquinic acids; 5-CQA, 5-O-
caffeoylquinic acid; CQLs, chlorogenic acid lactones; DMSO, dimethyl sulfoxide;
HSA, human serum albumin; Trp, tryptophan.
⇑
Corresponding authors. Tel.: +39 040 5583920; fax: +39 0405583903.
E-mail addresses: valentina.sinisi@phd.units.it (V. Sinisi), fberti@units.it
(F. Berti).
http://dx.doi.org/10.1016/j.foodchem.2014.07.080
0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

V. Sinisi et al. / Food Chemistry 168 (2015) 332–340
333
Fig. 1. Molecular structure of (–)-quinic acid (1), caffeic acid (2), ferulic acid (3), p-coumaric acid (4), 5-O-caffeoylquinic acid (5) (the IUPAC numbering system for chlorogenic
acid (IUPAC, 1976) is adopted and, to avoid confusion, the same numbering system of the carbon atoms both for lactones and for the acid precursors is used), 3,4-O-dicaffeoyl-
1,5-c-quinide (6), 3-O-[3,4-(dimethoxy)cinnamoyl]-1,5-c-quinide (7), 3,4-O-bis[3,4-(dimethoxy)cinnamoyl]-1,5-c-quinide (8), 1,3,4-O-tris[3,4-(dimethoxy)cinnamoyl]-1,5-
c
-quinide (9) and 3,4-dimethoxycinnamic acid (10).
Human serum albumin (HSA) is the most abundant protein in
(Kang et al., 2004; Min et al., 2004; Hu, Chen, Zhou, Bai, &
Ou-Yang, 2012). In this study the aim was to extend the knowledge
regarding the interaction between polyphenols present in coffee
and HSA. For this purpose we have synthesised four quinides of caf-
feic acid and of 3,4-O-dimethoxycinnamoyl acid: 3,4-O-dicaffeoyl-
human plasma, a monomeric 585-residue protein containing three
homologous helical domains (I–III), each divided into two subdo-
mains (A and B) (He & Carter, 1992). Two main binding sites for
small organic molecules are found, one located in subdomain IIA
and one in IIIA, that are known as Sudlow I and Sudlow II sites,
respectively (Sudlow, Birkett, & Wade, 1975a).
1,5-
3,4-O-bis[3,4-(dimethoxy)cinnamoyl]-1,5-
O-tris[3,4-(dimethoxy)cinnamoyl]-1,5- -quinide 9 (Fig. 1).
c
-quinide 6, 3-O-[3,4-(dimethoxy)cinnamoyl]-1,5-
c-quinide 7,
c-quinide 8, and 1,3,4-
The protein is able to bind a large variety of endogenous ligands,
as non-esterified fatty acids, bilirubin, heme, thyroxine, bile acids;
many drugs with acidic or electronegative moieties (including phe-
nols as paracetamol) also exploit the interaction with HSA to be
carried in human body (Ghuman et al., 2005; Varshney et al.,
2010). Recently an interactive association of multiple ligands with
the same binding site inside subdomain II of HSA has been pro-
posed (Yang et al., 2012). As to the binding of phenolic compounds
from dietary sources, flavonoids as flavanol, flavonol, flavone, iso-
flavone, flavanones, and anthocyanidins are known to interact with
HSA (Pal & Saha, 2014). The interactions of catechins [(–)-epigallo-
catechin-3-gallate, (–)-epigallocatechin, (–)-epicatechin-3-gallate],
flavones (kaempferol, kaempferol-3-glucoside, quercetin, naringe-
nin) and hydroxycinnamic acids (rosmarinic acid, caffeic acid,
p-coumaric acid) with bovine albumin has been reported in this
journal by Skrt, Benedik, Podlipnik, and Ulrih (2012). Resveratrol
binds to HSA and its interaction is modulated by stearic acid
(Pantusa, Sportelli, & Bartucci, 2012). Specific binding of caffeic,
ferulic, and 5-CQA acids inside Sudlow site I of HSA has been
c
Compound 6 is very abundant in roasted coffee, while the class
of dimethoxycinnamoylquinic acids, precursors of compounds 7, 8,
and 9, was recently found and characterised in green coffee beans
(Clifford, Knight, Surucu, & Kuhnert, 2006).
We have measured the dissociation constants to Sudlow site I of
our compounds in physiological conditions by fluorescence
spectroscopy following the quenching of the emission of the
unique fluorescent tryptophan residue within the binding site in
subdomain IIA (Trp-214) (Anna, 2002).
The absorption, distribution, albumin binding and excretion of
phenolic derivatives 10 in human plasma after coffee consumption
have been studied, but more complex derivatives were not consid-
ered yet (Farrell et al., 2012; Nagy et al., 2011). Our data may there-
fore be interesting to better understand the effects of coffee
consumption on the human body, as to the binding to albumin
and potential competition with drugs at the same site. Moreover,
we are also interested in the development of biosensing tools for
the rapid detection of coffee polyphenols in quality control of cof-
fee beverages. HSA could represent a valuable binder to be used in
studied, revealing KD about 6 lM, 40 lM and 25 lM respectively

334
V. Sinisi et al. / Food Chemistry 168 (2015) 332–340
such analytical devices for capturing coffee polyphenols. We have
recently obtained a functional 100 aminoacid fragment of HSA, to
be used as the starting scaffold to generate a library of albumin-
derived binders with improved affinity and selectivity (Luisi
et al., 2013).
2.3.2. 1-O-(2,2,2-trichloroethoxycarbonyl)-3,4-O-isopropyliden-1,5-c-
quinide (12)
Compound 11 (592 mg, 2.77 mmol) and pyridine (0.55 ml,
6.80 mmol) were dissolved in CH2Cl2 (6 ml). A solution of 2,2,2-tri-
chloroethyl chloroformate (0.42 ml, 3.09 mmol) in CH2Cl2 (2 ml)
was added dropwise to the reaction mixture at 0 °C. The solution
was stirred at 0 °C for 1 h and then at room temperature for 24 h.
The mixture was sequentially washed with 1 M HCl (two times,
5 ml at a time) and with brine (two times, 8 ml at a time) and
the organic layer was dried on anhydrous Na2SO4. The solvent
was eliminated under reduced pressure to obtain an orange oil.
The crude was dissolved in refluxing MeOH (6 ml) and cooled,
the solution was stored overnight at 4 °C to precipitate 12 as a
2. Materials and methods
2.1. Materials
HSA essentially fatty acid free (A3782, ꢀ99%), caffeic acid
(P98%), ferulic acid (99%), 3,4-dimethoxy cinnamic acid (predom-
inantly trans, 99%), chlorogenic acid hemihydrate (P98%) and all
the reagents for the synthesis were purchased from Sigma–
Aldrich Co. (St. Louis, MO, USA) and used without further purifica-
tion. Polyamide MN-SC-6 0.05–0.16 mm was purchased from
Macherey–Nagel (Düren, Germany).
white powder (yield 57%). M.p. 153–156 °C; [
a]
25 = ꢁ7.8 (c = 1,
D
CH2Cl2); 1H NMR (500 MHz, CDCl3): d 1.33 (CCH3, s, 3H), 1.52
(CCH3, s, 3H), 2.40 (C2-Heq
2.4 Hz), 2.56 (C2-Hax, ddd, 1H, Jgem = 14.7 Hz, JC2Hax-C3H = 7.7 Hz,
C2Hax-C6Heq = 2.3 Hz), 2.65 (C6-Hax, d, 1H, Jgem = 11.4 Hz,), 3.06
(C6-Heq, dddd, 1H, Jgem = 11.4 Hz JC6Heq-C5H = 6.5 Hz JC6Heq-C2Hax
2.3 Hz JC6Heq-C4H = 1.1 Hz), 4.34 (C4-H, ddd, 1H, JC4H-C3H = 5.3 Hz
, dd, 1H, Jgem = 14.7 Hz JC2eq-C3H =
J
=
2.2. Apparatus
J
C4H-C5H = 2.3 Hz
J
C4H-C6Heq = 1.1 Hz), 4.55 (C3-H, ddd, 1H,
C3H-C4H = 5.3 Hz C3H-C2Heq = 2.4 Hz), 4.72
JC3H-C2Hax = 7.7 Hz
J
J
Melting points were measured on a Sanyo Gallenkamp appara-
tus; the optical activity measurements ([a]) were performed with
(C10-H, d, 1H, Jgem = 11.8 Hz), 4.80 (C5-H, dd, 1H, JC5H-C6Heq = 6.5 Hz
J
= 2.3 Hz,), 4.82 (C10-H d, 1H, Jgem = 11.8 Hz); 13C NMR
(C15H2-C54H.4 MHz, CDCl3): d 24.41 (q, CH3), 27.08 (q, CH3), 30.34 (t,
a Perkin–Elmer 241 polarimeter at the wavelength of sodium D band
(k = 589 nm) using a quartz cuvette with a length of 10 cm
(l = 1 dm); 1H-NMR and 13C-NMR spectra were recorded on a Varian
500 spectrometer (scales settled on the solvents residual peaks:
7.26 ppm for CDCl3 and 3.31 ppm for CD3OD); Electrospray Ioniza-
tion (ESI) mass spectrometry measurements (MS) were performed
on a Esquire 4000 (Bruker-Daltonics) spectrometer; Infrared spectra
(IR) were recorded on a Avatar 320-IR FT-IR (Thermo-Nicolet) spec-
trometer with a thin film of sample on NaCl crystal windows;
Reverse Phase high-performance liquid chromatography (RP-HPLC)
analyses were run on Amersham Pharmacia Biotech liquid chroma-
tography equipped with UV Amersham detector, using a Gemini
C6), 35.42 (t, C2), 71.15 (d, C3), 72.46 (d, C4), 75.47 (d, C5),
77.09 (t,
C
10), 78.90 (s, C1), 94.03 (s, CCl3), 110.21 (s, C8),
151.50 (s, C9), 172.61 (s, C7); IR (cmꢁ1
)
2995, 2939, 1809,
1765, 1380, 1241, 1075, 736; MS (ESI+): 411 m/z (97, [M35Cl3-
ꢁNa]+,
413 m/z
(100,
[M35Cl237ClꢁNa]+,
415 m/z
(25,
[M35Cl37Cl2ꢁNa]+).
2.3.3. 1-O-(2,2,2-trichloroethoxycarbonyl)-1,5-c-quinide (13)
Trichloroacetic acid (373 mg, 2.28 mmol) was added to water
(42 l) and the mixture was heated until a clear solution was
l
obtained. When the acidic aqueous solution reached room temper-
ature, 12 (252 mg, 0.65 mmol) was added and reaction was stirred
for 4 h. Ice-cooled water (5.7 ml), ethyl acetate (11.4 ml) and 40%
aqueous NaHCO3 solution (11.4 ml) were added in order. The
organic layer was separated from the aqueous one, that was fur-
ther extracted with ethyl acetate (15 ml); the organic fractions
were collected and washed with 2% NaHCO3 (15 ml) and water
(12 ml). The organic phase was then dried over anhydrous Na2SO4,
filtered and the solvent was removed under vacuum to obtain a
powder. The residue was crystallized from toluene to afford the
product as white crystals (yield 61%). M.p. 132–133 °C;
C18 3
C18 5
l
l
m 2 * 150 mm column for the analytical runs and a Gemini
m 10 * 250 mm column for the semi-preparative ones.
2.3. Synthesis of 3,4-O-dicaffeoyl-1,5-c-quinide (6)
2.3.1. 3,4-O-isopropyliden-1,5-
c-quinide (11)
D
-(–)-Quinic acid (3.0 g, 15.6 mmol) and p-toluenesulfonic acid
(152 mg, 0.8 mmol) were suspended in distilled acetone (150 ml).
The mixture was heated under reflux (56 °C) for 48 h in a Soxhlet
apparatus, which was equipped with an extraction thimble filled
with molecular sieves (4 Å, Merck), activated overnight in an oven
at 120 °C. The reaction mixture was cooled to 0 °C using an
ice-bath; NaHCO3 (364 mg, 10.3 mmol) was added and the
suspension was stirred for 1 h. The mixture was filtered and the
organic phase was evaporated under vacuum to obtain the product
[a]
25 = ꢁ4.6 (c = 1, CH2Cl2); 1H NMR (500 MHz, CDCl3): d 1.67
D
(OH, br, 1H), 2.17 (C2-Hax, dd, 1H, Jgem = 11.6 Hz JC2Hax-C3H
=
11.2 Hz), 2.39 (C2-Heq, ddd, 1H, Jgem = 14.6 Hz JC2Heq-C3H = 6.7 Hz
JC2Heq-C6Heq = 3.1 Hz), 2.68 (C6-Hax, d, 1H, Jgem = 11.2 Hz), 2.84 (OH,
br, 1H), 3.06 (C6-Heq
6.3 Hz JC6Heq-C2Heq = 3.1 Hz), 4.03 (C3-H, ddd, 1H, JC3H-C2Hax = 11.2 Hz
JC3H-C2Heq = 6.7 Hz JC3H-C4H = 4.5 Hz), 4.18 (C4-H, dd, 1H, JC4H-
,
ddd, 1H, Jgem = 11.2 Hz JC6Heq-C5H =
as a white solid (yield 93%). M.p. 133–136 °C; [
a]
25 = ꢁ30.3 (c = 1,
D
CH3OH); 1H NMR (500 MHz, CD3OD): d 1.32 (CCH3, s, 3H), 1.49
(CCH3, s, 3H), 2.02 (C2-Heq, dd, 1H, Jgem = 14.6 Hz JC2Heq-C3H
3.0 Hz), 2.26 (C6-Heq, dddd, 1H, Jgem = 11.7 Hz JC6Heq-C5H = 6.1 Hz
=
C3H = 4.5 Hz JC4H-C5H = 4.5 Hz), 4.74 (C9-H, d, 1H, Jgem = 11.8 Hz), 4.81
(C9-H, d, 1H, Jgem = 11.8 Hz), 4.93 (C5-H, dd, 1H, JC5H-C6Heq = 6.3 Hz
J
J
C6Heq-C2Hax = 2.3 Hz
gem = 14.6 Hz JC2Hax-C3H=7.7 Hz JC2Hax-C6Heq = 2.3 Hz), 2.53 (C6-Hax
gem = 11.7 Hz), 4.30 (C4-H, ddd, 1H, C4H-C3H = 6.5 Hz
J
C6Heq-C4H = 1.4 Hz), 2.36 (C2-Hax
,
ddd, 1H,
J
C5H-C4H = 4.5 Hz); 13C NMR (125.4 MHz, CDCl3): d 32.65 (t, C6), 36.53
,
(t, C2), 65.86 (d, C3), 65.95 (d, C4), 76.20 (d, C5), 77.11 (s, C1), 79.04
(t, C9), 94.03 (s, CCl3), 151.58 (s, C8), 171.44 (s, C7); IR (cmꢁ1): 3412
(br), 1782, 1764, 1379, 1244, 1037, 665; MS (ESI-): 347 m/z (20,
[M35Cl3ꢁH]ꢁ), 349 m/z (20, [M35Cl237ClꢁH]ꢁ), 351 m/z (5, [M35Cl37
Cl2ꢁH]ꢁ), 383 m/z (80, [M35Cl3ꢁCl]ꢁ), 385 m/z (100, [M35Cl327ClꢁCl]ꢁ,
386 m/z (50, [M35Cl37Cl2ꢁCl]ꢁ), 389 (20, [M37Cl3ꢁCl]ꢁ).
d, 1H,
J
J
J
J
C4H-C5H = 2.5 Hz JC4H-C6Heq = 1.4 Hz), 4.52 (C3-H, ddd, 1H,
C3H-C2Hax) = 7.7 Hz JC3H-C4H = 6.5 Hz JC3H-C2Heq = 3.0 Hz), 4.67 (C5-H,
dd, 1H, JC5H-C6Heq = 6.1 Hz JC5H-C4H = 2.5 Hz); 13C NMR (125.4 MHz,
CD3OD): d 24.54 (q, CH3), 27.32 (q, CH3), 35.55 (t, C6), 38.98 (t,
C2), 72.28 (d, C3), 72.90 (d, C4), 73.62 (s, C1), 76.62 (d, C5), 110.74
(s, C8), 180.03 (s, C7); IR (cmꢁ1): 3583, 2932, 1777, 1315, 1161,
1075, 980; MS (ESI+): 215 m/z (50, [MꢁH]+), 237 m/z (100,
[MꢁNa]+).
2.3.4. 3,4-O-dimethoxycarbonyl caffeic acid (14)
Caffeic acid (2.01 g, 11 mmol) was dissolved in 1 M aqueous
NaOH (40 ml) and cooled to 0 °C. Methyl chloroformate (2.04 ml,

V. Sinisi et al. / Food Chemistry 168 (2015) 332–340
335
26.4 mmol) was added dropwise and the mixture was stirred for
1 h at 0 °C and for 1 h at room temperature: in a few time a yellow
powder began to precipitate. The reaction mixture was acidified
with 2 M aqueous HCl to pH 1, the solid was collected by filtration
and washed with water. Recrystallization from 50–50 v/v water–
ethanol (28 ml) gave 14 as a yellow-earth powder (yield 90%).
M.p.: 140–141 °C; 1H NMR (500 MHz, CDCl3): d 3.92 (OCH3, s,
3H), 3.93 (OCH3, s, 3H), 6.41 (C1-H, d, 1H, J = 15.9 Hz), 7.34 (C5-H,
d, 1H, Jortho = 8.4 Hz), 7.46 (C4-H, dd, 1H, Jortho = 8.4 Hz Jmeta = 1.9
Hz), 7.49 (C8-H, d, 1H, Jmeta = 1.9 Hz), 7.72 (C2-H, d, 1H,
J = 15.9 Hz); 13C NMR (125.4 MHz, CDCl3): d 56.10 (q, 2C, OCH3),
118.92 (d, C1), 122.71 (d, C8), 123.72 (d, C5), 127.03 (d, C4),
133.26 (s, C3), 142.86 (s, C7), 144.07 (s, C6), 144.91 (d, C2), 153.03
(s, OCOO), 153.20 (s, OCOO), 171.58 (s, COOH); IR (cmꢁ1): 2918.7,
1760.6, 1692.6, 1632.7, 1438.2, 1265.3, 932.1; MS (ESIꢁ): 295 m/z
(100, [MꢁH]ꢁ).
73.74 (d, C5), 78.78 (t, C9), 94.04 (s, CCl3), 114.2 (s, C1), 117.89
0
0
(d, C11), 118.11 (d, C11 ), 122.45 (d, C18), 122.51 (d, C18 ), 123.69 (d,
0
0
C
15), 123.81 (d, C15 ), 127.05 (d, C14), 127.23 (d, C14 ), 132.94
0
0
(s, C13), 133.16 (s, C13 ), 142.83 (s, C17), 142.93 (s, C17 ), 144.00
0
0
(s, C16), 144.21 (s, C16 ), 144.35 (d, C12), 144.94 (d, C12 ), 151.54 (s,
0
0
C8), 152.97 (s, C19), 152.99 (s, C19 ), 153.15 (s, C20), 153.21 (s, C20 ),
164.61 (s, C10), 164.78 (s, C10 ), 170.10 (s, C7); IR (cmꢁ1): 3583.1,
0
2918.0, 1771.3, 1722.2, 1441.2, 1259.7, 1146.3, 727.9; MS (ESI+):
929.2 m/z (100, [M37ClꢁNa]+).
2.3.7. 3,4-O-dicaffeoyl-1,5-c-quinide (6) (Blumberg, Frank, &
Hofmann, 2010)
Compound 16 (438 mg, 0.48 mmol) was suspended in dry pyr-
idine (stored on molecular sieves, 4.4 ml); LiCl (229 mg, 5.4 mmol)
was added and then the mixture was stirred for 7 days at 50 °C.
During the reaction time the suspension turned to a brown solu-
tion. The solvent was removed under vacuum and the residue
was dissolved in ethyl acetate (20 ml), then sequentially washed
with 2 M HCl (two times, 12 ml each one), 2% NaHCO3 (two times,
10 ml each one) and brine (8 ml); the organic phase was dried on
Na2SO4 and the vacuum removal of the solvent gave an orange res-
idue. The crude was treated by flash chromatography on polyam-
ide MN-SC-6 (glass column 2 ꢂ 30 cm, gradient elution from
ethyl acetate/methanol 80/20 to 50/50 v/v); the fractions rich in
the target molecule were purified by semi-preparative RP-HPLC
2.3.5. 3,4-O-dimethoxycarbonyl caffeic acid chloride (15)
A suspension of 14 (1.41 g, 4.76 mmol) in thionyl chloride
(2.4 ml, 33 mmol, added dropwise) was heated to 90 °C until the
formation of a homogeneous brown solution without gas develop-
ment (ꢀ2 h). Before stopping the reaction, the mixture was
checked by 1H-NMR in CDCl3 to control if the chlorination was fin-
ished. The unreacted thionyl chloride was removed under vacuum
and the brown solid residue was recrystallized from toluene
(10 ml) and filtered to obtain 15 as a yellow powder (yield 50%),
that was used immediately after its preparation. 1H NMR
(500 MHz, CDCl3): d 3.92 (OCH3, s, 3H), 3.93 (OCH3, s, 3H), 6.61
(C1-H, d, 1H, J = 15.6 Hz), 7.38 (C5-H, d, 1H, Jortho = 8.5 Hz), 7.48
(C4-H, dd, 1H, Jortho = 8.5 Hz Jmeta = 2.1 Hz), 7.52 (C8-H, d, 1H,
on a Phenomenex Gemini C18 5 lm 10 * 250 mm column (15 mg
of crude for each run, loop 10 ml), using a gradient of H2O + 0.1%
TFA (A) and MeOH + 0.1% TFA (B) (20 min A 80% B 20%, from 20
to 90 min increase of B until A 40% B 60%, from 90 to 110 min A
5% B 95%, from 110 to 125 min A 95% B 5%) at a flow rate of
2 ml/min. The elution was monitored with a UV/vis detector at k
214, 288 and 325 nm; the fractions corresponding to the peak of
interest were checked with ESI+-MS (molecular ion [MꢁH]+
499 m/z) and then freeze-dried: 6 was obtained as a white powder
(yield 20%). M.p. 134–136 °C; 1H NMR (500 MHz, CD3OD): d 2.16
J
meta = 2.1 Hz), 7.76 (C2-H, d, 1H, J = 15.6 Hz); 13C NMR
(125.4 MHz, CDCl3): d 56.16 (q, 2C, OCH3), 123.36 (d, C8), 123.89
(d, C1), 123.97 (d, C5), 127.74 (d, C4), 132.05 (s, C3), 143.01 (s, C7),
144.96 (s, C6), 148.21 (d, C2), 152.81 (s, OCOO), 153.07 (s, OCOO),
165.88 (s, COCl); MS (ESIꢁ): 334 m/z (100, [MOCH3-Na]+).
(C2-Hax, dd, 1H, Jgem = 11.8 Hz JC2Hax-C3H = 11.6 Hz), 2.28 (C2-Heq
ddd, 1H, gem = 11.8 Hz JC2Heq-C3H = 6.8 Hz JC2Heq-C6Heq = 2.4 Hz),
2.47 (C6-Heq ddd, 1H, Jgem = 11.9 Hz JC6Heq-C5H = 5.7 Hz
C6Heq-C2Heq = 2.4 Hz), 2.59 (C6-Hax, d, 1H, Jgem = 11.9 Hz), 4.92 (C5-
H, dd, 1H, JC5H-C6Heq = 5.7 Hz JC5H-C4H = 5.1 Hz), 5.18 (C3-H, ddd, 1H,
JC3H-C2Hax = 11.6 Hz JC3H-C2Heq = 6.8 Hz JC3H-C4H = 4.7 Hz), 5.61
(C4-H, dd, 1H, JC4H-C5H = 5.1 Hz JC4H-C3H = 4.7 Hz), 6.14 (C9-H, d,
,
2.3.6. 1-O-(2,2,2-trichloroethoxycarbonyl)-3,4-bis[3,4-O-
J
(dimethoxycarbonyl)caffeoyl]-1,5-
c
-quinide (16)
,
Compound 13 (136 mg, 0.39 mmol) was dissolved in CH2Cl2
(stored on CaCl2, 10 ml); DMAP (10 mg, 0.08 mmol) and Et3N
(0.35 ml, 2.5 mmol) were added and the solution was cooled to
0 °C. Chloride 15 (600 mg, 1.9 mmol) was slowly added and the
yellow solution was stirred for 1 h at 0 °C and then for 24 h at room
temperature. The reaction mixture was sequentially washed with
1 M HCl (two times, 15 ml at a time), 2% NaHCO3 (15 ml) and brine
(10 ml); the organic layer was dried over Na2SO4 and the solvent
was removed by vacuum evaporation. The crude was purified by
flash chromatography on silica gel (glass column 2.5 ꢂ 35 cm, gra-
dient elution from CH2Cl2/ethyl acetate 98/2 to 92/8 v/v) to obtain
16 as a pearly powder (yield 40%). 1H NMR (500 MHz, CDCl3): d 2.46
J
0
0
0
1H, JC9H-C10H = 15.9 Hz), 6.37 (C9 -H, d, 1H, JC9 H-C10 H = 15.8 Hz),
0
6.68 (C13-H, d, 1H, Jortho = 8.2 Hz), 6.80 (C12-H and C13 -H, m, 2H),
0
0
6.98 (C16-H and C12 -H, m, 2H), 7.08 (C16 -H, d, 1H, Jmeta = 2.0 Hz),
0
7.48 (C10-H, d, 1H, JC10H-C9H = 15.9 Hz), 7.63 (C10 -H, d, 1H,
0
0
J
C10 H-C9 H = 15.8 Hz); 13C NMR (125.4 MHz, CD3OD): d 37.36 (t,
C2), 38.70 (t, C6), 65.89 (d, C4), 67.79 (d, C3), 72.89 (s, C1), 75.07
0
(d, C5), 113.95 (d, C9), 114.07 (d, C9 ), 114.81 (d, C16), 115.34 (d,
0
0
C16 ), 116.42 (d, C13), 116.55 (d, C13 ), 123.41 (d, C12), 123.49 (d,
0
0
(C2-Hax, dd, 1H, Jgem = 11.8 Hz JC2Hax-C3H = 11.6 Hz), 2.56 (C2-Heq
ddd, 1H, gem = 11.8 Hz JC2Heq-C3H = 6.8 Hz JC2Heq-C6Heq = 2.7 Hz),
2.71 (C6-Hax d, 1H, Jgem = 11.6 Hz), 3.23 (C6-Heq ddd, 1H,
gem = 11.6 Hz JC6Heq-C5H = 5.9 Hz JC6Heq-C2Heq = 2.7 Hz), 3.89 (OCH3,
s, 3H), 3.90 (OCH3, s, 3H), 3.92 (OCH3, s, 3H), 3.93 (OCH3, s, 3H),
4.75 (C9-H, d, 1H, Jgem = 11.8 Hz), 4.85 (C9-H, d, 1H, Jgem = 11.8 Hz),
5.02 (C5-H, dd, 1H, JC5H-C6Heq = 5.9 Hz JC5H-C4H = 4.9 Hz), 5.36
,
C
C
C
12 ), 127.45 (s, C11), 127.50 (s, C11 ), 146.81 (s, C15), 146.89 (s,
0
0
J
15 ), 147.87 (d, C10), 148.55 (d, C10 ), 149.81 (s, C14), 150.00 (s,
0
0
,
,
14 ), 167.37 (s, C8), 167.54 (s, C8 ), 178.22 (s, C7); IR (cmꢁ1):
J
3405.7, 2950.7, 1790.6, 1633.0, 1269.35, 1020.7, 644.9; MS (ESI+):
499 m/z (100, [MꢁH]+).
2.4. Fluorescence spectroscopy
(C3-H, ddd, 1H, JC3H-C2Hax = 11.6 Hz JC3H-C2Heq = 6.8 Hz JC3H-C4H
=
4.7 Hz), 5.71 (C4-H, dd, 1H, JC4H-C5H = 4.9 Hz JC4H-C3H = 4.7 Hz), 6.28
Compounds 2, 3, 5, 6, 7, 8, 9, and 10 stock solutions (7 mM,
0
(C11-H, d, 1H, JC11H-C12H = 16 Hz), 6.45 (C11 -H, d, 1H,
1.4 mM, 350 lM and for 6 87.5 lM) were prepared in DMSO.
0
0
J
C11 H-C12 H = 15.9 Hz), 7.27 (C15-H, d, 1H, Jortho = 8.5 Hz), 7.34
(C14-H and C15 -H, m, 2H), 7.42 (C18-H and C14 -H, m, 2H), 7.52
Steady state fluorescence spectra were recorded at 25 °C on a
CARY Eclipse (Varian) spectrofluorimeter equipped with a 1 cm
quartz cuvette (kexc 280 nm, kem 340 nm). The emission corre-
sponding to kexc 280 nm was recorded in the kem range 300–
400 nm. Synchronous fluorescence spectra (SFS) were measured
by setting the excitation wavelength in the 240–320 nm range,
0
0
0
(C18 -H,
d,
1H,
Jmeta = 1.9 Hz),
7.59
(C12-H,
d,
1H,
0
0
0
J
C12H-C11H = 16 Hz), 7.67 (C12 -H, d, 1H, JC12 H-C11 H = 15.9 Hz); 13C
NMR (125.4 MHz, CDCl3): d 33.78 (t, C2), 33.87 (t, C6), 56.07 (q,
2C, 2OCH3), 56.11 (q, 2C, 2OCH3), 65.05 (d, C4), 66.12 (d, C3),

336
V. Sinisi et al. / Food Chemistry 168 (2015) 332–340
and the emission was recorded at
range. The slit width on the excitation was set to 10 nm, on the
emission to 20 nm. The concentration of HSA essentially fatty acid
D
= 60 nm in the 300–380 nm
then added at a 2.5 mM final concentration from a 250 mM refer-
ence solution in water, and the emission spectrum was recorded
upon excitation of bound warfarin at 320 nM. The emission maxi-
mum was observed at 380 nM. Lactone 6 was then added at
increasing concentrations by adding aliquots of its stock solution
in the 400 nM–72 mM range, and the emission spectrum was
recorded again at each addition.
free solutions was 0.5
buffer 10 mM in Na2HPO4 and 2 mM in KH2PO4 diluted in 215
mQ water, pH 7.4) for all the measurements; the ligand concentra-
tion was gradually increased during the titration from 1 M to
500 M by adding aliquots of their stock solutions; the final
lM in 350
ll of solvent (135
ll of phosphate
l
l of
l
l
amount of DMSO was always less than 8%, and it has been verified
that such amounts of the solvent do not affect the fluorescence of
HSA. For compound 6 the fluorescence quenching with a titration
3. Results and discussion
in a narrower concentration range (from 0.25 to 200 lM) was also
3.1. Synthesis of the quinide ligands
measured. After each addition of the ligand, the emission spectra,
the fluorescence intensity, and the SFS were recorded. All the anal-
yses were replicated three times.
The displacement of warfarin was studied with the same spec-
trofluorimeter and cell, in the same buffer described above for the
binding study. Warfarin was added to the buffer at a 10 mM final
concentration from a 1 mM reference solution in DMSO. HSA was
Quinide 6 has been synthesised as reported in Fig. 2, by revising
and improving a methodology reported by Blumberg (Blumberg
et al., 2010). Unfortunately, using the procedure reported in the lit-
erature, we always afforded a mixture of monoesters on carbons 3
or 4 of the quinide and only a small amount of the diester was
achieved. It was necessary to modify it in some steps to obtain
Fig. 2. Synthesis of compound 6.

V. Sinisi et al. / Food Chemistry 168 (2015) 332–340
337
the diester as the only coupling product: we have verified that it is
of the utmost importance to have the intermediates 13 and 15 in
the highest purity state before carrying out their coupling, and that
the maximum yield is obtained using Et3N and DMAP as base
instead of pyridine. All the intermediates were isolated as pure
compounds and completely characterised by NMR spectroscopy.
The synthesis of quinides 7, 8 and 9 have been described in
details in our previous work (Sinisi et al., 2014).
3.2. HSA fluorescence and quenching
The interactions of the eight ligands with essentially fatty acid
free HSA were studied monitoring the tryptophan-214 fluores-
cence intensity: the excitation and emission wavelengths were
set to 280 and 340 nm respectively, corresponding to the excita-
tion and emission maxima of the protein measured in the absence
of ligands. Emission spectra in the range 300–400 nm were also
recorded to monitor the eventual environmental changes near
the fluorophore by shifts in the emission maximum wavelength,
while synchronous spectra with a wavelength shift of 60 nm were
also recorded to distinguish between the tryptophan and the tyro-
sine residues (Dockal, Carter, & Rüker, 2000). Fluorescence quench-
ing titrations were carried out by increasing the ligand
concentration while keeping the concentration of protein constant.
Under these conditions only 3,4-O-dimethoxy cinnamic acid 10
showed interferences due to its intrinsic fluorescence emission,
which is greater at lower concentrations (such self-quenching is
most likely due to aggregation at higher concentrations). To avoid
this interference we subtracted the blank fluorescence of this
ligand from the experimental data. We have also verified that sim-
ilar corrections were not necessary in all the other measurements.
HSA fluorescence quenching was observed with all the tested
ligands in the same experimental conditions; in Fig. 3 the emission
spectra (A) and the synchronous spectra (B) of HSA for compound
5, taken as an example, are reported. Fig. 3A shows how the emis-
sion of the Trp residue, excited at 280 nm, changes during the titra-
tion (the fluorescence quenching full data for each ligand are
reported in the Supplementary data): the emission intensity
decreases while the emission maximum evidently moves to lower
wavelengths and the observed blue shifts are at least of 10 nm; this
shift is consistent with a change of the environmental polarity sur-
rounding the Trp residue, resulting from replacement of the sol-
vent in the active site by the less polar molecules of ligand (Liu,
Zheng, Yang, Wang, & Wang, 2009). The use of DMSO for the
ligands solutions does not contribute to the blue shift, as we have
verified by adding only DMSO in the same experimental conditions
of all the titrations.
Fig. 3. (A) Emission spectra of HSA (kex = 280 nm) in the presence of compound 5;
the concentrations of the ligands increase from top to bottom (0, 1, 5, 20, 60, 120,
180, 250 lM), while the protein concentration is fixed to 0.5 lM. (B) Synchronous
spectra of HSA fluorescence emission in the presence of compound 5; the
concentrations of the ligands increase from top to bottom (0, 1, 5, 10, 20, 40, 60,
80, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500
concentration in a and b is fixed to 0.5 M.
lM), while the protein
l
quenching kinetic constant, s0 is the lifetime of the fluorophore
(for the tryptophan fluorescence decay s0 is about 10ꢁ8 s) (Kragh-
Hansen, 1990), KSV is the Stern–Volmer quenching constant and
[Q] is the quencher concentration in mol/l; the protein concentra-
tion was fixed to 0.5
mined by linear regression of a plot of F0/F against [Q] (Fig. 4) in
the ligand concentration range 0–140 M, where all the plots were
lM. The KSV for all the ligands were deter-
l
linear: high concentrations of molecule indeed cause a deviation
from the linearity more or less evident in the different cases, prob-
ably because large amounts of ligand in solution complicate the
quenching mechanism (Min et al., 2004), with the progressive
increase of the dynamic quenching contribution; KSV and Kq (calcu-
lated using the equivalence Kq = KSV/s0) are reported in Table 1,
together with the binding parameters which will be discussed in
the next section.
A further confirmation of this microenvironmental modification
near the fluorophore is given by the effects of the ligand addition
on the synchronous emission spectra, as that shown in Fig. 3B, with
a slight blue shift similar in all the ligands (on average the maxi-
mum shifts from 280 to 278 nm). In both the emission and syn-
chronous spectra the fluorescence intensity decreases down to
zero with increasing ligand concentration and the amount of
quenching measured in the synchronous experiments replicates
those obtained in the corresponding fluorescence titration, sug-
gesting that the quenching phenomenon is related to tryptophan
emission.
3.3. Dissociation constants and binding sites
The number of binding sites and the ligand–protein dissociation
constants were extrapolated from the fluorescence data by a Hill
analysis (Goutelle et al., 2008), using Eq. (2):
To determine whether the observed quenching was due to
binding or collisional phenomena, the emission data were analysed
according to the Stern–Volmer equation (Eq. (1)):
ꢀ
ꢁ
Y
F0
F
log
¼ n log ½Qꢃ ꢁ log KD
ð2Þ
¼ 1 þ Kqs0½Qꢃ ¼ 1 þ KSV½Qꢃ
ð1Þ
1 ꢁ Y
In Eq. (1) F0 and F are the emission intensities before and after the
addition of the quencher, respectively, Kq is the bimolecular
where Y is the fraction of free binding sites (calculated as 1 ꢁ F/F0,
assuming that the ratio F/F0 gives the fraction of occupied binding

338
V. Sinisi et al. / Food Chemistry 168 (2015) 332–340
Fig. 5. Effect of the addition of compound 6 on the fluorescence intensity (the
protein concentration is fixed to 0.5 M, while the ligand concentration range is
0–200 M) (A) and the related Hill plot (B).
l
Fig. 4. Stern–Volmer plots of fluorescence quenching of HSA (kex = 280 nm,
kem = 340 nm) in the presence of compounds 3 (A), 9 (B).
l
an unusual event in protein–ligand interaction has been recently
reported for albumin, in the simultaneous binding of different drug
molecules at this binding site (Yang et al., 2012).
We have evaluated the two binding constants (Table 1) by
regression of the experimental data to Eq. (3) (see the Supplemen-
tary data for its derivation):
sites), [Q] is the quencher concentration in mol/l, n is the number of
binding sites, so it gives the stoichiometry of the interaction, and KD
is the dissociation constant.
All the ligands show linear Hill plots in the concentration range
0–140 lM, with the exception of lactone 6. The parameters n and
ꢁlog KD for all the other ligands (Table 1) were obtained by linear
½Lꢃ
½Lꢃ2
regression of the Hill plot.
D
F0
F
K1
K1K2
¼ x
½Lꢃ þ ð1 ꢁ xÞ
ð3Þ
Compound 6 halves the fluorescence intensity even at concen-
½Lꢃ2
1 þ K½Lꢃ þ K
1 þ K
trations as low as 1
we decided to investigate better the interaction in the ligand con-
centration range 0–200 M, keeping the protein concentration
fixed to 0.5 M. A double, consecutive sigmoidal behaviour can
lM, and to understand more about this case
1
1K2
1
l
where
DF is calculated as (F0 – F), F0 and F are the fluorescence
l
intensities before and after the addition of the quencher, respec-
tively, [L] is the quencher concentration in mol/l, K1 is the dissocia-
tion constant for the protein–ligand complex with stoichiometry
1:1, K2 the dissociation constant for that with stoichiometry 1:2, x
be clearly seen (Fig. 5A), while in the Hill plot the slope suddenly
shifts from 1 to 2 (Fig. 5B). This may be due to the binding of
two molecules of 6 inside the same albumin binding site. Such
Table 1
Quenching constants according to Stern–Volmer analysis: Stern–Volmer quenching constant (KSV) and bimolecular quenching kinetic constant (Kq).
Quenching constants
Binding parameters
SD
Ligand
KSV SD (104 L molꢁ1
)
Kq SD (1012 L molꢁ1 sꢁ1
)
R
n
KD SD (
l
mol Lꢁ1
)
R
Acid 2
Acid 3
Acid 5
Acid 10
Lactone 6
8.57 0.50
4.96 0.26
5.29 0.47
4.23 0.17
43.84 4.12
8.57 0.50
4.96 0.26
5.29 0.47
4.23 0.17
43.84 4.12
0.98
0.99
0.97
0.99
0.96
1.20 0.03
1.04 0.04
1.12 0.06
0.99 0.03
/
2.32 0.06
21.2 0.8
9.15 0.48
31.1 1.1
K1 0.967
0.99
0.99
0.99
0.99
K2 20.8
Lactone 7
Lactone 8
Lactone 9
13.75 1.11
9.81 0.18
6.86 0.17
13.75 1.11
9.81 0.18
6.86 0.17
0.97
0.99
0.99
1.08 0.06
1.08 0.02
1.02 0.02
12.1 0.8
4.31 0.06
13.3 0.2
0.96
0.99
0.99

V. Sinisi et al. / Food Chemistry 168 (2015) 332–340
339
is a coefficient that allows to take into account the different
quenching efficiency in the two possible complexes.
All the tested ligands cause the HSA fluorescence quenching and
both the emission and the synchronous spectra suggest that the
molecules enter in the subdomain IIA and interact with Trp-214.
Thebimolecularquenchingkineticconstants (Kq), showedin Table 1,
are at least 3–4 orders of magnitude higher than the higher value for
diffusion limited collisional quenching (2.0 ꢂ 1010 L molꢁ1 sꢁ1
)
(Eftink, 1991), thus the static quenching originating from the associ-
ation of the fluorophore and quenchers in a bimolecular complex is
the main contribution to the fluorescence quenching mechanism
within the 0–140 lM ligand concentration range. Higher ligand con-
centrations complicate the quenching mechanism because the
dynamic collision contribution becomes more significant.
Having thus established that the quenching data can be safely
used for measuring the binding parameters, we can evaluate the
results of the Hill analysis, showed in Table 1. The slope (n) in
the Hill plots is near 1 for all the ligands with the exception of com-
pound 6, which means a 1:1 interaction stoichiometry between
ligand and protein. In the case of compound 6, the trend of the
fluorescence emission intensity during the titration and the sudden
slope shift from 1 to 2 in the Hill plot suggest a 1:1 stoichiometry at
low ligand concentration, switching to a 1:2 ratio at higher concen-
trations, revealing the binding of two molecules of 6 inside the
same albumin binding site, as both the molecules of 6 must be in
proximity of Trp 214 in order to obtain fluorescence quenching
upon binding. All the calculated KD are in the micromolar range,
showing a remarkably high affinity of these molecules for the pro-
Fig. 6. Displacement of warfarin by compound 6: the fluorescence emission of HSA-
bound warfarin (excitation 320 nm/emission 380 nm) was measured on a 10
warfarin, 2.5 M HSA solution. The emission decays upon addition of lactone 6 in
the 400 nM–72 M range.
lM
l
l
4. Conclusions
In summary, we have demonstrated that HSA is able to bind all
the considered ligands, with the formation of a bimolecular com-
plex within the Sudlow site I. The dissociation constants show a
very high affinity of the protein towards this family of compounds,
moreover minimal changes in the chemical structure lead to signif-
icant changes in binding. A reference drug warfarin is fully dis-
placed from albumin by low concentrations of lactone 6: this
result suggest that the dietary assumption of polyphenols from
coffee and other sources could affect the pharmacokinetic profile
of drugs binding to serum albumin.
tein. The binding constants of 2, 3 and 5 (2.3, 21.2 and 9.2
respectively) are comparable in magnitude and trend to the pub-
lished ones (6, 40 and 25 M) (Hu et al., 2012; Kang et al., 2004;
lM
l
Min et al., 2004); among the carboxylic acids, compound 10 shows
the lowest affinity, probably due to the replacement of both pheno-
lic hydroxyl groups with methoxyl ones: this leaves only the acid
moiety capable to form hydrogen bonds, that can strengthen the
ligand–protein interaction (Bartolomè, Estrella,
& Hernández,
Acknowledgments
2000), with the polypeptide chain within the binding site. For lac-
tones 7, 8 and 9, the second has a better affinity and this may be
explained as a consequence of the free hydroxyl group in position
1 on the quinide core and of the presence of two aromatic rings
instead of one as in lactone 7, enhancing the ability to establish
hydrophobic interactions, and of its lower dimension compared
to lactone 9, that makes easier the entrance of the molecule in
the active site.
We thank Dr. Filomena Guida and Professor Alessandro Tossi
(Department of Life Science, University of Trieste, Italy) for the pos-
sibility to use their HPLC and for their precious help during the
analysis. The University of Trieste is gratefully acknowledged for
financial support.
We want to highlight the very interesting case of lactone 6: HSA
seems capable to host two molecules of it in the same binding site,
and the K1 is even in the hundred nanomolar range. To our knowl-
edge, this is the lowest affinity ever reported for albumin, and by
far more favourable than binding of most drugs to this protein.
We have evaluated also the ability of lactone 6 to displace a drug
from the binding site of albumin. We have chosen warfarin as this
drug is the reference ligand of Sudlow site I; moreover, the intrinsic
fluorescence of warfarin is strongly enhanced by the interactions
with albumin, and decreases upon competition with other drugs
for the protein. This phenomenon has been exploited to set up a
well established method to study drug association to HSA
(Sudlow, Birkett, & Wade, 1975b). The experiment was carried on
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2014.
07.080.
References
Anna, S. (2002). Interaction of drugs with bovine and human serum albumin. Journal
of Molecular Structure, 614, 227–232.
Bartolomè, B., Estrella, I., & Hernández, M. T. (2000). Interaction of low molecular
weight phenolics with proteins (BSA). Journal of Food Science, 65, 617–621.
Blumberg, S., Frank, O., & Hofmann, T. (2010). Quantitative studies on the influence
of the bean roasting parameters and hot water percolation on the
concentrations of bitter compounds in coffee brew. Journal of Agricultural and
Food Chemistry, 58, 3720–3728.
Clifford, M. N. (1985). Chlorogenic acids. In R. J. Clarke & R. Macrae (Eds.). Coffee,
Vol. 1, Chemistry (pp. 153–202). London: Elsevier Applied Science Publ..
Clifford, M. N. (2000). Chlorogenic acids and other cinnamates: Nature, occurrence,
dietary burden, absorption, and metabolism. Journal of the Science of Food and
Agriculture, 80, 1033–1043.
a 10
2.5
l
M solution of warfarin in phosphate buffer, containing
l
M HSA. By adding increasing concentrations of lactone 6,
the displacement of warfarin from the albumin binding site can
be clearly seen from the decrease of its fluorescence emission
(Fig. 6). 50% of the initially bound drug is displaced at a 12
centration of lactone 6, while warfarin is fully squeezed out at
70 M lactone. Data regression to a hyperbolic binding isothermal
gives an apparent dissociation constant for lactone 6 of 12.4 M at
10 M warfarin.
lM con-
Clifford, M. N., Knight, S., Surucu, B., & Kuhnert, N. (2006). Characterization by LC–
MSn of four new classes of chologenic acids in green coffee beans:
l
Dimethoxycinnamoylquinic
acids,
diferuloylquinic
acids,
caffeoyl-
l
dimethoxycinnamoylquinic acids, and feruloyl-dimethoxycinnamoylquinic
acids. Journal of Agricultural and Food Chemistry, 54, 1957–1969.
l

340
V. Sinisi et al. / Food Chemistry 168 (2015) 332–340
de Paulis, T., Schmidt, D. E., Bruchey, A. K., Kirby, M. T., McDonald, M. P., Commers,
P., et al. (2002). Dicinnamoylquinides in roasted coffee inhibit the human
adenosine transporter. European Journal of Pharmacology, 442, 215–223.
Dockal, M., Carter, D. C., & Rüker, F. (2000). Conformational transitions of the three
recombinant domains of human serum albumin depending on pH. Journal of
Biological Chemistry, 275, 3042–3050.
Eftink, M. R. (1991). Fluorescence quenching reactions: Probing biological macro-
molecular structures. In T. G. Dewey (Ed.). Biophysical and biochemical aspects of
fluorescence spectroscopy (pp. 105–133). New York: Plenum.
Farah, A., de Paulis, T., Trugo, L. C., & Martin, P. R. (2005). Effect of roasting on the
formation of chlorogenic acid lactones in coffee. Journal of Agricultural and Food
Chemistry, 53, 1505–1513.
Farah, A., Monteiro, M., Donangelo, C. M., & Lafay, S. (2008). Chlorogenic acids from
green coffee extract are highly bioavailable in humans. Journal of Nutrition, 138,
2309–2315.
Monteiro, M., Farah, A., Perrone, D., Trugo, L. C., & Donangelo, C. (2007). Chlorogenic
acid compounds from coffee are differentially absorbed and metabolized in
humans. Journal of Nutrition, 137, 2196–2201.
Nagy, K., Redeuil, K., Williamson, G., Rezzi, S., Dionisi, F., Longet, K., et al. (2011).
First identification of dimethoxycinnamic acids in human plasma after coffee
intake by liquid chromatography–mass spectrometry. Journal of
Chromatography A, 1218, 491–497.
Nardini, M., Cirillo, E., Natella, F., & Scaccini, C. (2002). Absorption of phenolic acids
in humans after coffee consumption. Journal of Agricultural and Food Chemistry,
50, 5735–5741.
Navarini, L., Colomban, S., Lonzarich, V., Rivetti, D., Brollo, G., & Suggi-Liverani, F.
(2008). Hyper Espresso Coffee Extraction: Adding Physics to Chemistry. 22nd
International Studies on Coffee Science, Proceedings, Campinas (SP, Brazil):296–
309.
Olthof, M. R., Hollman, P. C. H., & Katan, M. B. (2001). Chlorogenic acid and caffeic
acid are absorbed in humans. Journal of Nutrition, 131, 66–71.
Pal, S., & Saha, C. (2014). A review on structure-affinity relationship of dietary
flavonoids with serum albumins. Journal of Biomolecular Structure and Dynamics,
32, 1132–1147.
Farrell, T. L., Gomez-Juaristi, M., Poquet, L., Redeuil, K., Nagy, K., Renouf, M., et al.
(2012). Absorption of dimethoxycinnamic acid derivatives in vitro and
pharmacokinetic profile in human plasma following coffee consumption.
Molecular Nutrition & Food Research, 56, 1413–1423.
Gloess, A. N., Schönbächler, B., Klopprogge, B., D’Ambrosio, L., Chatelain, K.,
Bongartz, A., et al. (2013). Comparison of nine common coffee extraction
methods: Instrumental and sensory analysis. European Food Research and
Technology, 236, 607–627.
Ghuman, J., Zunszain, P. A., Petipas, I., Bhattacharya, A. A., Otagiri, M., & Curry, S.
(2005). Structural basis of the drug-binding specificity of human serum
albumin. Journal of Molecular Biology, 353, 38–52.
Pantusa, M., Sportelli, L., & Bartucci, R. (2012). Influence of stearic acids on
resveratrol–HSA interaction. European Biophysics Journal, 41, 969–977.
Renouf, M., Guy, P. A., Marmet, C., Fraerinf, A. L., Longet, K., Moulin, J., et al. (2010).
Measurement of caffeic and ferulic acid equivalents in plasma after coffee
consumption: Small intestine and colon are key sites for coffee metabolism.
Molecular Nutrition & Food Research, 54, 760–766.
Scholz, B. M.,
& Maier, H. G. (1990). Isomers of quinic acid and quinide in
Goutelle, S., Maurin, M., Rougier, F., Barbaut, X., Bourguignon, L., Ducher, M., et al.
roasted coffee. Zeitschrift für Lebensmittel-Untersuchung und -Forschung, 190,
132–134.
(2008). The Hill equation:
a review of its capabilities in pharmacological
modeling. Fundamental & Clinical Pharmacology, 22, 633–648.
He, X. M., & Carter, D. C. (1992). Atomic structure and chemistry of human serum
albumin. Nature, 358, 209–215.
Schrader, K., Kiehne, A., Engelhardt, U. H., & Maier, H. G. (1996). Determination of
chlorogenic acids with lactones in roasted coffee. Journal of the Science of Food
and Agriculture, 71, 392–398.
Hu, Y. J., Chen, C. H., Zhou, S., Bai, A. M., & Ou-Yang, Y. (2012). The specific binding of
chlorogenic acid to human serum albumin. Molecular Biology Reports, 39,
2781–2787.
Skrt, M., Benedik, E., Podlipnik, C., & Ulrih, N. P. (2012). Interactions of different
polyphenols with bovine serum albumin using fluorescence quenching and
molecular docking. Food Chemistry, 135, 2418–2424.
IUPAC Commission on the Nomenclature of Organic Chemistry (CNOC) and IUPAC-
IUB Commission on Biochemical Nomenclature (CBN), (1976). Biochemical
Journal, 153, 23–31.
Kang, J., Yuan, L., Meng-Xia, X., Song, L., Min, J., & Ying-Dian, W. (2004). Interactions
of human serum albumin with chlorogenic acid and ferulic acid. Biochimica et
Biophysica Acta, 1674, 205–214.
Kragh-Hansen, U. (1990). Structure and ligand binding properties of human serum
albumin. Danish Medical Bulletin, 37, 57–84.
Liu, Z., Zheng, X., Yang, X., Wang, E., & Wang, J. (2009). Affinity and specificity of
levamlodipine–human serum albumin interactions: Insights its carrier
function. Biophysical Journal, 96, 3917–3925.
Sinisi, V., Boronová, K., Colomban, S., Navarini, L., Berti, F., & Forzato, C. (2014).
Synthesis of mono, di and tri-3,4-dimethoxycinnamoyl-1,5-c-quinides.
European Journal of Organic Chemistry, 1321–1326. http://dx.doi.org/10.1002/
ejoc.201301657.
Sudlow, G., Birkett, D. J., & Wade, D. N. (1975a). The characterization of two specific
binding sites on the human serum albumin. Molecular Pharmacology, 11,
824–832.
Sudlow, G., Birkett, D. J., & Wade, D. N. (1975b). Spectroscopic techniques in the
study of protein binding. A fluorescence technique for the evaluation of the
albumin binding and displacement of warfarin and warfarin–alcohol. Clinical
and Experimental Pharmacology and Physiology, 2, 129–140.
Varshney, A., Sen, P., Ahmad, E., Rehan, M., Subbaro, N., & Khan, R. H. (2010). Ligand
binding strategies to human serum albumin: How can the cargo be utilized?
Chirality, 22, 77–87.
Luisi, I., Pavan, S., Fontanive, G., Tossi, A., Benedetti, F., Sgarra, R., et al. (2013). An
albumin-derived peptide scaffold capable of binding and catalysis. PLoS One, 8,
e56469.
Min, J., Meng-Xia, X., Dong, Z., Yuan, L., Xiao-Yu, L., & Xing, C. (2004). Spectroscopic
studies on the interaction of cinnamic acid and its hydroxyl derivatives with
human serum albumin. Journal of Molecular Structure, 692, 71–80.
Yang, F., Yue, J., Ma, L., Ma, Z., Li, M., Wu, X., et al. (2012). Interactive associations of
drug–drug and drug–drug–drug with IIA subdomain of human serum albumin.
Molecular Pharmaceutics, 9, 3259–3265.