Hydroxycinnamoyl amino acids conjugates: A chiral pool to distinguish commercially exploited Coffea spp.

Article abstract of DOI:10.3390/molecules25071704

The synthesis of five hydroxycinnamoyl amides (HCAs) was accomplished and their identification and quantification in the green coffee bean samples of Coffea arabica, Coffea canephora, and Coffea liberica was performed. The HCAs p-coumaroyl-N-tyrosine 1b,

Full text of DOI:10.3390/molecules25071704

molecules
Article
Hydroxycinnamoyl Amino Acids Conjugates: A
Chiral Pool to Distinguish Commercially Exploited
Coffea spp.
Federico Berti , Luciano Navarini , Silvia Colomban and Cristina Forzato 1,*
1
2
2
1
Dipartimento di Scienze Chimiche e Farmaceutiche, Università degli Studi di Trieste, via L. Giorgieri 1,
3
4127 Trieste, Italy; fberti@units.it
2
Illycaffè S.p.A., via Flavia 110, 34147 Trieste, Italy; Luciano.Navarini@illy.com (L.N.);
Silvia.Colomban@illy.com (S.C.)
*
Correspondence: cforzato@units.it
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editor: Fernanda Borges
Received: 15 March 2020; Accepted: 3 April 2020; Published: 8 April 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: The synthesis of five hydroxycinnamoyl amides (HCAs) was accomplished and their
identification and quantification in the green coffee bean samples of Coffea arabica, Coffea canephora,
and Coffea liberica was performed. The HCAs p-coumaroyl-N-tyrosine 1b, caffeoyl-N-phenylalanine
2b, caffeoyl-N-tyrosine 3b, and p-coumaroyl-N-tryptophan 4b were characteristic of the C. canephora
species while caffeoyl-N-tryptophan 5b was present in both C. canephora and C. arabica, but with
higher content in C. canephora. The HCAs presence was also analyzed in C. liberica for the first time
and none of the targeted compounds was found, indicating that this species is very similar to C.
arabica species. Between C. canephora samples from various origins, significant differences were
observed regarding the presence of all the HCAs, with C. canephora from Tanzania containing all
five derivatives.
Keywords: hydroxycinnamoyl amides; coffee beans; LC-MS
1
. Introduction
Hydroxycinnamic acid conjugates are secondary metabolites widely present in the plant kingdom,
such as endive (Cichorium endivia) [
Hypoxis hemerocallidea) [ ], lady fern (Athyrium filix-femina) [
and cocoa (Theobroma cacao) [ ]. Hydroxycinnamic acids can be esterified with quinic acid forming
1
], purple coneflower (Echinacea purpurea) [
2
], African star grass
(
3
4
], Robusta coffee (Coffea canephora) [
5],
6
the class of chlorogenic acids, esterified with monosaccharides forming the cinnamoyl glycosides, or
can give origin to amides with different amino acids, named hydroxycinnamoyl amides (HCAs) or
N-phenylpropenoyl-amino acids. The hydroxycinnamoyl acids are usually conjugated with the amino
acids phenylalanine, tyrosine, tryptophan, or aspartic acid, and although their role in the plant is not
known, they exhibit biological activity, such as anti-neuroinflammatory activity [
7], hepatoprotective
activity [ ], HIV-1 integrase inhibitors [ ], and antidiabetic effects [10]. Moreover, from a sensory point
8
9
of view, HCAs have been indicated as possible contributors to the sensory properties of some food [11].
More than 124 species are known of the genus Coffea and the two most important ones are Coffea
arabica L., simply known as Arabica, and Coffea canephora Pierre ex Froehner, simply known as Robusta,
which correspond to 57% and 43% of the international coffee trade, respectively, according to the
International Coffee Organization (ICO, 2019). Coffea liberica Bull ex Hiern, contributing less than one
per cent of the marketed coffee, is the third commercially exploited coffee species, well known for its
larger cherries, when compared with those of C. arabica or C. canephora. C. liberica is less cultivated, due
to its lower caffeine content (1.8% dry matter basis), and to its sensitivity to Fusarium xyloriodes, despite
Molecules 2020, 25, 1704; doi:10.3390/molecules25071704
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1704
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the fact that this species was of great economic importance during the 1930–1950 period [12]. Due to
its low economic importance, C. liberica is also the less studied of the three considered species.
The HCAs can contribute to distinguish the two mainly commercially exploited coffee species,
Arabica and Robusta, since in the literature it is shown that HCAs are manly present in the Robusta
species [5,13–18]. Moreover, their identification could also contribute in differentiating the two
species, depending on their geographical origin. To our knowledge, HCAs presence in C. liberica
have not yet been evaluated and their analysis could contribute in the chemical characterization of
this species. Caffeoyl-N-tryptophan was first isolated in 1987 by Takai et al. [19] from coffee beans
of C. canephora from Java, while caffeoyl-N-tyrosine was first isolated in 1989 by Kellard et al. [20],
from C. canephora from Angola. Successively, caffeoyl-N-phenylalanine, p-coumaroyl-N-tyrosine, and
p-coumaroyl-N-tryptophan were identified in green coffee beans, analyzing both Robusta and Arabica
coffee beans by LC-MS [
was determined by UV detection using the available standard 5-caffeoylquinic acid (5-CQA) and
assuming a similar molar absorption coefficient for 5-CQA and each of the cinnamoyl amides [ ].
5,14]. Since these HCAs are not commercially available, their quantification
5
Usually, chlorogenic acids are the major phenolic compounds present in coffee (30.0–44.4 g/kg) while
caffeoyl-N-tyrosine and p-coumaroyl-N-tyrosine were estimated in lower amount (1.3–4.8 g/kg and
1
.9–3.7 g/kg, respectively) [5].
HCAs are normally present in plants as enantiomerically enriched compounds, as found, for
0
0
example, by Hofmann in cocoa beans [11] who isolated N-[3 ,4 -dihydroxy-(E)-cinnamoyl]-L-aspartic
acid and verified the absolute configuration of the amino acid by means of polarimetry. Murata et al. [17]
isolated from coffee beans p-coumaroyl-N-tryptophan and demonstrated that L-tryptophan was part
of the HCA.
In the present work, 23 samples of green coffee beans (10 of C. canephora, 10 of C.
arabica, and 3 of C. liberica) of different geographical origin were analyzed by LC-MS using
synthetized p-coumaroyl-N-tyrosine 1b, caffeoyl-N-phenylalanine 2b, caffeoyl-N-tyrosine 3b
,
p-coumaroyl-N-tryptophan 4b, and caffeoyl-N-tryptophan 5b. The availability of authentic standards is
useful to unequivocally determine their presence in the examined green coffee samples and contribute
in the identification of a chiral pool to distinguish the two commercially relevant species.
2
. Results and Discussion
To identify and quantify the HCAs in green coffee beans, the amides 1b–5b were synthetized,
which are the compounds of interest in differentiating the two coffee species (Figure 1). Different
syntheses of the compounds are reported in the literature [ 11], but the best results have been
obtained with the procedure of Wang et al. [ ], which performed the condensation reaction between the
6,9,
7
hydroxycinnamic acid and the methyl ester amino acid in pyridine with DCC as the condensing agent.
The hydroxycinnamoyl amides methyl esters were obtained from 41% to 78% yield after purification
by flash chromatography. These results are in line with the results reported in literature using the
same synthetic procedure [7], although for other HCAs. The deprotection reaction with LiOH was a
crucial step, and each deprotection was performed at room temperature and was monitored by thin
layer chromatography (TLC) in order to determine the end of the reaction, while in the literature, it
◦
was performed at 40 C overnight [
7]. In fact, for longer times, we observed a rapid degradation
of the products. In this case, better yields were obtained with respect to the literature ranging from
6% to 94%, depending on the purification procedure. For compounds 1b 4b a recrystallization from
5
–
petroleum ether was performed, while for 5b it was necessary to purify it by flash chromatography at
the expense of a lower yield (56%). All products were amorphous solids. The complete synthesis is
1
reported in Scheme 1. All compounds 1a
–
5a and 1b
–
5b were fully characterized by 1D and 2D H and
13
C NMR using acetone-d as the solvent for all the products, while in the literature, different solvents
6
were used (Figure 1). Moreover, chiroptical properties, as optical rotatory power and circular dichroism,
were determined for 1b 5b. Circular Dichroism (CD) spectra are reported in Figure 2. Although the
isolation of the pure compounds from coffee beans was not the purpose of this work, it was considered
–

Molecules 2020, 25, 1704
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important to analyze the chiroptical properties of the synthetized compounds to evaluate, in the future,
the correspondence of the chirality of the products with the one isolated from plants.
Figure 1. Chemical structures of 1a–5a and 1b–5b.
Scheme 1. Synthesis of the hydroxycinnamoyl amides.
1
3
0
1
b
1
2b
3
4
5
b
b
b


5
0
-
4
2
00
250
300
350

(nm)
Figure 2. CD spectra of hydroxycinnamoyl amides 1b–5b.

Molecules 2020, 25, 1704
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The synthetized HCAs where analyzed by LC-MS, both in positive and negative mode, to
determine their retention time and obtain their mass spectra, in order to unequivocally determine their
presence in green coffee bean extract (Table 1).
Table 1. HPLC retention times, MS analysis, and specific rotation.
Dimer + Na
[
M + H] (m/z)
Compound
tR (min)
(m/z)
Exp.
Specific Rotation
Exp.
2
0
[
α] −39.9 (c 0.1 MeOH) (lit.
D
2
0
p-coumaroyl-N-tyrosine 1b
26.9
328.1
677.1 (positive mode)
[α] −26.9 (MeOH) [11] and
D
2
D
0
[
α]
−
17.7 (c 0.1 MeOH) [
7])
2
0
caffeoyl-N-phenylalanine 2b
caffeoyl-N-tyrosine 3b
30.6
24.6
328.0
344.1
677.2 (positive mode)
709.2 (positive mode)
[α] −48.8 (c 0.1 MeOH)
D
2
5
[
α]
−
41.7 (c 0.1, MeOH) (lit.
D
2
0
[
α] −35.6 [11])
D
2
D
2
D
5
p-coumaroyl-N-tryptophan 4b
caffeoyl-N-tryptophan 5b
33.0
31.1
351.0
367.1
723.2 (positive mode)
731.1 (negative mode)
[α] −27.8 (c 0.1, MeOH)
[α] −33.2 (c 1.0, MeOH)
5
As can be seen in Figure 3, all mass spectra of HCAs 1b–5b present, besides the quasi molecular
ion, an intense peak at higher m/z ratio, which can be attributed to a dimer that is formed during
ionization. The structure of the dimer formed from 1b is reported in Figure 4 and the assignment of
this structure is based on what was found by Yuan et al. in 2014 [8], who isolated these compounds
from Abrus mollis Hance. Analogues dimers can be expected from the other cinnamoyl amides as
confirmed by their m/z value.
Figure 3. Cont.

Molecules 2020, 25, 1704
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Figure 3. Mass spectra of cinnamoyl amides 1b–5b.
Figure 4. Structure of 1b dimer.
Although only a few works reported the presence of HCAs in coffee beans, it was evidenced in
the literature that these compounds were characteristic of the Robusta species [ 13 18]. In the present
work, compounds 1b 5b have been quantified in green coffee beans of C. arabica and C. canephora of
5,
–
–
different geographical origin, and for the first time of C. liberica, using an aqueous extraction procedure,
as was already used in our previous works for the quantification of chlorogenic acids [21,22].

Molecules 2020, 25, 1704
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Quantification of the HCAs 1b and 4b was reported to p-coumaric acid, and quantification of
HCAs 2b 3b, and 5b was reported to caffeic acid, as suggested by Alonso-Salces [14]. Results are
,
summarized in Table 2 and are given on dry weight basis (DW). A 10% moisture content in green
coffee beans was assumed, as it has been done before by others authors [23].
Table 2. Concentrations of cinnamoyl amides in green coffee beans (mg/kg of DW, standard deviation
in brackets).
Sample
Species
1b
2b
3b
4b
5b
Total
1
17
117
(2)
1
2
3
4
5
C. arabica Nicaragua
C. arabica Peru
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
(2)
<LOD
-
5
1
51
(1)
C. arabica Rwanda
C. arabica El Salvador
C. arabica India
(1)
<LOD
-
1
(
25
3)
125
(3)
3
7
37
(1)
6
7
8
9
C. arabica Brazil
C. arabica Guatemala
<LOD
<LOD
<LOD
<LOD
<LOD
n.q. a
<LOD
<LOD
<LOD
<LOD
<LOD
n.q. b
n.q. b
n.q. b
<LOD
n.q. b
n.q. b
n.q. b
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
(
(
(
(
(
1)
97
97
(2)
2)
59
59
(1)
C. arabica – lot 1 Ethiopia
C. arabica – lot 2 Ethiopia
C. arabica Burundi
1)
75
75
(2)
2)
40
40
(1)
1
1
1
1
1
1
1
1
1
1
2
0
1
2
3
4
5
6
7
8
9
0
1)
1
(
19
4)
878
(26)
997
(30)
C. canephora Cameroon
C. canephora Ivory Coast
C. canephora Tanzania
C. canephora Indonesia
C. canephora – lot 1 India
C. canephora – lot 2 India
C. canephora – lot 3 India
C. canephora Brazil var. Conilon
C. canephora Uganda
1
(
16
4)
712
(21)
828
(25)
<LOD
n.q. a
3
(
48
5)
319
(3)
1042
(14)
1709
(22)
1
(
13
2)
590
(12)
703
(14)
<LOD
<LOD
<LOD
<LOD
n.q. a
<LOD
<LOD
<LOD
<LOD
1
(
03
3)
790
(24)
893
(27)
2
(
12
6)
1205
(48)
1417
(54)
1
(
28
4)
780
(23)
908
(27)
47
182
(4)
470
(9)
699
(14)
<LOD
(1)
1
(
40
3)
195
(5)
717
(21)
1052
(29)
n.q. a
n.q. b
n.q. b
1
(
36
3)
702
(20)
838
(23)
C. canephora Vietnam
<LOD
<LOD
2
2
2
1
2
3
C. liberica – lot 1
C. liberica – lot 2
C. liberica – lot 3
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
<LOD
-
-
-
n.q.: not quantifiable: a overlapped with unknown compound with m/z 557, 499, and 279; b overlapped with
unknown compound with m/z 513 and 186; DW: dry weight; LOD: limit of detection.
As can be seen from Table 2, the cinnamoyl amides 1b–4b were present only in the Robusta
species, although their presence depended on the geographical origin. Compounds 1b and 3b are
not present in Robusta green coffee beans from India, Vietnam, and Ivory Coast, while only 3b is not
present in Robusta from Cameroon. Although 1b and 2b have been detected in Robusta samples, they
could not be quantified, since, in the mass spectra, two other mass peaks were present, indicating
that the peak corresponding to the cinnamoyl amides 1b and 2b was overlapped with the one of two

Molecules 2020, 25, 1704
7 of 14
unknown compounds with mass spectra reported in Figure 5 (positive mode). For this reason, their
quantification is not reported in Table 2, and is indicated as not quantifiable (n.q.).
Figure 5. Mass spectra of overlapped peaks of 1b (A) and 2b (B) with unknown compounds.
LC-MS analysis of C. liberica did not show the presence of any cinnamoyl amide, indicating that
it is more similar to the Arabica species than to Robusta, but more samples of Liberica or different
extraction methods are needed to verify that HCAs are completely absent in this species.
In Figure 6, the LC chromatograms referred to the Arabica sample from India, Robusta sample
from Tanzania, and Liberica sample—lot 1, are reported as an example, which clearly show the
differences between the three species regarding the content of HCAs.
In order to compare the results obtained in the present work with the ones reported in the
literature, the extraction method used must be taken into consideration. All methods previously
reported, regarding the extraction of HCAs, use an organic solvent, such as methanol [
5,17], ethanol [16],
or acetone [18], and sometimes, acidic conditions are used [13 14]. Only Rezende et al. [15] and
,
Clifford et al. [24] used hot water for the extraction of HCAs and, in particular, Clifford analyzed the
presence of 1b and 3b, also in coffee brew. Although the usage of an organic solvent can improve
the extraction process, it was interesting to perform the extraction with only hot water to mimic the
coffee preparation. The results obtained with this method will be compared in the future with the
results obtained in the analysis of coffee brew, to highlight the presence of HCAs also in the final
beverage. Nevertheless, results obtained in the present work can be related to the ones reported in
the literature by Alonso-Salces [14] and Babova et al. [16] for the two varieties, Arabica and Robusta,
although the extraction procedure was different. Moreover, 5-caffeoylquinic acid (5-CQA) is used
as standard, assuming that there is a similar molar absorption coefficient for 5-CQA and each of the
HCAs [5]; this aspect has to also be taken into consideration when comparing the results with literature
data. The results obtained in the present work are in line with the results found by Alonso-Salces [14
]
since the same compounds for calibration curves were used. The 144 mg/kg of 4b and 789 of 5b as
a mean value for Robusta samples were obtained, while Alonso-Salces found 225 mg/kg and 1557,
respectively. These lower values are probably due to the extraction method, since Alonso-Salces used
methanol/water/acetic acid 30:67.5:2.5. The results obtained are also in line with the results found by
Babova et al. [16], who obtained 110 mg/kg for 4b and 400 mg/kg for 5b, although, using ethanol:water
5
0:50 as the extraction solvent.

Molecules 2020, 25, 1704
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Figure 6. Cont.

Molecules 2020, 25, 1704
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Figure 6. Chromatograms of samples from Arabica India (A), Robusta Tanzania (B), and Liberica
lot 1 (C).
3
. Materials and Methods
3
.1. Chemicals
L-tyrosine methyl ester, L-phenylalanine methyl ester, L-tryptophan methyl ester, caffeic acid,
0
p-coumaric acid, N,N -Dicyclohexylcarbodiimide (DCC), LiOH, silica gel were from Sigma-Aldrich (St.
Louis, MO, USA). All solvents were used without further purification.
3
.2. Instrumentation
NMR spectra were recorded on a Varian 400 (Agilent Technologies, Santa Clara, CA, USA)
spectrometer using acetone-d as the solvent. Coupling constants are given in Hz. LC-MS analyses
6
were run on an Agilent Infinity 1200 (Agilent Tech.), with autosampler and a diode array detector set
at 320 nm, coupled with an Agilent Technologies 6120 Quadrupole (Agilent Tech.) equipped with an
electrospray ionization source (ESI) operating in both positive and negative mode. The column was a
Kinetex C18 (150
×
2.1 mm, 100 Å, 5 µm) (Phenomenex, Torrance, CA, USA), and the eluent was a
mixture of water + 0.1% formic acid (FA) (v/v) (solvent A) and acetonitrile + 0.1% FA (v/v) (solvent B)
with the gradient reported in Table 3. The flow was 0.25 mL/min.
Table 3. HPLC gradient method.
%
A (H O + 0.1% FA)
% B (CH CN + 0.1% FA)
Time (min)
2
3
9
8
6
6
9
7
5
0
0
7
3
0
15
40
40
3
10
30
40
45

Molecules 2020, 25, 1704
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Optical rotations were determined on a Jasco P2000 (Jasco, Tokyo, Japan) polarimeter at the
wavelength of sodium D ( = 589 nm) using a quartz cell of 1 dm length. Circular dichroism spectra
were recorded on a Jasco J-710 spectropolarimeter using a quartz cell of 0.1 cm optical path.
λ
3
.3. Samples
A total of 23 samples of green coffee beans from different geographical origins and commercial lots,
0 of C. arabica, 10 of C. canephora, and 2 of C. liberica were analyzed. A wild C. liberica (named lot 3) was
1
also used. In particular, all C. arabica samples were “wet processed” with zero primary and secondary
defects, and the beverages prepared by using the samples were not affected by off-flavors (clean cup).
C. canephora from India, all “in parchment”, were kindly supplied by Allanasons Private Limited
(
(
India), C. canephora var. Conilon “dry processed” from Brazil was kindly supplied by Fazendas Dutra
Brazil); all of the other C. canephora samples were kindly provided by Sandalj Trading Company S.p.A.,
Trieste (Italy). Two different lots of commercial C. liberica “dry processed” from India were kindly
provided by Allanasons Private Limited (India), whereas C. liberica, named lot 3 from the Costa Rica
CATIE germplasm (code T.03476) “in parchment”, was kindly provided by Dr. William Solano S
Agriculture, Livestock and Agroforestry Program, CATIE, Turrialba, Costa Rica).
ánchez
(
3
.4. General Procedure for the Preparation of the Hydroxycinnamoyl Amides Esters 1a–5a
The syntheses of all hydroxycinnamoyl amides were performed according to a literature
procedure [7].
In a three-necked round bottom flask, 1.58 mmol of the amino acid were dissolved in 12.8 mL of
anhydrous pyridine. 2.35 mmol of the hydroxycinnamic acid, and 2.28 mmol of DCC were subsequently
added. The reaction mixture was stirred under argon atmosphere for 72 h. At the end of the reaction,
the crude was filtered, and the liquid phase was acidified with H SO 3 M till pH = 2 and washed
2
4
with a NaHCO 5% solution. After extraction with ethyl acetate, the organic phase was dried over
3
anhydrous Na SO and finally evaporated in vacuum. The crude product was purified by silica gel
2
4
column chromatography using mixtures of petroleum ether/ethyl acetate as eluent.
N-[4 -Hydroxy-(E)-cinnamoyl]-L-tyrosine methyl ester 1a: Yield 67%. 1H NMR (400 MHz,
0
0
Acetone-d )
δ
8.84 (1H, s, OH), 8.27 (1H, s, OH), 7.49 (1H, d, J 15.7, H-7 ), 7. 47 (1H, NH), 7.43 (2H, d, J
6
0
0
0
0
=
8.6, H-2 , H-6 ), 7.06 (2H, d, J 8.5, H-2, H-6), 6.86 (2H, d, J 8.6, H-3 , H-5 ), 6.76 (2H, d, J = 8.5, H-3,
0
H-5), 6.62 (1H, d, J 15.7, H-8 ), 4.81 (1H, dt, J 7.7, 5.8, H-8), 3.66 (s, 3H, CH ), 3.08 (1H, dd, J 13.9, 5.8,
3
13
0
δ 172.06 (C-9), 165.8 (C-9 ), 159.0
H-7), 2.98 (1H, dd, J 13.9, 7.7, H-7); C NMR (100 MHz, Acetone-d )
6
0 0 0 0 0 0
C-4 ), 156.3 (C-4), 140.4 (C-7 ), 130.2 (C-2, C-6), 129.5 (C-2 , C-6 ), 127.6 (C-1), 126.7 (C-1 ), 117.9 (C-8 ),
(
1
0 0
15.7 (C-3 , C-5 ), 115.2 (C-3, C-5), 54.1 (C-8), 51.4 (CH ), 36.8 (C-7).
3
0
0
1
N-[3 ,4 -Dihydroxy-(E)-cinnamoyl]-L-phenylalanine methyl ester 2a: Yield 41%. H NMR and
13
C NMR are in accordance with the literature [9].
N-Caffeoyl-tyrosine methyl ester 3a: Yield 58%. H NMR is in accordance with the literature [
C NMR (100 MHz, Acetone-d )
1
9];
13
0 0 0
δ 172.0 (C-9), 166.0 (C-9 ), 156.3 (C-4), 147.2 (C-4 ), 145.4 (C-3 ), 140.9
6
0
0
0
0
0
(
C-7 ), 130.2 (C-2 + C-6), 127.5 (C-1), 127.3 (C-1 ), 120.9 (C-6 ), 117.8 (C-8 ), 115.4 (C-5 ), 115.2 (C-3 +
0
C-5), 114.1 (C-2 ), 54.9 (C-8), 52.2 (COOCH ), 36.8 (C-7).
3
0
1
N-[4 -Hydroxy-(E)-cinnamoyl]-L-tryptophan methyl ester 4a: Yield 72%. H NMR (400 MHz,
0
Acetone-d6)
δ
10.15 (1H, s, OH), 8.82 (1H, s, NH), 7.65 (1H, d, J 7.8, H-4), 7.54 (1H, d, J 15.7, H-7 ), 7.50
0
0
(
2H, d, J 8.5, H-2 + H-6 ), 7.45 (1H, d, J 8.1, H-7), 7.27 (1H, s, H-1), 7.17 (1H, t, J 7.9, H-6), 7.09 (1H,
t, J 7.9, H-5), 6.93 (2H, d, J 8.6, H-3 + H-5 ), 6.67 (1H, d, J 15.7, H-8 ), 5.01 (1H, m, H-10), 3.72 (3H, s,
0
0
0
1
3
OCH ), 3.36 (2H, dd, J 20.0, J 6.4, H-9); C NMR (100 MHz, Acetone-d ) δ 173.2 (COOCH ), 166.5
3
3
1
2
6
0 0 0 0 0 0
(
1
C-9 ), 159.8 (C-4 ), 141.0 (C-7 ), 137.5 (C-8), 130.2 (C-2 + C-6 ), 128.5 (C-1 ), 127.6 (C-3), 124.3 (C-1),
0
0
0
22.2 (C-6), 119.6 (C-5), 119.1 (C-4), 118.9 (C-8 ), 116.5 (C-3 + C-5 ), 112.2 (C-7), 110.8 (C-2), 54.1 (C-10),
5
2.2 (COOCH ), 28.5 (C-9).
3
0
0
1
N-[3 ,4 -Dihydroxy-(E)-cinnamoyl]-L-tryptophan methyl ester 5a: Yield 78%. H NMR (400 MHz,
Acetone-d6) 8.33 (1H, s, OH), 8.11 (1H, s, OH), 7.57 (1H, d, J 7.6, H-4), 7.50 (1H, NH), 7.45 (1H, d, J
δ

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0
0
1
6
3
1
1
5.6, H-7 ), 7.37 (1H, d, J 8.1, H-7), 7.20 (1H, s, H-1), 7.08 (2H, m, H-2 + H-5), 7.03 (1H, t, J 8.1, H-6),
0
0
0
.92 (1H, dd, J 8.2, J 2.0, H-6 ), 6.82 (1H, d, J 8.2, H-5 ), 6.56 (1H, d, J 15.6, H-8 ), 4.93 (1H, m, H-10),
.65 (3H, s, COOCH ), 3.30 (2H, ddd, J 21.8, J 14.7, J 6.4, H-9); C NMR (100 MHz, Acetone-d6)
72.4 (C-11), 165.9 (C-9 ), 147.3 (C-7 ), 145.4 (C-3 ), 140.7 (C-4 ), 136.7 (C-8), 127.6 (C-3), 127.6 (C-1 ),
23.5 (C-1), 121.3 (C-4), 121.3 (C-6 ), 120.9 (C-5), 118.8 (C-6), 118.8 (C-8 ), 115.4 (C-5 ), 114.0 (C-2 ), 111.3
1
2
13
δ
3
1
2
3
0 0 0 0 0
0
0
0
0
(
C-7), 109.9 (C-2), 53.4 (C-10), 51.42 (COOCH ), 27.6 (C-9).
3
3
.5. General Procedure for the Preparation of the Hydroxycinnamoyl Amides 1b–5b
The 1.11 mmol of the cinnamoylamide ester were dissolved in 68 mL of a THF/H O 1:1 v/v
2
solution and cooled in an ice bath. Subsequently, 5.53 mmol of LiOH H O were added and the mixture
·
2
was stirred at room temperature. TLC analyses were performed to check the end of the reaction.
The mixture was diluted with 50 mL of distilled water and acidified with HCl 3 M till pH 2. After
extraction with ethyl acetate, the organic phase was dried over anhydrous Na SO . Evaporation of the
2
4
solvent under vacuum gave the crude product which was first purified by column chromatography on
silica gel using a 99:1 mixture of ethyl acetate: acetic acid as the eluent or by recrystallization from
petroleum ether.
0
-)-N-[4 -Hydroxy-(E)-cinnamoyl]-L-tyrosine 1b: Yield 85%. H NMR (400 MHz, Acetone-d ) δ
6
1
(
0 0 0
.80 (1H, bs, OH), 8.20 (1H, bs, OH), 7.50 (1H, d, J 15.7, H-7 ), 7.43 (2H, d, J 8.6, H-2 , H-6 ), 7.31 (1H, d,
8
0
0
NH), 7.10 (2H, d, J 8.5, H-2, H-6), 6.85 (2H, d, J 8.6, H-3 , H-5 ), 6.75 (2H, d, J 8.5, H-3, H-5), 6.45 (1H,
0
d, J 15.7, H-8 ), 4.81 (1H, dt, J = 7.8, 5.3, H-8), 3.17 (1H, dd, J 14.0, 5.3, H-7), 3.00 (1H, dd, J 14.0, 7.8,
H-7); 13C NMR (100 MHz, Acetone-d )
0 0 0
δ 172.3 (C-9), 165.9 (C-9 ), 158.9 (C-4 ), 156.1 (C-4), 140.4 (C-7 ),
6
0
0
0
0
0
0
1
30.3 (C-2, C-6), 129.5 (C-2 , C-6 ), 127.8 (C-1), 126.7 (C-1 ), 117.9 (C-8 ), 115.6 (C-3 , C-5 ), 115.1 (C-3,
20
20
D
C-5), 53.8 (C-8), 36.5 (C-7); [α]D
−
39.9 (c 0.1 MeOH) (lit. [α]
= +13.05; LC-MS (ESI ), m/z 328 ([M + H] ), (ESI ), m/z 326 ([M-H] ).
-)-N-[3 ,4 -Dihydroxy-(E)-cinnamoyl]-L-phenylalanine 2b: Yield 94%. H NMR (400 MHz,
−
26.9) [
7
]; CD (CH OH) ∆ε309nm
=
−
−
2.94,
3
+
+
−
∆
ε
= +2.17, ∆ε
2
58nm
234 nm
0
0
1
(
0
Acetone-d6)
δ
8.30 (OH), 7.44 (1H, d, NH), 7.42 (1H, d, J 15.6 Hz, H-7 ), 7.35–7.19 (5H, m), 7.08 (1H, d, J
.0 Hz, H-2 ), 6.95 (1H, dd, J 8.2, 2.0 Hz, H-6 ), 6.84 (1H, d, J 8.2 Hz, H-5 ), 6.56 (1H, d, J 15.6 Hz, H-8 ),
0
0
0
0
2
4
13
.88 (1H, dt, J 8.0, 5.4 Hz, H-8), 3.27 (1H, dd, J 13.9, 5.4 Hz, H-7), 3.09 (1H, dd, J 13.9, 8.0 Hz, H-7);
C
0 0
δ 172.2 (C-9), 165.7 (C-9 ), 147.0 (, 145.2, 140.6 (H-7 ), 137.4 (Ph), 129.3
NMR (100 MHz, Acetone-d6)
0 0 0 0
Ph), 128.3 (Ph), 127.4 (Ph), 126.6 (Ph), 120.9 (C-6 ), 118.0 (C-8 ), 115.4 (C-5 ), 114.0 (C-2 ), 53.5 (C-8), 37.3
(
(
C-7); [α]2
0
−
48.8 (c 0.1, MeOH); CD (MeOH) ∆ε
=
−
1.1, ∆ε
= 1.0, ∆ε250nm = +1.8, ∆ε230nm
−
D
330nm
−
297nm
−
+
+
=
+3.9; LC-MS (ESI ), m/z 328 ([M + H] ), (ESI ), m/z 326 ([M-H] ).
0
0
1
(
-)-N-[3 ,4 -Dihydroxy-(E)-cinnamoyl]-L-tyrosine 3b: yield 84%. H NMR (400 MHz, Acetone-d )
6
0
0
δ
7.36 (1H, d, J 15.6, H-7 ), 7.26 (1H, NH), 7.05 (2H, d, J 8.6, H-2 + H-6), 7.03 (1H, d, J 2.0, H-2 ), 6.90
0 0
1H, dd, J 8.2, J 2.0, H-6 ), 6.78 (1H, d, J 8.2 Hz, H-5 ), 6.70 (2H, d, J 8.6, H-3 + H-5), 6.51 (1H, d, J 15.6,
1 2
(
0
H-8 ), 4.75 (1H, dt, J 7.8, J 5.3, H-8), 3.10 (1H, dd, J 14.0, J 5.3, H-7), 2.95 (1H, dd, J 14.0, J 7.8, H-7);
1
2
1
2
1
2
13
0 0 0
C NMR (100 MHz, Acetone-d )
C-7 ), 131.1 (C-2 + C-6), 128.7 (C-1), 128.3 (C-1 ), 121.7 (C-6 ), 119.0 (C-8 ), 116.3 (C-5 ), 115.9 (C-3 +
C-5), 114.9 (C-2 ), 54.7 (C-8), 37.7 (C-7); [α]
δ 173.2 (C-9), 166.6 (C-9 ), 157.0 (C-4), 147.9 (C-4 ), 146.1 (C-3 ), 141.4
6
0
0
0
0
0
(
0
25
20
D
−
41.7 (c 0.1, MeOH) (lit. [α]
−
35.6) [11]; CD (MeOH):
D
+
+
−
∆
3
ε
=
31
−
1.5, ∆ε269 = +0.4, ∆ε235 = +6.5, ∆ε221
=
−
0.2; LC-MS (ESI ), m/z 344.1 ([M + H] ), (ESI ), m/z
3
−
42.0 ([M-H] ).
0
1
(
-)-N-[4 -Hydroxy-(E)-cinnamoyl]-L-tryptophan 4b: yield 91%. H NMR (400 MHz, Acetone-d )
0.05 (1H, s, OH), 8.90 (1H, bs, COOH), 7.61 (1H, d, J 7.9, H-4), 7.51 (2H, m, H-7 +NH), 7.37 (2H, d, J
.6, H-6 + H-2 ), 7.33 (1H, d, J 8.1, H-7), 7.20 (1H, s, H-1), 7.04 (1H, t, J 7.9, H-6), 6.96 (1H, t, J 7.9, H-5),
.82 (2H, d, J 8.6, H-5 + H-3 ), 6.60 (1H, d, J 15.7, H-8 ), 4.99 (1H, m, H-10), 3.42 (1H, dd, J 22.2, J
δ
6
0
1
8
6
1
(
0
0
0
0
0
1
2
1
3
4.8, H-9), 3.28 (1H, dd, J 22.2, J 6.3, H-9); C NMR (100 MHz, Acetone-d )
δ
172.9 (COOH), 166.4
1
2
6
0 0 0 0 0 0
C-9 ), 159.2 (C-4 ), 140.6 (C-7 ), 136.7 (C-8), 129.6 (C-2 + C-6 ), 127.9 (C-1 ), 126.7 (C-3), 123.6 (C-1),
0
0
0
1
2
21.4 (C-6), 118.8 (C-5), 118.5 (C-4), 117.9 (C-8 ), 115.8 (C-3 + C-5 ), 111.4 (C-7), 110.1 (C-2), 53.4 (C-10),
7.6 (C-9); [α]2
5
−
27.8 (c 0.1, MeOH); CD (MeOH): ∆ε323
1.2, ∆ε231 = +11.7, ∆ε = −3.5; LC-MS (ESI ), m/z 351.0 ([M + H] ), (ESI ), m/z 349.1 ([M-H] ).
=
−
1.4, ∆ε
=
−
0.1, ∆ε270
=
−
0.5, ∆ε
=
250
D
292
+
+
−
−
+
217

Molecules 2020, 25, 1704
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0
0
(
-)-N-[3 ,4 -Dihydroxy-(E)-cinnamoyl]-L-tryptophan 5b: Yield 56%. 1H NMR (400 MHz,
0
Acetone-d6)
δ
10.06 (1H, s, OH), 7.59 (1H, d, J 7.6, H-4), 7.40 (1H, d, J 15.6, H-7 ), 7.36 (1H, NH), 7.32
0
(
1H, d, J 8.2, H-7), 7.17 (1H, d, J 2.0, H-1), 7.02 (2H, m, H-5 + H-2 ), 6.96 (1H, t, J 7.6, H-6), 6.88 (1H, dd,
J 8.2, J 2.0, H-6 ), 6.77 (1H, d, J 8.2, H-5 ), 6.53 (1H, d, J 15.6, H-8 ), 4.94 (1H, m, H-10), 3.37 (1H, dd,
J 14.8, J 5.3, H-9), 3.24 (1H, dd, J 14.8, J 7.3, H-9); C NMR (100 MHz, Acetone-d6)
66.8 (C-9 ), 147.9 (C-7 ), 146.1 (C-3 ), 141.4 (C-4 ), 137.5 (C-8), 128.7 (C-3), 128.3 (C-1 ), 124.3 (C-1),
22.1 (C-4), 121.7 (C-6 ), 119.5 (C-5), 119.3 (C-6), 119.0 (C-8 ), 116.2 (C-5 ), 114.9 (C-2 ), 112.1 (C-7), 111.0
C-2), 54.0 (C-10), 28.3 (C-9). [α]D
+10.2, ∆ε216 = +2.4; LC-MS (ESI ), m/z 367.1 ([M + H] ), (ESI ), m/z 365.1 ([M-H] ).
0
0
0
1
2
13
δ
0
173.5 (C-11),
1
2
1
2
0
0
0
0
1
1
(
0
0
0
0
25
−
33.2 (c 1.0, MeOH); CD (MeOH): ∆ε325
=
−
2.3, ∆ε
= −2.1, ∆ε232
298
+
+
−
−
=
3
.6. Extraction of Hydroxycinnamoyl Amides and Sample Preparation
Green coffee beans were ground to a fine powder in a mixer ball mill MM400 (Retsch, Haan,
Germany) and extraction was performed in duplicate by dynamic heat-assisted water extraction [15 16].
,
For this purpose, 1 g of powdered green coffee from each species was added to 100 mL of boiling water
and the mixture was stirred for 10 min at 200 rpm on a heated plate (Arex Velp Scientifica, Usmate,
Italy) and filtered. The aqueous extract was frozen with liquid nitrogen and freeze dried for 3 days.
HPLC samples were prepared dissolving the lyophilized extracts in MilliQ water (obtained with
a Milli-Q Reference Water Purification System, Merck, Darmstadt, Germany) at a concentration of
6
mg/mL. Subsequently the solutions were filtered with 0.2
µm PTFE syringe filters (from VWR
International Ltd., Radnor, PA, USA), preconditioned with methanol and injected to LC-MS.
3
.7. Quantitative Analyses
To quantify the hydroxycinnamoyl amides, caffeic acid and p-coumaric acid were used as
standard compounds, exploiting their UV absorption at 320 nm for detection, as already reported
in the literature [14]. Calibration curves of caffeic acid and p-coumaric acid were built reporting
the integration signal (mAu*s) versus concentration (µg/mL) as mean value of triplicate analyses
in the range of concentration 0.035–3.5 g/mL and 0.041–4.1
curves showed a good response linearity with a coefficient of correlation (R ) of 0.997. Limits of
µ
µ
g/mL, respectively. Both calibration
2
detection (LOD) and quantification (LOQ) were, respectively, 0.14
µg/mL and 0.20 µg/mL calculated as
signal-to-noise ratio with S:N = 3 for LOD and S:N = 10 for LOQ.
4
. Conclusions
The results obtained in the present work are in accordance with the literature demonstrating
that the HCAs p-coumaroyl-N-tyrosine 1b, caffeoyl-N-phenylalanine 2b, caffeoyl-N-tyrosine 3b, and
p-coumaroyl-N-tryptophan 4b, and caffeoyl-N-tryptophan 5b, are characteristic of the Robusta green
coffee beans, although their distribution between the samples depends on the geographical origin.
Samples from Tanzania and Uganda contain all five HCAs while Robusta from Indonesia has only
p-coumaroyl-N-tryptophan 4b and caffeoyl-N-tryptophan 5b. The only HCA present in Arabica
is caffeoyl-N-tryptophan 5b, although in a very small amount with respect to Robusta. Liberica
didn’t show the presence of any HCAs, although a small number of samples were considered.
The investigation on more samples of Liberica of different origins is necessary to confirm the absence
of HCAs. Preliminary studies on roasted coffee beans of both Robusta and Arabica were carried out by
Kim et al. [13], where it was evidenced that the HCAs content decreased. A future development of the
HCAs analysis could consider coffee brews from different commercial sources to evaluate if the HCAs
content can discriminate on the coffee species used.
Author Contributions: Conceptualization: C.F.; Investigation: C.F.; Formal analysis: C.F., F.B.; Methodology: C.F.;
Validation and supervision: C.F., F.B., L.N.; Writing—original draft, C.F.; Writing—review and editing, C.F., F.B.,
L.N., S.C. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.

Molecules 2020, 25, 1704
13 of 14
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
CD
Circular Dichroism
5
-CQA
5-caffeoylquinic acid
N,N -Dicyclohexylcarbodiimide
Dry weight
Formic acid
0
DCC
DW
FA
HCA
Hydroxycinnamoyl amide
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Sample Availability: Samples of the compounds are not available from the authors.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).