Peptides N-connected to hydroxycoumarin and cinnamic acid derivatives: Synthesis and fluorescence spectroscopic, antioxidant and antimicrobial properties

Article abstract of DOI:10.1039/c8nj00383a

The tripeptide Tyr-Gly-Ser and a series of conjugations to coumarin, cinnamic and gallic acid were synthesized in salt form and their antioxidant and antimicrobial activities were investigated. The N-connecting hydroxycoumarin, cinnamic and gallic acid derivatives to peptides and the use of BBr₃ as a demethylating agent for peptides was reported. Their activities were investigated based on the conjugated moiety structures. Studies of their activities showed that conjugated tripeptides 7,8-dihydroxycoumarin-peptide (17), caffeic acid-peptide (22) and gallic acid-peptide (28) were found to be superior to ascorbic acid with respect to their antioxidant activity, and 12, 14, 24, and 25 exhibited the most antimicrobial activity in the series compared to amoxicillin. Additionally, the incredible florescence intensity and brightness of 17 in water and DMSO, compared to other synthesized compounds, qualified this peptide as a suitable probe in the human body.

Full text of DOI:10.1039/c8nj00383a

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The role of atropisomers on the photo-reactivity and fatigue of
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Peptides N-Connected to Hydroxycoumarin and cinnamic acid derivatives:
Synthesis and Fluorescence Spectroscopic, Antioxidant and Antimicrobial
Properties
Hajar G. Ghalehshahi,a Saeed Balalaie,*a,b Atousa Aliahmadic
a Peptide Chemistry Research Center, K. N. Toosi University of Technology, P. O. Box
15875-4416, Tehran, Iran
b Medical Biology Research Center, Kermanshah University of Medical Sciences,
Kermanshah, Iran
c Medicinal Plant and Drug Research Institute, Shahid Beheshti University, Tehran, Iran
ABSTRACT: The tripeptide TyrꢀGlyꢀSer and a series of conjugations to coumarin, cinnamic
and gallic acid were synthesized in salt form and their antioxidant and antimicrobial activities
were investigated. The Nꢀconnecting hydroxycoumarin, cinnamic and gallic acid derivatives
to peptide and using of BBr3 as demethylating agent on peptide was reported. Their activity
was investigated based on the conjugated moiety structures. Studying of their activities
showed that, conjugated tripeptides 7,8ꢀdihydroxycoumarinꢀpeptide (17), caffeic acidꢀpeptide
(22) and gallic acidꢀpeptide (28) were found to be superior to ascorbic acid with respect to
their antioxidant activity, and 12, 14, 24, and 25 exhibited the most antimicrobial activity in
the series compared to amoxicillin. Additionally, incredible florescence intensity and
brightness of 17 in water and DMSO compare to other synthesized compounds, qualified this
peptide as a suitable probe in human body.
Keywords: Hydroxycoumarinꢀpeptide, Hydroxycinnamicꢀpeptide, Gallic acidꢀpeptide,
Antioxidant activity, Antimicrobial activity, Fluorescence spectroscopy.
1

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Introduction
Several reports have described the antioxidant activity of peptides extracted from foods such
as whey, casein, soy, egg yolk and other sources, and these peptides can be categorized as
safe, clean, and economical natural antioxidants.1 The antioxidative properties of a peptide
depends on its amino acid composition, structure, sequence and hydrophobicity.2 An
important feature of bioactive peptides (BAP), defined as peptides with molecular masses of
<6000 Da, is that the human intestine easily absorbs these peptides, especially diꢀ and
tripeptides.3 Absorption of small peptides is 70ꢀ80% higher than that of free amino acids.4
Antimicrobial peptides (AMPs) are another important class of peptides with extended
activities that represent a large group of endogenous compounds widely distributed in nature
and found in animals, fungi, plants, and bacteria. AMPs have become a model for the
discovery of novel antimicrobial drugs that can answer the problem of the increasing
antibiotic resistance of pathogenic microorganisms.5
Due to the high bioactivities of coumarins, some efforts have focused on the development of
novel coumarinꢀbased therapeutic agents.6 The antioxidant activity of coumarins may include
different molecular mechanisms and is probably related to their structural analogy with
flavonoids. The position and type of substituent attached to the aromatic ring on the structure
of coumarin have a great influence on the antioxidant activity of these compounds. The
number of hydroxyl groups on the ring and their positions strongly affect the antioxidant
activity of coumarins.7 Coumarins have low antibacterial activity, but compounds having
longꢀchain hydrocarbons such as ostruthine 1 and ammoresinol 2 with coumarin structures
show activity against a wide spectrum of Gramꢀpositive bacteria (Figure 1).8 Coumarins are
also widely used as additives in optical brighteners. They can disperse fluorescent and laser
dyes and are used as fluorophore labels on peptides and proteins due to their highꢀemission
quantum yields, photostability, and extended spectral range.9 Due to the importance of
peptides and coumarins separately, the conjunction of these two moieties is an interesting
approach in medicinal chemistry. In this regard, some approaches have focused on the
development of conjugated peptideꢀcoumarins, Moroder reported the connection of coumarin
to the Nꢀterminus of a dipeptide containing thiol in a solution for labeling peptides.10
Figure 1. The structures of antibacterial compounds
2

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In another approach, using cinnamic acid as an open analog of coumarin carboxylic acid,
researchers used it as a linker to connect the Cꢀ and Nꢀ termini of a peptide to form a cyclic
peptide for the metabolic oxidation of CYP3A4.11 Some methods for solidꢀphase peptide
synthesis of Cꢀterminalꢀmodified peptides that also include the coumarin skeleton have been
reported.12 Cuꢀcatalyzed macrocyclization was performed to obtain a macrocyclic coumarinꢀ
containing tripeptide for use in thrombin activity measurements.13 Both hydroxyl and orthoꢀ
methoxy groups at the coumarin ring connected to the peptide enhanced the activity of
coumarinꢀ3ꢀacylamino derivatives in protecting DNA against OH and AAPHꢀinduced
oxidation.14 Recently Freitas et al used a triazole as a linker for the synthesis of conjugated
coumarinꢀpeptide via copper(I)ꢀcatalyzed azideꢀalkyne cycloaddition. The reported approach
has some merits such as low diversity for the starting material; only 7ꢀdiethylaminocoumarin
was used as sole starting material. Meanwhile, antimicrobial activity of the synthesized
conjugated peptide is related to triazole linker, and coupling of coumarin to peptide was done
in solution phase and the selected peptide was an amidated Cꢀterminal octapeptide.15
Additionally, a tripeptide TyrꢀGlyꢀSer, isolated from peanut showed high antioxidant activity
with strong oxygen radical absorbance capacity (ORAC). 16 It displayed a stronger protective
effect on linoleic acid peroxidation and H2O2ꢀinduced oxidative injury in rat
pheochromocytoma line PC12 cells than GSH. However, it showed no radical scavenging
(DPPH) and no metal cleating (FRAP) activities. In continuation of our research for the
synthesis of bioactive peptides,17 herein, we report an efficient approach for the synthesis of
conjugated hydroxycoumarinꢀ, gallicꢀ and ꢀcinnamic acidꢀtripeptides (TyrꢀGlyꢀSer) to
improve antioxidant activity through DPPH and FRAP methods and their antimicrobial
activities and fluorescence spectra were compared.
Results and discussion
Chemistry
To attain the goal for the synthesis of conjugated coumarinꢀpeptides, different coumarin
carboxylic acid derivatives 3aꢀk were prepared by the reaction of different oꢀhydroxy
carbaldehydes and Meldrum’s acid refluxing in water18 (purified yields 82ꢀ99%). Compound
4 was synthesized through the nucleophilic addition of salicylaldehyde and maleic anhydride,
followed by a cyclization reaction.19 Compound 8 was selected as a candidate to investigate
the effect of the phenolic group. However, for carboxylic acid derivatives containing a
hydroxyl moiety in their structure, different additional protection approaches were required
3

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because it is essential to couple them to the tripeptide sequence. For compounds 5, 6, 7, 9a
and 10a, protection of the free hydroxyl group was done (Table 1). Different known methods
were examined for protection of the hydroxyl group in carboxylic acid derivatives, and based
on our results, we found that using a methyl ether protected form was the most effective
approach.
Table 1. Structures of the starting compounds
3a, 93%
3b, 94%
3c, 91%
3d, 97%
3e, 90%
3f, 98%
3g, 95%
3h, 95%
3i, 99%
3j, 97%
3k. 91%
4, 71%
5, 87%
6, 82%
7, 90%
8
9
9a, ,89%
10
10a, 88%
The linear protected peptide (FmocꢀTyr (tBu)ꢀGlyꢀSer (tBu)) was synthesized on the surface
of the 2ꢀchloroꢀtritylchloride (2ꢀCTCꢀresin) through a standard solid phase peptide synthesis
(SPPS) strategy. After the completion of the peptide sequence, coumarin carboxylic acid 3a
was coupled to the resinꢀprotected peptide. Finally, cleavage from the surface of the resin and
final deprotection was performed, yielding desired peptide 11 (Scheme 1). The structure of
the synthesized conjugated coumarinꢀtripeptide 11 was confirmed by NMR spectroscopy,
LCꢀMass and HRMS spectrometry.
4

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Scheme 1. Synthesis of conjugated coumarin peptide
The data to distinguish the compounds by 13C NMR are related to 3ꢀamidic carbonyls that
resonate at 160.5, 168.1, 170.8 ppm and the carbonyl of ester moiety that resonates at 173.2
ppm. The methylene of the tyrosine resonated at two different chemical shifts as
diastereotopic hydrogens with different coupling constants (8.6 Hz and 4.6 Hz) with the
adjacent hydrogen .
Table 2. Structures of synthesized products
11, 94%
12, 92%
13, 95%
14, 85%
15, 94%
16, 95%
17, 82%
20, 89%
23, 96%
18, 93%
21, 88%
24, 95%
19, 96%
22, 86%
25, 93%
5

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26, 91%
27, 90%
28, 80%
29, 84%
30, 87%
31, 88%
32, 90%
Additionally, the HRMS confirmed the synthesis of compound 11, with a peak at [M+H]
498.1517. The same strategy was used for the synthesis of all conjugated tripeptides 11ꢀ28. In
the case of peptides 17, 21, 26 and 28, their coumarin starting materials 4, 5, 8, 9a were
protected by a methyl ether group. Because the free hydroxyl has an essential role in the
antioxidant activity of the product, different methods were investigated for deprotection of
the protecting group, and the best approach was demethylation using BBr3 in DCM. This step
was done before the final deprotection to avoid removing the hydroxyl group of the serine.
We tried to do debenzylation of peptide 24 using catalytic hydrogenation reaction conditions,
however, in addition to the debenzylation, reduction of the double bond occurred. Using
BF3.Et2O for debenzylation was not successful either. Conjugation of the coumarin
carbocyclic acid to the three amino acids in the tripeptide sequence separately (Ser, Gly, and
Tyr) lead to compounds 30ꢀ32, and we compared their activity with the synthesized
conjugated coumarinꢀtripeptide. In this way, our aim was to investigate the activity of the
products and to determine the role of the individual amino acids in this activity. Table 2
summarizes the structures of synthesized products 11ꢀ32 (purified yields 80ꢀ96%).
Biological test
The in vitro antioxidant activity of the starting carboxylic acid derivatives 3aꢀk and 4ꢀ10 and
conjugatedꢀtripeptides, amino acids 11ꢀ32 were screened using (1,1ꢀdiphenylꢀ2ꢀ
picrilhydrazyl) DPPH and ferrous ionꢀchelating capacity (FRAP) methods. The results
obtained from the two approaches were consistent. The ferric reducing antioxidant power
assay is based on the reduction of the ferric tripyridyltriazine (Fe3+ꢀTPTZ) complex to ferrous
tripyridyltriazine (Fe2+ꢀTPTZ) at low pH. The production of Fe2+ꢀTPTZ generates a blue
color with a specific sd at 593 nm. The absorbance was plotted against ferrous ion
concentrations. Lꢀascorbic acid was used as the standard antioxidant. The results are
expressed as mM ascorbic acid equivalents per mM of each sample.20 The starting materials
6

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were divided into two sets based on their FRAP activity. The first set consisted of compounds
with lower FRAP effects than ascorbic acid: compounds 3a, 3b, 3f, 7, and 9 have no FRAP
effect, and naphtalenecoumarin carboxylic acid 3e, 7ꢀhydroxycoumarin carboxylic acid 3c
and trimethoxybenzoic acid 8 have low FRAP effects. These data indicated the important role
of coumarin and the position of the methoxy group. 8ꢀMethoxycoumarin 3i and 8ꢀethoxy
coumarin carboxylic acid 3g were similar and more effective than 7ꢀmethoxycoumarin
carboxylic acid 7. 8ꢀHydroxycoumarin carboxylic acids 3k and 4 exhibited the highest
antioxidant activity within the first set.
14
12
10
8
6
4
2
0
3a 3b 3c 3e 3f 3g 3h 3i 3j 3k
4
5
7
8
9
10
Compounds
Figure 2. Ferrous ionꢀchelating capacity activity
In the second set, the compounds that exhibited the highest capacity compared to ascorbic
acid was 7,8ꢀdihydroxy coumarin carboxylic acid 3h, which was 13 times stronger than
ascorbic acid and a little better than 7,8ꢀdimethoxy coumarin 5. The coumarin ring conferred
an enhancement of FRAP activity of both compounds compared to dihydroxycinnamic acid
10. (Figure 2) Tripeptide 11 and 29 had a negligible FRAP effect and peptides 14, 16, 18, 19,
24, 25 and 31 had a weak to moderate FRAP effect (Figure 3). Compound 19, with a
dimethylamino group at position 7 on the coumarin ring, had the highest effect among the
peptides that had a lesser effect than ascorbic acid. Peptides containing caffeic acid 22,
trihydroxybenzoic acid 28, and dihydroxycoumarin carboxylic acid 17 exhibited the highest
ferrous ionꢀchelating capacity, 5ꢀ6 times more than ascorbic acid, which revealed the
contribution of a free phenolic OH group. pꢀHydroxycinnamic acid 21 is twice as strong as
ascorbic acid, but its methoxy analog 27 had a weak effect. The DPPH freeꢀradical inhibition
activity assay was performed in accordance with standard protocols.
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6
5
4
3
2
1
0
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
Synthesized peptides
Figure 3. Ferrous ionꢀchelating capacity activity of peptides
The percent of freeꢀradical inhibition (IC50%) was calculated.21 Among the compounds in
Table 1, only five exhibited considerable radicalꢀscavenging activity (Figure 4). 3ꢀOxoꢀ3Hꢀ
benzo[f]chromeneꢀ2ꢀcarboxylic acid 3e and 8ꢀhydroxycoumarin carboxylic acid 3k
demonstrated moderate to high DPPH capacity. The orthoꢀdihydroxy arrangement in 7,8ꢀ
dihydroxy carboxylic acid 3h and caffeic acid 10 and the dimethoxy arrangement in
coumarin carboxylic acid 5 enhanced the DPPH activity of these compounds 1.5ꢀ3 times
compared to ascorbic acid. The great difference in DPPH activity of trimethoxybenzoic acid
peptide 20 and its triꢀhydroxyl analog 28 reveal the effect of phenolic groups, which was
consistent with the high activity of dihydroxy coumarinꢀtripeptide 17 and cinnamicꢀtripeptide
22.
ρ<0.005
1000
859.7
800
600
400
132.8
200
4.7
0.20
0,50 0.44
0.72
AS
0
3e
3h
3k
5
10
BHT
Compounds
Figure 4. Radicalꢀscavenging activity of the starting compounds
The higher radicalꢀscavenging activity of conjugatedꢀtripeptide 25 compared to BHT and
conjugatedꢀtripeptide 18 was conferred by the high stability of the resulting radical.
Conjugated coumarinꢀserine 31 had more powerful antioxidant effect than BHT. The tyrosine
analog 32 had the same effect, but due to unexpected interaction in 96ꢀcell plates during the
DPPH test, the absorption was not readable (Figure 5).
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ρ<0.005
1400
1200
1000
800
600
400
200
0
Compounds
Figure 5. Radicalꢀscavenging activity of synthesized peptides
The in vitro antibacterial activity of samples was assessed against E. coli ATCC 25922 and S.
aureus ATCC25923 strains as Gramꢀnegative and Gramꢀpositive bacteria, respectively.
Minimum inhibitory concentration (MIC) values were determined using the standard broth
microꢀdilution technique recommended by the Clinical and Laboratory Standard Institute.22
The results are shown in Table 3. The MIC results showed that all tested coumarin peptide
salts had weak antimicrobial effects against E. coli and good to moderate effects against S.
aureus. Conjugated peptides 12, 14, 16, 24, 25 and 26 had good antimicrobial effects against
the Gramꢀpositive bacterium. Peptides 14 and 24, with the extra aromatic ring on coumarin
backbone, had the highest antibacterial effects. The lowest inhibition of S. aureus was related
to 13, 17 and 18. In the case of the 7ꢀbenzyloxy conjugated coumarinꢀpeptide 24, the effect
against S. aureus was half as strong as cefixime and higher than amoxicillin. A comparison of
the data revealed that the antibacterial effect of peptide 25 is five times more than the
conjugated one 18. Conjugatedꢀtripeptide 25, containing a methoxy group, demonstrated
higher inhibition than 26, with a hydroxyl group in the same position, and 16, with ethoxy
group, had higher antibacterial activity than 18, which had a methoxy group at the same
position.
Table 3. Antimicrobial activity of synthesized peptides
MIC a
(mM)
MIC b
(mM)
MIC a
(mM)
MIC b
(mM)
Sample
Sample
11
13
16
18
22
24
26
10
10
10
10
1
5
12
14
17
19
23
25
27
10
10
10
10
10
10
20
0.625
0.312
>5
>5
1.25
>5
2.5
1
1.25
0.625
5
10
10
0.01
1.25
Amoxicilline
0.0082
0.170
Cefixime
0.0088
0.0022
a MIC against E. coli (ATCC 25922). b Minimum inhibition concentration (MIC) against S. aureus (ATCC25923)
Because some coumarin derivatives are used as fluorophores in peptides and proteins, we
were interested in the fluorescence properties of the novel synthesized compounds in DMSO
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and H2O. Conjugated coumarinꢀpeptides have absorbance between 215 and 450 nm, and their
emission is between 330 and 500 nm, depending on the substituents of the coumarin ring.
Absorption (λAbs.) with molar extinction coefficients (ε) and fluorescence (λEm.) wavelength
maxima were measured for the synthesized conjugated peptides (Table 4, supporting
information).
Table 4. Fluorescence and absorbance data for the conjugated peptides
Noa
λAbs
.
b (nm) (ε)c
λAbs.d (nm) ) (ε)c
λEm.
b (nm) (φ)
λEmd (nm) (φ)
11
12
13
14
15
16
290 (2.71)
275(5.20)
216(11.89),301(3.09)
218(12.82),274(5.98)
218(12.05),350(4.39)
221(12.51),377(2.89)
218(11.95), 302(3.01)
218(12.34),314(3.50)
436 (0.0268)
424(0.0657)
416(0.0275)
446(0.0622)
438(0.0245)
484(0.0343)
-
-
348(4.07)
408(0.1085)
262(1.33),373(0.89)
298(2.87)
458(0.1733)
-
-
313(2.96)
269(1.91),364(2.49),
446(1.56)
219(12.18),264(2.02),
348(3.47)
17
18
498(0.2125)
460(0.1993)
218(12.21),253(2.51),
314(4.15)
312(3.61)
478(0.0346)
-
217(12.24),266(2.36),
19
20
21
263(2.69),424(8.36)
263(1.84)
473(0.1637)
426(0.0292)
434(0.0230)
485(0.0468)
432(8.62)
218(12.27),260(2.36)
-
-
218(11.46),282(1.14),
297(1.04)
281(1.55)
218(12.34),296(2.00),
319(2.13)
22
289(1.88),325(1.96)
418(0.0497)
-
219(11.82),294(2.25),
345(0.93)
23
24
25
288(2.29)
348(3.86)
291(2.35)
434(0.0300)
432(0.0547)
446(0.0314)
-
216(11.94),348(3.54)
410(0.0894)
-
219(12.47),252(1.94),
293(2.48)
217(12.17),253(1.75),
293(2.37)
26
288(2.69)
422(0.1310)
-
27
28
29
31
289(2.44)
273(1.50)
263(1.62)
300(2.10)
216(11.94),308(2.58)
218(12.01), 272(1.48)
218(11.10),274(0.42)
217(11.44),299(2.09)
434(0.0229)
424(0.0555)
-
-
-
-
434(0.0372)
413(0.0304)
217(12.15),266(2.13),
429(7.15)
32
263(2.91),423(7.61)
474(0.1569)
480(0.0783)
a Synthesized conjugated peptide. b λ(nm) in DMSO. c cmꢀ1Mꢀ1×104 d λ(nm) in H2O
In all cases more fluorescence intensity observed in DMSO compared to H2O. Compound 17
had the most fluorescence intensity in both solvents. Conjugated coumarinꢀpeptides showed
quantum yields (φ) higher than the original coumarinꢀ3ꢀcarboxylic acid derivetives. Emission
of compounds in DMSO had an extra peak around 335 nm compare to the same compound in
water. Additionally, absorption of compounds in water had an extra peak around 218 nm that
is related to amide bonds and their hydrogen bonding with solvent.
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Conclusions
In conclusion, hydroxycoumarins, ꢀcinnamic acids and gallic acid were conjugated to
peptides to improve the biological properties and offer interesting opportunity for
fluorometric applications. The conjugation of 7, 8ꢀdihydroxy coumarin to the tripeptide 17
had strongly modified its ferrous ionꢀchelating and radical scavenging activity and also had
the best florescence intensity in water and DMSO. The same antioxidant effect was shown in
other compounds 22, 28 containing two and three hydroxyl groups proved that the number
and position of phenolic hydroxyl have important effective roles. The synthesized
conjugatedꢀpeptides had well to moderate antimicrobial activity against S. aureus especially
for those that are more lipophilic. Compound 24 had better antimicrobial activity compared to
amoxicillin. Despite the need for further research, these compounds could be considered as
suitable antioxidants for the prevention of oxidative reaction.
Experimental section
General information
1H, 13C NMR spectra were recorded on a Bruker Advance 300 and 400 spectrometers.
Chemical shifts are given in ppm from TMS as an internal reference. Flash column
chromatography was carried out using silica Gel 60 (particle size 0.04–0.06 mm/230–400
mesh). The abbreviations are given in separate place. The mass spectra were recorded by EIꢀ
mass (70 eV), mass (ESIꢀtriple quadrupole), mass (ESIꢀion trap), HRMS (ESIꢀFTꢀICR),
HRMS (MALDIFTꢀICR). Mass spectra are reported as m/z (% of relative intensity). ACN
(H2O < 5 ppm) was purchased from SdSꢀCarlo Erba and stored under a nitrogen atmosphere.
The purification of peptides was done using preparative HPLC (column C18, 7 µm). IR
spectra were obtained on an ABB FTꢀIR FTLA 2000 spectrometer. Fluorescence data were
recorded by JASCO spectrofluorometer FPꢀ6500. Absorption of compounds was read with
ELISA Microplate Reader Elxꢀ800 BIOꢀTEK, BioꢀTek Instruments, Inc. Freeze Dryer (Alpha
1ꢀ2 LD plus) for drying purified peptide. All starting materials were purchased from Merck
and Aldrich and were used without further purification.
General procedure for the synthesis of 3-coumarin carboxylic acid derivatives (3aꢀk) 18
In a 25 mL round flask, salicylic aldehyde (1mmol, 152.15 mg) and Meldrum’s acid (1.2
mmol, 144 mg) were suspended in 2 mL water, and refluxed for 1h. The precipitated product
was filtered and recrystallized in methanol. (Yield 90ꢀ99% depending on compound)
Process for the synthesis of 2-(5-methoxy-2-oxo-2H-chromen-3-yl) acetic acid (4)19
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A stirred mixture of succinic anhydride (2mmol, 0.2 gr), 3ꢀmethoxy salicylic aldehyde
(1mmol, 0.152 gr) and triethylamin (3 mmol, 0.021 mL) was heated to 100 °C. Stirring was
continued for 30 minutes. The mixture was cooled and washed with HCl 10% (10 mL). The
resulted participates were filtered and purified by column chromatography. (Yellow powder,
yield 71%)
Process for the methylation of hydroxyl groups of 7, 8-dihydroxy coumarin-3-carboxylic
acid 5
In a 25 mL round flask, 7,8ꢀdihydroxy coumarinꢀ3ꢀcarboxylic acid (1mmol, 224 mg) was
solved in DMF in an ice bath, NaH in mineral oil (3 mmol, 240 mg) was added slowly and
the mixture was stirred. Dimethyl sulfate (285 µl, 3 mmol) was added slowly to the resulted
mixture and stirred at room temperature for 24h. Then, 5mL HCl (10%) was added to the
reaction mixture, and the product was filtered. The filtrated solid was refluxed in NaOH 1M
(10 mL) for 1 hour and then cooled and acidify with 5 mL HCl (10%). (Yellow powder, Yield
87%) For 7 and 9a methylation was performed in the same way .
Synthesis of 7-benzyloxy coumarin 3-carboxilc acid
A mixture of 7ꢀhydroxy coumarin 3ꢀcarboxylic acid (10 mmol, 2.06 g) in 30 mL methanol
was added to a 50 mL round flask and stirred in an ice bath. Thionyl chloride (30 mmol, 2.19
mL) was added dropwise to the cold mixture to make it completely soluble. The solution was
stirred at room temperature overnight, and the solvent was evaporated under vacuum. The
remaining solid was recrystallized in methanol. (Blue crystal, yield 90%) The 7ꢀhydroxyꢀ3ꢀ
coumarin carboxylic acid methyl ester (1 mmol, 0.22 g) was dissolved in N, N'ꢀdimethyl
formamide (2 mL), NaH in mineral oil (1.2 mmol, 48 mg) was added slowly and then benzyl
bromide (0.23 mL, 2 mmol) was added. The mixture was stirred overnight. After adding 10
mL of water, the light gray solid was filtered and recrystallized in ethanol (Yield 80%).To
methyl 7ꢀbenzyloxy coumarin 3ꢀcarboxylic acid (1 mmol, 310 mg) in 3 mL methanol, NaOH
(3 mmol, 120 mg) in 1 mL H2O was added and refluxed for 6 hours. The solvent was
removed under reduced pressure and the viscous product was acidified with HCl 10% to pH
3. The solid was filtered and recrystallized in ethanol. (Yield 82%, green crystal)
Synthesis of protected caffeic acid 10a23
A mixture of caffeic acid methyl ester (2.02 mmol, 392 mg), acetone (9.7 mL), toluene (36
mL), 2, 2ꢀdimethoxy propane (1 mL) and PTSA (19 mg) was refluxed in an apparatus
equipped deanꢀstark trap for 3h. The progress of the reaction was monitored by TLC. The
solvent was removed under vacuum, resulting in a green powder. Then, KOH in 10 mL water
12

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was added to the reaction mixture and was refluxed for 3h. The reaction mixture was cooled
using small pieces of ice and the pH of the mixture was adjusted to pH 6 using 2 mL of 10%
citric acid.
General process for the synthesis of conjugated peptide coumarin in solid phase
Synthesis was carried out using 2ꢀchlorotrityl chloride resin (2.0 mmol/g) following the
standard Fmoc strategy. FmocꢀSer (tꢀBu)ꢀOH (1,92 gr, 3.4 mmol) was attached to the swelled
2ꢀCTC resin (2.000 g) with N, Nꢀdiiopropylethylamine (DIEA, 3.6 mL, 20.8 mmol) in
anhydrous DCMꢀDMF (50 mL, 1:1) at room temperature for 2 h. After filtration, the
remaining trityl chloride groups were capped by a solution of DCM/MeOH/DIEA (20:2.5:1.2,
47.5 mL) for 30 min in 3 steps. Then, it was filtered and washed thoroughly with DCM (10
mL), DMF (10 mL) and MeOH (10 mL). The loading capacity was determined by weight
after drying the resin under vacuum and was 1.3. The resinꢀbound Fmocꢀamino acid was
washed with DMF (10 mL) and treated with 25 % piperidine in DMF (28 mL) for 20 min and
the resin was washed with DMF (8 mL) 3 times and DCM (8 mL). Then a solution of Fmocꢀ
GlyꢀOH (3.4 mmol, 1.0 gr), TBTU (1.1 g, 3.4 mmol), and DIEA (1.58 mL, 9.1 mmol) in 13
mL DMF was added to the resinꢀbound free amine and shaken for 1 h at room temperature.
After completion of coupling, resin was washed with DMF (10 mL) two times. The coupling
was repeated as in the same way as for other amino acid of the sequence. In all cases for the
presence or absence of free primary amino groups, Kaiser test was used. Fmoc determination
was done using UV spectroscopy method. After completion of couplings, resin was washed
with DMF (10mL, two times) and DCM (10mL, two times). The produced tripeptide was
cleaved from resin by treatment of TFA (1 %) in DCM (200 mL) and neutralization with
pyridine (2 %) in MeOH (100 mL) in 5 steps. The solvent was removed under reduced
pressure until 2 mL of the solution remained and was added droplet to flask containing 10mL
pure water and magnet stirrer. After 30 minutes stirring at room temperature, the precipitated
clean protected conjugated peptide was filtered and dried under vacuum in water .
Process for synthesis of 17
In an inert media, to a stirred solution of protected form of conjugatedꢀpeptide 17 (0.67 gr,
1mmol) in 3 mL DCM, BBr3 (1.7 mL, 0.161 mmol) in 1mL DCM was added slowly and for
6h was stirred at room temperature. Then solvent removed under vacuumed and protected
peptide was used for next step. The methylation for 21, 26, and 28 was done in the same way.
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General process for final deprotection:
Reagent K (TFA 9.1mL: Thioanisol 1mL: Phenol 1mg: H2O 1mL: EDT 0.6mL: TES 0.3mL)
was prepared and was added slowly to the protected conjugated peptide in a 25mL flask in
ice bath. After 2 hours of stirring, the solution was evaporated under vacuum. The reminded
solution (2mL) was slowly added to a flask containing 20 mL Et2O and the precipitated
peptide was separated by filtration. The dried peptide was converted to its sodium salt by
using an equivalent amount of diluted NaOH solution, the further purification was done by
preparative HPLC.
Sodium (2-Oxo-2H-chromene-3-carbonyl)-
(488 mg, 94%); 1H NMR (300 MHz, DMSOꢀ d6):
1H, ꢀCH2), 3.08 (dd = 14.3, 4.2 Hz, 1H, ꢀCH2), 3.27 (dd
3.55 (dd = 9.5, 5.4 Hz, 1H, ꢀCH2), 3.72ꢀ3.78 ( , 3H, ꢀCH, ꢀCH2), 4.73 (td
4.5 Hz, 1H, ꢀCH), 5.70 ( ,1H, OH), 6.62 ( = 8.1 Hz, 2H, HꢀAr), 7.02 (
Hz, 2H, HꢀAr ), 7.43 ( = 7.5 Hz, 1H, HꢀAr), 7.50 (
= 7.9 Hz, 1H, HꢀAr), 7.97 (dd = 7.8, 1.5 Hz,1H, NH), 8.60 (
NH), 8.85 ( , 1H, =CH), 8.98 ( , J = 7.7 Hz, 1H, NH), 9.32 (
, 1H, OH); 13C NMR (75
MHz, DMSOꢀ d6): (ppm) = 37.4, 42.3, 54.7, 58.5, 62.6, 115.1, 116.2, 118.2, 118.4,
125.3, 126.9, 130.3, 130.4, 134.3, 134.4, 148.0, 153.9, 156.1, 160.5, 168.1, 170.8,
173.2; HRꢀMS (ESI, 0.5eV) m/z= Calculated for C24H24N3O9 [M+
]+ = 498.1513,
L
-tyrosylglycyl-
(ppm) = 2.85 (dd
=9.5, 5.4 Hz, 1H, ꢀCH2),
L
-serinate (11) White powder
δ
, J
= 14.0, 8.0 Hz,
,
J
, J
,
J
m
,
J
= 8.0,
= 8.1
s
d,
J
d, J
t
,
J
d,
J
= 8.1 Hz, 2H, HꢀAr) 7.78 (
t,
J
,
J
t, J
= 5.8 Hz, 1H,
s
d
s
δ
H
Found = 498.1518. Calculated for C24H23N3NaO9 [M+Na]+ = 520.1332, found =
520.1336.
Sodium (6-Nitro-2-oxo-2H-chromene-3-carbonyl)-L-tyrosylglycyl-L-serinate (12) Light
yellow powder (519 mg. 92%); 1H NMR (300 MHz, DMSOꢀd6):
δ(ppm) = 2.86 (dd, J = 14.0,
7.8 Hz, 1H, ꢀCH2), 3.04 (dd, J = 14.0, 4.6 Hz, 1H, ꢀCH2), 3.62 (dd, J = 10.9, 4.3 Hz, 1H, ꢀ
CH2), 3.70 (dd, J = 10.9, 4.3 Hz, 1H, ꢀCH2), 3.82 (d, J = 5.7 Hz, 2H, ꢀCH2), 4.29 (dt, J = 8.5,
4.7 Hz, 1H, ꢀCH), 4.76 (td, J = 7.7, 4.7 Hz, 1H, ꢀCH), 6.62 (d, J = 8.3 Hz, 2H, HꢀAr), 7.01 (d,
J = 8.2 Hz, 2H, HꢀAr), 7.71 (d, J = 9.2 Hz, 1H, NH), 8.07 (d, J = 7.8 Hz, 1H, NH), 8.49 (d, J
= 2.9 Hz, 1H, HꢀAr), 8.52 (d, J = 2.5 Hz, 1H, HꢀAr), 8.89 (d, J = 7.7 Hz, 1H, NH), 8.98 (d, J
= 2.8 Hz, ꢀHꢀAr), 9.00 (s, 1H, =CH), 9.21 (s, 1H, OH); 13C NMR (75 MHz, DMSOꢀd6):
δ(ppm) = 37.4, 41.9, 54.6, 54.7, 61.4, 115.1, 117.8, 118.9, 120.3, 126.2, 126.9, 128.4, 130.4,
144.0, 146.9, 156.0, 157.4, 159.6, 160.0, 168.8, 170.7, 171.9; HRꢀMS (ESI, 0.5eV) m/z=
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Calculated for C24H23N4O11 [M+ H]+ = 543.1363, Found = 543.1366. Calculated for
C24H22N4NaO11 [M+Na]+ = 565.1183, found = 565.1184.
Sodium (7-Methoxy-2-oxo-2H-chromene-3-carbonyl) tyrosylglycylserinate (13) White
1
powder (522 mg, 95%); H NMR (300 MHz, DMSOꢀd6):
δ(ppm) = 2.83 (dd, J = 14.0, 8.3
Hz, 1H, ꢀCH2), 3.06 (dd, J = 14.0, 4.6 Hz, 1H, ꢀCH2), 3.20ꢀ3.46 (m, 1H, ꢀCH2, covered by
H2O), 3.56 (dd, J = 9.5, 5.3 Hz, 1H, ꢀCH2), 3.67 – 3.80 (m, 3H, ꢀCH2, ꢀCH), 3.89 (s, 3H, ꢀ
OMe), 4.72 (td, J = 8.0, 4.6 Hz, 1H, ꢀCH), 6.61 (d, J = 8.3 Hz, 2H, HꢀAr), 6.98 – 7.05 (m,
3H, HꢀAr), 7.10 (d, J = 2.4 Hz, 1H, HꢀAr), 7.48 (d, J = 6.0 Hz, 1H, HꢀAr), 7.87 (d, J = 8.7
Hz, 1H, NH), 8.55 (t, J = 5.9 Hz, 1H, NH), 8.79 (s, 1H, ꢀCH), 8.91 (d, J = 7.8 Hz, 1H, NH),
9.25 (s, 1H, OH); 13C NMR (75 MHz, DMSOꢀd6):
δ(ppm) = 35.0, 42.2, 54.4, 54.5, 56.3,
62.6, 100.3, 112.1, 113.8, 114.2, 115.0, 118.9, 120.5, 127.1, 130.3, 155.9, 156.3, 160.9,
160.9, 164.6, 168.0, 170.9, 172.7; HRꢀMS (ESI, 0.5eV) m/z = Calculated for C25H24N3O10
[MꢀH]ꢀ = 526.1467, Found = 526.1465.
Sodium (3-Oxo-3H-benzo[f]chromene-2-carbonyl)-
L-tyrosylglycyl-
L-serinate (14) Light
yellow powder (484 mg, 84%); 1H NMR (300 MHz, DMSOꢀd6):
δ(ppm) = 2.89 (dd, J = 14.1,
8.5 Hz, 1H, ꢀCH2), 3.12 (dd, J = 14.2, 4.5 Hz, 1H, ꢀCH2), 3.27ꢀ3.47 (m, 1H, ꢀCH2, covered by
H2O), 3.59(dd, J = 10.0, 5.2 Hz, 1H, ꢀCH2), 3.78 (t, J = 5.6 Hz, 2H, ꢀCH2), 3.80 (q, J = 6.3
Hz, 1H, ꢀCH), 4.79 (td, J = 7.8, 4.5 Hz, 1H, ꢀCH), 5.48 (s, 1H, OH), 6.67 (d, J = 7.8 Hz, 2H,
HꢀAr), 7.08 (d, J = 7.9 Hz, 2H, HꢀAr), 7.59 (d, J = 9.4 Hz, 2H, HꢀAr), 7.65 (t, J = 7.6 Hz, 1H,
HꢀAr), 7.73 (t, J = 7.6 Hz, 1H, HꢀAr), 8.04 (d, J = 8.1 Hz, 1H, HꢀAr), 8.27(d, J = 9.0 Hz, 1H,
HꢀAr), 8.49 (d, J = 8.5 Hz, 1H, NH), 8.73 (t, J = 5.9 Hz, 1H, NH), 9.05 (d, J = 7.4 Hz, 1H,
NH), 9.38 (s, 1H,=CH), 9.45 (s, 1H, OH); 13C NMR (75 MHz, DMSOꢀd6):
δ(ppm) = 34.1,
42.5, 55.0, 55.4, 62.7, 112.6, 115.6, 116.2, 116.3, 116.7, 122.2, 123.7, 125.5, 127.1, 128.9,
129.9, 130.4, 135.9, 143.1, 154.4, 156.2, 160.4, 161.1, 168.3, 171.1, 173.6; HRꢀMS (ESI,
0.5eV) m/z= Calculated for C28H26N3O9 [M+H]+ = 548.1669, Found = 548.1669. Calculated
for C28H25N3NaO9 [M+Na]+ = 570.1488, found = 570.1486.
Sodium (6-Methyl-2-oxo-2H-chromene-3-carbonyl)-
L-tyrosylglycyl-
L-serinate (15)
White powder (501mg, 94%); 1H NMR (300 MHz, DMSOꢀd6):
δ(ppm) = 2.37 (s, 3H, ꢀCH3),
2.83 (dd, J = 14.0, 8.4 Hz, 1H, ꢀCH2), 3.07 (dd, J = 14.0, 4.6 Hz, 1H, ꢀCH2), 3.28 (dd, J = 9.5,
5.4 Hz, 1H, ꢀCH2), 3.55 (dd, J = 9.5, 5.4 Hz, 1H, ꢀCH2), 3.70 – 3.78 (m, 3H, ꢀCH2, ꢀCH), 4.71
(td, J = 8.0, 4.5 Hz, 1H, CH), 5.6 (s, 1H, OH), 6.59 (d, J = 8.6 Hz, 2H, HꢀAr), 7.0 (d, J = 8.5
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Hz, 2H, HꢀAr), 7.39 (d, J = 8.5 Hz, 1H, HꢀAr), 7.49 (d, J = 6.0 Hz, 1H, NH), 7.56 (dd, J =
8.6, 2.1 Hz, 1H, HꢀAr), 7.74 (d, J = 2.1 Hz, 1H, HꢀAr), 8.59 (t, J = 5.9 Hz, 1H, NH), 8.76 (s,
13
1H, =CH), 8.99 (d, J = 7.7 Hz, 1H, NH), 9.29 (s, 1H, OH); C NMR (75 MHz, DMSOꢀd6):
27.2, 37.5, 42.1, 53.0, 54.6, 61.9, 115.2, 116.0, 118.0, 118.1, 126.94, 129.9, 130.3, 130.4,
134.7, 148.0, 152.2, 156.1, 160.6, 168.7, 168.8, 170.9, 172.0; HRꢀMS (ESI, 0.5eV) m/z=
Calculated for C25H26N3O9 [M+H]+ = 512.1669, Found = 512.1674, C25H25N3NaO9
[M+Na]+ = 534.1488, Found = 534.1492.
Sodium (8-Ethoxy-2-oxo-2H-chromene-3-carbonyl)-
L-tyrosylglycyl-
L-serinate (16)
White powder (545 mg, 95%); 1H NMR (300 MHz, DMSOꢀd6):
δ(ppm) = 1.41 (t, J = 6.9 Hz,
3H, ꢀCH3), 2.85 (dd, J = 13.9, 8.2 Hz, 1H, ꢀCH2), 3.07 (dd, J = 14.0, 4.6 Hz, 1H, ꢀCH2), 3.30
(dd, J = 9.6, 5.3 Hz, 1H, ꢀCH2), 3.56 (dd, J = 9.6, 5.3 Hz, 1H, ꢀCH2), 3.67 – 3.82 (m, 3H, ꢀ
CH2, ꢀCH), 4.19 (q, J = 7.0 Hz, 2H,ꢀCH2), 4.73 (td, J = 8.0, 4.6 Hz, 1H, ꢀCH), 5.63 (s, 1H,
OH), 6.62 (d, J = 8.5 Hz, 2H, HꢀAr), 7.02 d, J = 8.5 Hz, 2H, HꢀAr), 7.34 (AB-q, J = 7.8 Hz,
1H, HꢀAr), 7.40 (dd, J = 6.0, 1.7 Hz, 1), 7.48 (dd, J = 7.7, 1.5 Hz, 1H, HꢀAr), 7.51 (d, J = 6.1
Hz, 1H, NH), 8.61 (t, J = 5.8 Hz, 1H, NH), 8.80 (s, 1H, =CH), 8.98 (d, J = 7.7 Hz, 1H, NH),
9.33 (s, 1H, OH); 13C NMR (75 MHz, DMSOꢀd6): 14.6, 30.3, 42.8, 54.7, 54.9, 62.0, 64.6,
115.1, 117.2, 118.2, 119.1, 121.3, 125.3, 127.0, 130.4, 143.4, 145.6, 156.1, 159.8, 160.3,
160.6, 168.1, 170.9, 173.1; HRꢀMS (ESI, 0.5eV) m/z= Calculated for C26H26N3O10 [MꢀH]ꢀ
=
540.1624, Found = 540.1620.
Sodium (7, 8-Dihydroxy-2-oxo-2H-chromene-3-carbonyl)-
L-tyrosylglycyl-
L-serinate
(17) Brown powder (452 mg, 82%); 1H NMR (300 MHz, DMSOꢀd6):
δ(ppm) = 2.78 (dd, J =
14.0, 8.4 Hz, 1H, ꢀCH2), 2.99 (dd, J = 14.0, 4.6 Hz, 1H, ꢀCH2), 3.10ꢀ3.70 (m, 5H, ꢀCH2,
phenolic OH, alcoholic OH), 3.72 (d, J = 5.7 Hz, 2H, CH2), 3.84 (q, J = 6.2 Hz, 1H, ꢀCH),
4.64 (td, J = 8.2, 4.5 Hz, 1H, ꢀCH), 6.46 (d, J = 8.7 Hz, 1H, HꢀAr), 6.62 (d, J = 8.1 Hz, 2H,
HꢀAr), 7.01 (d, J = 8.2 Hz, 2H, HꢀAr), 7.06 (d, J = 8.6 Hz, 1H, HꢀAr), 7.60 (d, J = 6.3 Hz,
1H, NH), 8.35 (s, 1H, =CH), 8.46 (t, J = 5.8 Hz, 1H, NH), 8.99 (d, J = 7.6 Hz, 1H, NH); 13C
NMR (75 MHz, DMSOꢀd6): δ(ppm) = 37.4, 42.3, 54.7, 58.5, 62.6, 115.1, 118.2, 118.4, 125.3,
127.0, 130.0, 132.4, 134.4, 148.0, 153.9, 156.1, 160.5, 168.1, 171.9, 173.2; HRꢀMS (ESI,
0.5eV) m/z= Calculated for C24H22N3O11 [MꢀH]ꢀ = 528.1260, Found = 528.1258.
Sodium (8-Methoxy-2-oxo-2H-chromene-3-carbonyl)-
L
-tyrosylglycyl-L-serinate (18)
1
White powder (511mg, 93%); H NMR (300 MHz, DMSOꢀd6):
δ(ppm) = 2.86 (dd, J = 14.0,
8.2 Hz, 1H, ꢀCH2), 3.07 (dd, J = 14.0, 4.7 Hz, 1H, ꢀCH2), 3.40ꢀ 3.50 (m, 1H, ꢀCH2, covered
16

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by H2O), 3.58 (dd, J = 9.8, 5.2 Hz, 1H, ꢀCH2), 3.75 (dd, J = 5.8, 2.3 Hz, 2H, ꢀCH2), 3.83 (q, J
= 6.4 Hz, 1H, ꢀCH), 3.90 (s, 3H, ꢀOCH3), 4.74 (td, J = 7.9, 4.6 Hz, 1H, ꢀCH), 6.63 (d, J = 8.5
Hz, 2H, HꢀAr), 7.01 (d, J = 8.5 Hz, 2H, HꢀAr), 7.35 (dd, J=15.0, 6.07 Hz, 1H, HꢀAr), 7.40
(dd, J = 8.3, 1.5 Hz, 1H, HꢀAr), 7.47 (dd, J = 7.5, 1.6 Hz, 1H, HꢀAr), 7.52 (d, J = 6.4 Hz, 1H,
NH), 8.60 (t, J = 5.8 Hz, 1H, NH), 8.78 (s, 1H, =CH), 8.98 (d, J = 7.7 Hz, 1H, NH), 9.38 (s,
1H, OH); 13C NMR (75 MHz, DMSOꢀd6): 37.4, 42.4, 54.6, 54.9, 56.3, 62.6, 101.0, 111.1,
115.1, 119.0, 121.3, 127.0, 130.3, 143.3, 146.3, 148.2, 156.1, 160.2, 160.6, 168.1, 170.8,
173.1, 174.7; HRꢀMS (ESI, 0.5eV) m/z= Calculated for C25H26N3O10 [M+H]+ = 528.1618,
Found = 528.16171, Calculated for C25H25N3NaO10 [M+Na]+ = 550.1438, Found =
550.14353.
Sodium (7-(Diethylamino)-2-oxo-2H-chromene-3-carbonyl)-
L-tyrosylglycyl-
L-serinate
1
(19) Yellow powder (566mg, 96%); H NMR (300 MHz, DMSOꢀd6):
δ(ppm) = 1.13 (t, J =
7.0 Hz, 6 H, ꢀCH3), 2.80 (dd, J = 14.0, 8.5 Hz, 1H, ꢀCH2), 3.05 (dd, J = 14.0, 4.6 Hz, 1H, ꢀ
CH2), 3.31 (dd, J = 9.6, 5.3 Hz, 1H, ꢀCH2), 3.47 (q, J = 7.1 Hz, 4H, ꢀCH2), 3.57 (dd, J = 9.6,
5.3 Hz, 1H, ꢀCH2 ), 3.72 (d, J = 6.0 Hz, 2H,ꢀCH2 ), 3.78 (dd, J = 9.8, 3.8 Hz, 1H, ꢀCH), 4.70
(td, J = 8.1, 4.6 Hz, 1H, ꢀCH), 5.59 (brs, 1H, OH), 6.59 (d, J = 2.3 Hz, 1H. HꢀAr), 6.62 (d, J
= 8.6 Hz, 2H, HꢀAr), 6.78 (dd, J = 9.1, 2.4 Hz, 1H, HꢀAr), 7.00 (d, J = 8.6 Hz, 2H, HꢀAr),
7.50 (d, J = 6.1 Hz, 1H, NH), 7.63 (d, J = 9.1 Hz, 1H, HꢀAr), 8.55 (t, J = 6.0 Hz, 1H, NH),
8.59 (s, 1H, =CH), 8.92 (d, J = 7.7 Hz, 1H, NH), 9.34 (s, 1H, OH); 13C NMR (75 MHz,
DMSOꢀd6): 12.3, 37.5, 42.3, 44.4, 54.5, 54.6, 62.6, 96.0, 107.60 108.9, 110.2, 115.0, 127.2,
130.2, 131.7, 147.8, 152.6, 155.9, 157.3, 161.6, 161.8, 168.1, 171.2, 172.9; HRꢀMS (ESI,
0.5eV) m/z= Calculated for C28H31N4O9 [MꢀH]ꢀ = 567.2097, Found = 567.2094.
Sodium (3, 4, 5-Trimethoxybenzoyl)-L-tyrosylglycyl-
L-serinate (20) White powder
1
(481mg, 89%); H NMR (300 MHz, DMSOꢀd6):
δ(ppm) = 2.92 (dd, J = 13.8, 10.8 Hz, 1H, ꢀ
CH2), 3.06 (dd, J = 14.0, 4.1 Hz, 1H, ꢀCH2), 3.45ꢀ3.52 (m, 1H, ꢀCH2, covered by H2O), 3.59
(dd, J = 10.0, 5.2 Hz, 1H, ꢀCH2), 3.68 (s, 3H, ꢀOCH3), 3.75 (d, J = 4.2 Hz, 2H, ꢀCH2), 3.80 (s,
6H, 2ꢀOCH3), 3.93 (q, J = 6.2 Hz, 1H, ꢀCH), 4.62 (ddd, J = 12.3, 8.0, 4.0 Hz, 1H, ꢀCH), 5.28
(brs, 1H, OH), 6.64 (d, J = 8.3 Hz, 2H, HꢀAr), 7.13 (s, 2H, HꢀAr), 7.16 (d, J = 8.5 Hz, 2H, Hꢀ
Ar), 7.60 (d, J = 6.9 Hz, 1H, NH), 8.61 (t, J = 5.9 Hz, 1H, NH), 8.85 (d, J = 8.2 Hz, 1H, NH),
9.43 (s, 1H, OH); 13C NMR (75 MHz, DMSOꢀd6): 36.6, 42.6, 55.3, 55.9, 56.0, 60.1, 62.8,
105.2, 115.0, 128.6, 129.4, 130.2, 140.0, 152.5, 155.9, 166.1, 168.3, 172.3, 173.8; HRꢀMS
(ESI, 0.5eV) m/z= Calculated for C24H28N3O10 [MꢀH]ꢀ = 518.1780, Found = 518.1777.
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Sodium ((E)-3-(4-hydroxyphenyl) acryloyl)-L-tyrosylglycyl-
L-serinate (21) White powder
1
(434mg, 88%); H NMR (300 MHz, DMSOꢀd6): (ppm) = 2.73 (dd, J = 13.9, 10.2 Hz, 1H, ꢀ
δ
CH2), 3.01 (dd, J = 14.1, 4.1 Hz, 1H, ꢀCH2), 3.45ꢀ3.55 (m, 1H, ꢀCH2, covered by H2O), 3.58
(dd, J = 9.8, 5.3 Hz, 1H,ꢀCH2), 3.68 (dd, J = 9.8, 5.3 Hz, 1H,ꢀCH2), 3.73 (d, J = 5.5 Hz, 2H, ꢀ
CH2), 3.87 (q, J = 6.5 Hz, 1H, ꢀCH), 4.57 (ddd, J = 10.3, 8.2, 4.2 Hz, 1H, ꢀCH), 5.37 (brs,
1H, OH), 6.56 (d, J = 15.8 Hz, 1H, =CH), 6.64 (d, J = 8.5 Hz, 2H, HꢀAr), 6.95 (d, J = 8.8 Hz,
2H, HꢀAr), 7.07 (d, J = 8.5 Hz, 2H, HꢀAr), 7.30 (d, J = 15.7 Hz, 1H, =CH), 7.48 (d, J = 8.8
Hz, 2H, HꢀAr), 7.54 (d, J = 6.6 Hz, 1H, NH), 8.41 (d, J = 8.3 Hz, 1H, NH), 8.54 (t, J = 5.8
Hz, 1H, NH), 9.40 (brs, 2H, OH);, 13C NMR (75 MHz, DMSOꢀd6): 37.0, 42.4, 54.8, 55.1,
62.7, 114.4, 115.3, 119.5, 127.5, 128.1, 129.2, 130.1, 138.8, 155.9, 160.4, 165.4, 168.2,
172.0, 173.4; HRꢀMS (ESI, 0.5eV) m/z= Calculated for C23H24N3O8 [MꢀH]ꢀ = 470.1569,
Found = 470.1569.
Sodium ((E)-3-(3, 4-Dihydroxyphenyl) acryloyl)-
L
-tyrosylglycyl-L-serinate (22) Brown
powder (438 mg, 86%); 1H NMR (300 MHz, DMSOꢀd6):
δ(ppm) = 2.71 (dd, J = 14.0, 10.2
Hz, 1H, ꢀCH2), 3.00 (dd, J = 14.0, 4.0 Hz, 1H, CH2), 3.10ꢀ3.70 (m, 2H, ꢀCH2 covered by
H2O), 3.72 (d, J = 5.1 Hz, 2H, ꢀCH2), 3.85 (q, J = 6.2 Hz, 1H, ꢀCH), 4.53 (td, J = 9.1, 8.5, 4.0
Hz, 1H, ꢀCH), 6.39 (d, J = 15.7 Hz, 1H, =CH), 6.63 (d, J = 8.1 Hz, 2H, HꢀAr), 6.69 – 6.83
(m, 2H, HꢀAr), 6.93 (d, J = 1.9 Hz, 1H, HꢀAr), 7.07 (d, J = 8.3 Hz, 2H, HꢀAr), 7.15 (d, J =
15.7 Hz, 1H, =CH), 7.52 (d, J = 6.7 Hz, 1H, NH), 8.28 (d, J = 8.2 Hz, 1H, NH), 8.42 (d, J =
5.9 Hz, 1H, NH), 9.27 (brs, 3H, OH); 13C NMR (75 MHz, DMSOꢀd6): 37.2, 42.5, 54.8, 54.9,
62.6, 114.1, 114.9, 116.0, 118.2, 120.5, 126.3, 128.2, 130.1, 139.6, 145.7, 147.6, 155.8,
165.5, 168.2, 172.1, 173.0; HRꢀMS (ESI, 0.5eV) m/z= Calculated for C23H24N3O9 [MꢀH]ꢀ =
486.1518, Found = 486.1515.
Sodium
(6-Bromo-2-oxo-2H-chromene-3-carbonyl)-
L-tyrosylglycyl-
L
-serinate
(23)
White powder (573 mg, 96%); 1H NMR (300 MHz, DMSOꢀd6):
δ(ppm) = 2.85 (dd, J = 13.9,
8.2 Hz, 1H, ꢀCH2), 3.07 (dd, J = 14.0, 4.7 Hz, 1H, CH2), 3.40ꢀ3.51 (m, 1H, CH2, covered by
H2O), 3.56 (dd, J = 9.6, 5.3 Hz, 1H, ꢀCH2), 3.73 (d, J = 5.8 Hz, 2H, ꢀCH2), 3.75 – 3.84 (m,
1H, ꢀCH), 4.73 (td, J = 7.9, 4.6 Hz, 1H, ꢀCH), 6.62 (d, J = 8.3 Hz, 2H, HꢀAr), 7.01 (d, J = 8.3
Hz, 2H, HꢀAr), 7.46 (d, J = 9.0 Hz, 1H, HꢀAr), 7.50 (d, J = 6.4 Hz, 1H, NH), 7.87 (dd, J =
8.9, 2.4 Hz, 1H, HꢀAr), 8.23 (d, J = 2.4 Hz, 1H, HꢀAr), 8.58 (t, J = 5.9 Hz, 1H, NH), 8.79 (s,
1H, ꢀCH), 8.93 (d, J = 7.7 Hz, 1H, NH), 9.30 (brs, 1H, OH); 13C NMR (75 MHz, DMSOꢀd6):
37.4, 42.3, 54.6, 54.7, 62.5, 110.6, 115.1, 116.7, 118.5, 119.3, 120.3, 127.0, 130.3, 132.2,
136.4, 153.0, 156.0, 160.0, 160.2, 168.0, 170.7, 172.7; HRꢀMS (ESI, 0.5eV) m/z= Calculated
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for C24H23BrN3O9 [M+H]+ = 576.0618, Found = 576.0620, Calculated for C24H22BrN3NaO9
[M+Na]+ = 598.0437, Found = 598.0439.
Sodium (7-(Benzyloxy)-2-oxo-2H-chromene-3-carbonyl)-
L-tyrosylglycyl-L-serinate (24)
White powder (594 mg, 95%); 1H NMR (300 MHz, DMSOꢀd6):
δ(ppm) = 2.86 (dd, J = 14.0,
8.2 Hz, 1H, ꢀCH2), 3.07 (dd, J = 14.2, 4.8 Hz, 1H, ꢀCH2), 3.30ꢀ3.60 (m, 3H, ꢀCH2O, CH2N, ,
covered by H2O), 3.76 (qd, J = 16.6, 5.7 Hz, 2H, ꢀCH2), 4.00 (dt, J = 7.8, 4.1 Hz, 1H, ꢀCH),
4.77 (td, J = 7.8, 4.5 Hz, 1H, ꢀCH), 5.24 (s, 2H, ꢀCH2), 6.65 (d, J = 8.1 Hz, 2H, HꢀAr), 7.01
(d, J = 8.3 Hz, 2H, HꢀAr), 7.08 (dd, J = 8.7, 2.3 Hz, 1H, HꢀAr), 7.16 (d, J = 2.4 Hz, 1H, Hꢀ
Ar), 7.29 – 7.52 (m, 6H, HꢀAr), 7.86 (d, J = 8.8 Hz, 1H, NH), 8.64 (t, J = 6.0 Hz, 1H, NH),
8.79 (brs, 1H, =CH), 8.96 (d, J = 7.6 Hz, 1H, NH), 9.86 (brs, 1H, OH); 13C NMR (75 MHz,
DMSOꢀd6): δ(ppm) = 34.7, 42.4, 54.6, 55.2, 62.6, 70.3, 101.2, 112.2, 114.3, 115.2, 126.9,
128.1, 128.3, 128.6, 130.2, 131.8, 136.0, 148.2, 156.1, 160.9, 161.0, 163.6, 167.6, 170.9,
172.8; HRꢀMS (ESI, 0.5eV) m/z= Calculated for C31H30N3O10 [M+H]+ = 604.1931, Found =
604.1941, Calculated for C31H29N3NaO10 [M+Na]+ = 626.1751, Found = 626.1752.
Sodium (2-(5-Methoxy-2-oxo-2H-chromen-3-yl) acetyl)-
L-tyrosylglycyl-L-serinate (25)
White powder (524 mg, 93%); 1H NMR (300 MHz, DMSOꢀd6):
δ(ppm) = 2.71 (dd, J = 14.0,
10.0 Hz, 1H, ꢀCH2), 2.98 (dd, J = 14.0, 4.2 Hz, 1H, ꢀCH2), 3.37 (d, J = 4.3 Hz, 2H, ꢀCH2),
3.40ꢀ3.60 (m, 2H, ꢀCH2, covered by H2O), 3.77 (t, J = 5.3 Hz, 2H, ꢀCH2), 3.88 (s, 3H, ꢀOMe),
3.95 (q, J = 5.9 Hz, 1H, ꢀCH), 4.48 (td, J = 8.9, 4.2 Hz, 1H, ꢀCH), 5.24 (brs, 1H, OH), 6.65
(d, J = 8.2 Hz, 2H, HꢀAr), 7.06 (d, J = 8.3 Hz, 2H, HꢀAr), 7.12 (dd, J = 6.5, 2.8 Hz, 1H, Hꢀ
Ar), 7.18 – 7.31 (m, 2H, HꢀAr), 7.60 (d, J = 6.9 Hz, 1H, NH), 7.65 (s, 1H, ꢀCH), 8.50 (t, J =
5.9 Hz, 1H, NH), 8.56 (d, J = 8.1 Hz, 1H, NH), 9.57 (brs, 1H, OH); 13C NMR (75 MHz,
DMSOꢀd6): δ(ppm) = 36.7, 37.8, 43.3, 55.0, 55.7, 56.2, 62.8, 113.6, 115.1, 119.4, 119.7,
123.8, 124.7, 128.1, 130.2, 141.5, 142.1, 146.4, 156.0, 160.7, 168.3, 168.8, 172.0, 173.9; HRꢀ
MS (ESI, 0.5eV) m/z= Calculated for C26H26N3O10 [MꢀH]ꢀ = 540.1624, Found = 540.1622.
Sodium (2-(5-Hydroxy-2-oxo-2H-chromen-3-yl) acetyl)-
L-tyrosylglycyl-L-serinate (26)
1
White powder (500mg, 91%); H NMR (300 MHz, DMSOꢀd6):
δ(ppm) = 2.69 (dd, J = 14.0,
10.1 Hz, 1H, ꢀCH2), 2.98 (dd, J = 14.0, 4.2 Hz, 1H, ꢀCH2), 3.41ꢀ3.52 (m, 1H, ꢀCH2 , covered
by H2O), 3.37 (s, 2H, ꢀCH2), 3.57 (dd, J = 9.7, 5.3 Hz, 1H, ꢀCH2), 3.72 (d, J = 5.8 Hz, 2H, ꢀ
CH2), 3.80 (dd, J = 7.2, 5.5 Hz, 1H, ꢀCH), 4.46 (td, J = 9.3, 4.2 Hz, 1H, ꢀCH), 6.63 (d, J = 8.5
Hz, 2H, HꢀAr), 7.05 (d, J = 8.3 Hz, 2H, HꢀAr), 7.16 (dd, J = 5.7, 3.5 Hz, 1H, HꢀAr), 7.23ꢀ
7.26(m, 2H, HꢀAr), 7.50 (d, J = 6.2 Hz, 1H, NH), 7.68 (s, 1H, =CH), 8.40 (d, J = 6.1 Hz, 1H,
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NH), 8.45 (d, J = 8.6 Hz, 1H, NH), 9.20 (brs, 1H, OH); 13C NMR (75 MHz, DMSOꢀd6):
(ppm) = 36.5, 42.4, 54.8, 56.1, 62.6, 113.6, 114.9, 119.3, 119.7, 123.8, 124.5, 128.1, 130.1,
δ
141.4, 142.0, 146.4, 155.8, 157.4, 160.5, 168.1, 168.7, 171.7, 172.9; HRꢀMS (ESI, 0.5eV)
m/z= Calculated for C25H24N3O10 [MꢀH]ꢀ = 526.1467, Found = 526.1468.
Sodium ((E)-3-(4-methoxyphenyl) acryloyl)-
L-tyrosylglycyl-
L-serinate (27) White
1
powder (456 mg, 90%); H NMR (300 MHz, DMSOꢀd6):
δ(ppm) = 2.73 (dd, J = 14.1, 10.2
Hz, 1H, ꢀCH2), 3.01 (dd, J = 14.0, 4.1 Hz, 1H, ꢀCH2), 3.41ꢀ3.49 (m, 1H, ꢀCH2 , covered by
H2O), 3.58 (dd, J = 9.8, 5.3 Hz, 1H,ꢀCH2), 3.73 (d, J = 5.5 Hz, 2H, ꢀCH2), 3.76 (s, 3H, ꢀOMe)
3.87 (q, J = 6.5 Hz, 1H, ꢀCH), 4.57 (ddd, J = 10.3, 8.2, 4.2 Hz, 1H, ꢀCH), 5.37 (brs, 1H, OH),
6.56 (d, J = 15.8 Hz, 1H, =CH), 6.64 (d, J = 8.5 Hz, 2H, HꢀAr), 6.95 (d, J = 8.8 Hz, 2H, Hꢀ
Ar), 7.07 (d, J = 8.5 Hz, 2H, HꢀAr), 7.30 (d, J = 15.7 Hz, 1H, =CH), 7.48 (d, J = 8.8 Hz, 2H,
HꢀAr), 7.54 (d, J = 6.6 Hz, 1H, NH), 8.41 (d, J = 8.3 Hz, 1H, NH), 8.54 (t, J = 5.8 Hz, 1H,
NH), 9.61 (brs, 1H, OH); 13C NMR (75 MHz, DMSOꢀd6):
δ(ppm) = 36.9, 42.5, 55.1, 55.3,
62.7, 102.3, 114.4, 114.9, 115.3, 119.5, 127.5, 128.1, 129.2, 129.4, 130.1, 138.8, 155.9,
160.4, 165.4, 168.2, 172.1, 173.4; HRꢀMS (ESI, 0.5eV) m/z= Calculated for C24H26N3O8 [Mꢀ
H]ꢀ = 484.1725, Found = 484.1723.
Sodium (3, 4, 5-Trihydroxybenzoyl)-
L
-tyrosylglycyl-
L-serinate (28) Red powder (399 mg,
1
80%); H NMR (300 MHz, DMSOꢀd6):
δ(ppm) = 2.65ꢀ4.00 (m, 7H, ꢀCH2, ꢀCH, covered by
H2O), 4.50ꢀ4.60 (m, 1H, ꢀCH), 6.62 (d, J = 7.9 Hz, 2H, HꢀAr), 6.81 (s, 2H, HꢀAr), 7.07 (d, J
= 8.0 Hz, 2H, HꢀAr ), 7.54 (brs, 1H, NH), 8.09 (d, J = 8.7 Hz, 1H,NH), 8.27 (s, 1H, =CH),
9.13 (brs, 1H, OH); 13C NMR (75 MHz, DMSOꢀd6):
δ(ppm) = 36.5, 42.6, 55.3, 56.0, 60.1,
105.2, 115.0, 128.6, 129.4, 130.2, 140.0, 152.5, 155.9, 166.1, 168.3, 172.3, 173.8; HRꢀMS
(ESI, 0.5eV) m/z= Calculated for C21H22N3O10 [MꢀH]ꢀ = 476.1311, Found = 476.1308.
(S)-1-((2-(((S)-1-carboxy-2-hydroxyethyl)
hydroxyphenyl)-1-oxopropan-2-aminium 2,2,2-trifluoroacetate (29) White powder (369
mg, 84%); 1H NMR (300 MHz, DMSOꢀd6):
(ppm) = 2.84 (dd, J = 13.9, 7.4 Hz, 1H, ꢀCH2),
amino)-2-oxoethyl)
amino)-3-(4-
δ
3.01 (dd, J = 14.2, 5.3 Hz, 1H, ꢀCH2), 3.66 (qd, J = 10.9, 4.8 Hz, 2H, ꢀCH2), 3.85 (dd, J = 5.6,
2.9 Hz, 2H, ꢀCH2), 3.99 (t, J = 6.6 Hz, 1H, ꢀCH), 4.26 (dt, J = 8.4, 4.8 Hz, 1H, ꢀCH), 6.20
(brs, 4H, ꢀOH, NH2), 6.70 (d, J = 8.0 Hz, 2H, HꢀAr), 7.06 (d, J = 8.2 Hz, 2H, HꢀAr), 8.14 (d,
J = 7.7 Hz, 1H, NH), 8.74 (t, J = 5.7 Hz, 1H, NH); 13C NMR (75 MHz, DMSOꢀd6):
δ(ppm) =
36.5, 42.0, 54.0, 55.2, 61.7, 115.6, 125.1, 130.7, 156.8, 158.6, 159.0, 159.1, 168.4, 168.9,
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172.3; HRꢀMS (ESI, 0.5eV) m/z= Calculated for C14H20N3O6 [M+H]+ = 326.1352, Found =
326.1347.
Sodium (7-(Diethylamino)-2-oxo-2H-chromene-3-carbonyl) glycinate (30) Yellow
powder (296 mg, 87%); 1H NMR (300 MHz, DMSOꢀd6):
δ(ppm) = 1.14 (t, J = 7.0 Hz, 6H, ꢀ
2CH3), 3.40 – 3.55 (m, 6H, ꢀ2CH2N, ꢀCH2NH), 6.61 (d, J = 2.4 Hz, 1H, HꢀAr), 6.78 (dd, J =
9.1, 2.5 Hz, 1H, HꢀAr), 7.66 (d, J = 8.9 Hz, 1H, HꢀAr), 8.62 (s, 1H, =CH), 9.01 (t, J = 4.4 Hz,
1H, NH);13C NMR (75 MHz, DMSOꢀd6): low solubility, HRꢀMS (ESI, 0.5eV) m/z =
Calculated for C16H17N2O5 [MꢀH]ꢀ = 317.1143, Found = 317.1142.
Sodium (2-Oxo-2H-chromene-3-carbonyl)-
L-serinate (31) White powder (263 mg, 88%);
1H NMR (300 MHz, DMSOꢀd6):
δ(ppm) = 3.46 (dd, J = 9.8, 7.1 Hz, 1H, ꢀCH2), 3.75 (dd, J =
9.7, 4.9 Hz, 1H, ꢀCH2), 3.99 (dt, J = 6.9, 5.2 Hz, 1H, ꢀCH), 5.44 (brs, 1H, OH), 7.42 (t, J =
7.5 Hz, 1H, HꢀAr), 7.49 (d, J = 8.4 Hz, 1H, HꢀAr), 7.73 (t, J = 7.1 Hz, 1H, HꢀAr), 7.97 (dd, J
= 7.8, 1.6 Hz, 1H, HꢀAr), 8.89 (s, 1H, =CH), 9.32 (d, J = 5.9 Hz, 1H, NH); 13C NMR (75
MHz, DMSOꢀd6): δ(ppm) = 55.8, 62.4, 116.1, 118.5, 118.8, 125.1, 130.3, 134.0, 147.6,
154.0, 160.1, 160.2, 172.9; HRꢀMS (ESI, 0.5eV) m/z= Calculated. for C13H10NO6 [MꢀH]ꢀ =
276.0508, Found = 276.0501.
Sodium (7-(Diethylamino)-2-oxo-2H-chromene-3-carbonyl)-
L-tyrosinate (32) Yellow
powder ( 381 mg, 90%); 1H NMR (300 MHz, DMSOꢀd6):
δ(ppm) = 1.13 (t, J = 7.0 Hz, 6H, ꢀ
2CH3), 2.91 (dd, J = 13.4, 5.1 Hz, 1H, ꢀCH2), 3.03 (dd, J = 13.4, 5.4 Hz, 1H, ꢀCH2), 3.46 (q, J
= 7.2 Hz, 4H, ꢀCH2), 4.20 (q, J = 5.2 Hz, 1H, ꢀCH), 6.53 (d, J = 8.4 Hz, 2H, HꢀAr), 6.58 (d, J
= 2.3 Hz, 1H, HꢀAr), 6.77 (dd, J = 9.0, 2.4 Hz, 1H, HꢀAr), 6.89 (d, J = 8.4 Hz, 2H, HꢀAr, ),
7.64 (d, J = 9.0 Hz, 1H, HꢀAr ), 8.62 (s, 1H, =CH), 9.01 (d, J = 6.8 Hz, 1H, NH), 9.25 (brs,
1H, OH); 13C NMR (75 MHz, DMSOꢀd6):
δ(ppm) =12.3, 36.9, 44.3, 56.2, 95.9, 107.6,
109.9, 110.3, 114.5, 129.2, 130.3, 131.4, 133.9, 147.2, 152.2, 155.4, 157.1, 160. 6, 161.1,
173.1; HRꢀMS (ESI, 0.5eV) m/z= Calculated for C23H23N2O6 [MꢀH]ꢀ = 423.1556, Found =
423.1559.
Sodium
N-(((S)-2-(6-bromo-2-oxo-2H-chromene-3-carboxamido)-3-(4-
1
(tertbutoxy)phenyl) propanoyl)glycyl)-O-(tert-butyl)-
L
-serinate White powder, H NMR
(300 MHz, DMSOꢀd6):
δ(ppm) 0.81 (s, 9H, tꢀBu), 1.08 (s, 9H, tꢀBu), 2.87 (dd, J = 14.0, 7.8
Hz, 1H,ꢀCH2), 3.05 (dd, J = 14.0, 4.8 Hz, 1H, ꢀCH2), 3.50 (dd, J = 9.5, 4.0 Hz, 1H, ꢀCH2),
3.63 (t, J = 4.9 Hz, 1H, ꢀCH2), 3.83 (t, J = 5.5 Hz, 2H, ꢀCH2), 4.38 (dt, J = 8.7, 4.3 Hz, 1H, ꢀ
CH), 4.77 (q, J = 7.0 Hz, 1H,ꢀCH), 5.41 (brs, 1H, OH), 6.63 (d, J = 8.0 Hz, 2H, HꢀAr), 7.01
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(d, J = 8.0 Hz, 2H, HꢀAr), 7.42 (d, J = 8.8 Hz, 1H, HꢀAr), 7.83 (dd, J = 9.0, 3.0, 1H, HꢀAr),
7.91 (d, J = 8.0 Hz, 1H, HꢀAr), 8.18 (s, 1H, HꢀAr), 8.52 (t, J = 5.8 Hz, 1H, NH), 8.78 (s, 1H,
=CH), 8.96 (d, J = 7.7 Hz, 1H, NH); 13C NMR (75 MHz, DMSOꢀd6):
δ(ppm) = 27.2, 37.5,
42.0, 53.0, 54.6, 61.8, 72.9, 115.2, 116.9, 118.5, 119.3, 120.3, 126.9, 130.4, 132.3, 136.6,
146.8, 153.0, 156.1, 160.3, 168.8, 170.8, 171.9.
DPPH Assay: 21
Ninetyꢀsixꢀwell plates were used to evaluate five different concentrations of each sample.
Each concentration was evaluated in triplicate. A 200 µM solution of DPPH was prepared in
methanol and added to different concentrations of samples to final concentrations of 40 µM.
Sample blanks were prepared by adding methanol instead of DPPH solution to different
concentrations of samples. Plates were incubated at 25 ºC for 45 min (Heidolph titramax
1000 and incubator 1000, Germany), and absorbances were recorded at 517 nm (Powerwave
XS Microplate spectrophotometer, BioꢀTek Instruments, Inc.). The percent of freeꢀradical
inhibition (In %) was calculated as below :
(A
− A
)
blank
A
sample
In% =
×100
blank
Where Ablank is the absorbance of the control reaction (DPPH solution), and Asample is the
mean absorbance of test compounds minus the mean absorbance of each corresponding
blank. Subsequently, the concentration of solution that resulted in 50% inhibition (IC50) was
calculated by plotting inhibition percentage against each sample concentration. Butylated
hydroxytoluene (BHT) was assessed as a standard antioxidant.
FRAPAssay:20
The ferric reducing antioxidant power assay is based on the reduction of ferric
tripyridyltriazine (Fe3+–TPTZ) complex to ferrous tripyridyltriazine (Fe2+–TPTZ) at low pH.
The production of Fe2+–TPTZ generates a blue color at 593 nm absorbance (Chen et al.
2006). FRAP reagent was prepared prior to each experiment by mixing solutions a, b, and c
(a: Acetate buffer 300 mM pH 3.6, b: 10 mM TPTZ (2, 4, 6ꢀtripyridylꢀsꢀ triazine) in 40 mM
HCl, c: 20 mM FeCl3. 6 H2O) at a 10:1:1 ratio. Then, 20 µL of 20 mM stock solution of each
sample was mixed with 200 µl of FRAP reagent. The mixture was maintained at room
temperature for 10 min. Absorbance was then recorded at 593 nm (Powerwave XS
Microplate spectrophotometer, BioꢀTek Instruments, Inc.). Different concentrations of
FeSO4.7H2O (200, 400, 800, 1200, and 1600 ꢁM) were used as standard solutions and
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reacted with TPTZ reagent. Absorbance was plotted against ferrous ion concentrations. Lꢀ
ascorbic acid was used as the standard antioxidant. Results were expressed as mM ascorbic
acid equivalents per mM of each sample (Chen et al. 2006).
Antibacterial activity assay:22
Briefly, 20 mM sample stock solution in DMSO was used to prepare a serial dilution of 8–
0.06 mM in sterile Mueller Hinton broth medium in 96ꢀwell plates. An overnight culture of
each strain on nutrient agar plate was used to prepare bacterial suspensions equal to 0.5
McFarland standards. The suspensions were further diluted prior to addition to each well.
Thus, the final bacterial cell concentration in each well was approximately 0.5ꢀ1×106
cfu/mL. Cefixime and Amoxicillin were used as the reference antibiotic, and all experiments
were conducted in triplicate.
ACKNOWLEDGMENT
We thank the National Institute for Medical Research Development (NIMAD, project No
943185) for financial support.
References and notes:
1
R. J. Elias, S. S. Kellerby, E. A. Decker, Food Sci. Nutr., 2008, 48, 430ꢀ441.
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DOI: 10.1039/C8NJ00383A
Graphical Abstract
Peptides N-Connected to Hydroxycoumarin and cinnamic acid derivatives: Synthe-
sis and Fluorescence Spectroscopic, Antioxidant and Antimicrobial Properties
Hajar Golshadi Ghalehshahi, Saeed Balalaie, * Atousa Aliahmadi
OH
OH
OH
28
X4
O
O
OH
H
X3
X2
O
O
N
O Na
H
X1
X2
N
H
N
H
N
O Na
N
N
22
OH
O
O
O
H
H
O
O
O
O
X1
17
X1, X2, X3, X4 = OH, Br, NO2, MeO, EtO, BzO, Benzo,H
OH
Ascorbic
acid
If X1,X2 = OH (22)
X1,X2 = OMe, OH, H
if X3 = NO2; X1, X2, X4 = H (
)
H
If X1, X2=OH; X3, X4= H (17)
If X2= BzO; X2, X3, X4 = H (
12
N
O
3
4
1
2
H2
N
N
14
If X , X = Benzo; X , X = H (
OH
)
24
)
OH
H
O
O
OH
O
O
O
O
H
OH
N
O
Na
O
O
H
N
H
N
H
N
O Na
X
X
N
N
O
O
X
H
H
O
O
X = OH, OMe
25
If X = OH (
)
X = OH, OMe
28
)
if X = OH (
X
In this study, we developed an efficient synthesis of tripeptide Tyr-Gly-Ser and a series of conjugations
to coumarin, cinnamic and gallic acid and their antioxidant and antimicrobial activities were investigat-
ed. The first application of BBr3 as demethylating agent in synthesis of N-connecting peptide in solid
phase peptide synthesis was reported. The activity results showed that, conjugated tripeptides 7,8-
dihydroxycoumarin-tripeptide (17), caffeic acid- tripeptide (22) and gallic acid- tripeptide (28) were
found to be superior to ascorbic acid with respect to their antioxidant activity, and 12, 14, 24, and 25
exhibited the most antimicrobial activity in the series compared to amoxicillin. Additionally, fluores-
cence spectra of these conjugated peptides was investigated.