Concise synthesis of catechin metabolites 5-(30,40-dihydroxyphenyl)-γ-valerolactones (dhpv) in optically pure form and their stereochemical effects on skin wrinkle-reducing activities

Article abstract of DOI:10.3390/molecules25081970

A concise and scalable synthetic route for optically pure (4S) and (4R)-5-(3⁰,4⁰-dihydroxyphenyl)-γ-valerolactones (DHPVs), catechin metabolites, has been developed via the efficient construction of a γ-valerolactone moiety from hexe

Full text of DOI:10.3390/molecules25081970

molecules
Article
Concise Synthesis of Catechin Metabolites
5-(30,40-Dihydroxyphenyl)-γ-valerolactones (DHPV)
in Optically Pure Form and Their Stereochemical
Effects on Skin Wrinkle-Reducing Activities
Joonseong Hur 1, A-Ram Kim 2, Hyun Su Kim 3, Changjin Lim 4, Taewoo Kim 3, Tae-Aug Kim 2,
Jaehoon Sim 5,
*
and Young-Ger Suh 3,
*
1
Natural Products Research Institute, Korea Institute of Science and Technology (KIST), 679 Saimdang-ro,
Gangneung 25451, Korea; hjs1120@kist.re.kr
2
3
Department of Biochemistry, School of Medicine, CHA University, 335 Pangyo-ro, Bundang-gu,
Seongnam 13496, Gyeonggi-do, Korea; tmfdkdjssl@hanmail.net (A.-R.K.); kimuara@msn.com (T.-A.K.)
Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Sciences, CHA University,
120 Haeryong-ro, Pocheon 11160, Gyeonggi-do, Korea; khs8812@snu.ac.kr (H.S.K.);
taewookim@snu.ac.kr (T.K.)
College of Pharmacy, Jeonbuk National University, Jeonju 54896, Korea; limcj@jbnu.ac.kr
College of Pharmacy, Chungnam National University, Daejeon 34134, Korea
Correspondence: jsim@cnu.ac.kr (J.S.); ygsuh@snu.ac.kr or ygsuh@cha.ac.kr (Y.-G.S.);
Tel.: +82-42-821-5938 (J.S.); +82-31-850-9300 (Y.-G.S.)
4
5
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 3 April 2020; Accepted: 21 April 2020; Published: 23 April 2020
Abstract: A concise and scalable synthetic route for optically pure (4S) and (4R)-5-(30,40-
dihydroxyphenyl)-
efficient construction of a
γ
-valerolactones (DHPVs), catechin metabolites, has been developed via the
-valerolactone moiety from hexenol. Noticeably, the different skin
γ
wrinkle-reducing activities of each metabolite were revealed via our unique syntheses of DHPVs in
an enantiomerically pure form.
Keywords: catechin metabolite; γ-valerolactone; concise synthesis; skin wrinkle-reducing activity
1. Introduction
Flavan-3-ols are the most abundant class of naturally occurring polyphenols widely found in
dietary plants, especially green tea, red wine, and cocoa extract [1]. The monomeric flavan-3-ols
structurally possess a 2-phenyl-3,4-dihydro-2H-chromen-3-ol skeleton with two chiral centers at the
C2 and C3 position (Figure 1), and each natural product including (epi)afzelechin, (epi)catechin, and
(epi)gallocatechin contains a different number of hydroxyl groups on the aromatic ring. This class
of compounds has received significant attention for their protective effects against various chronic
diseases, such as cancer, cardiovascular disease, diabetes, and neurodegenerative disease [
after consumption of polyphenol-rich foods, only a few flavan-3-ols are able to directly reach the
circulatory system due to their poor absorbability [ ]. Instead, substantial quantities of intact
2]. However,
3,4
flavan-3-ols are degraded into simple metabolites by the microbiota in the colon and then absorbed into
the blood stream [5]. Therefore, synthetic and biological studies focused on each single metabolite of
the flavan-3-ols are essential for understanding the beneficial effects of polyphenols on human health.
5-(30,40-Dihydroxyphenyl)-
generated by microbial degradation in the colon (Figure 1) [
enantiomer is derived from the C3 configuration of parent flavan-3-ols and both enantiomers are
naturally found. Upon incubation of naturally abundant (+)-catechin and ( )-epicatechin with rat
γ-valerolactone (DHPV;
1) is a major metabolite of (epi)catechin
6,7
]. The C4-stereochemistry of each
−
Molecules 2020, 25, 1970; doi:10.3390/molecules25081970
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1970
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intestinal microbiota, the C3 configuration of each flavan-3-ol was retained during the catabolism
(Figure 1) [ ]. Since the first report on the metabolic fate of (+)-catechin in rabbits by Watanabe et al. [9]
there have been intensive pharmacokinetic studies on DHPV [10 12] after the intake of a single
flavan-3-ol or polyphenol-rich foods in various living organisms for the past half century. However,
investigation on its biological effects were relatively rare, except for a few reports [13 16]. Despite
8
,
–
–
the lack of studies on DHPVs’ role in the biological system, several papers recently reported that
DHPV exhibits a protective effect against UV-induced skin inflammation [17] or photoaging [18].
Particularly, Lee and Kim et al. [18] recently reported that oral administration of DHPV reduced
UV-induced skin-wrinkling via the regulation of genes involved in dermal matrix maintenance. These
observations encouraged us to develop a novel protocol for the synthesis of DHPV to secure optically
pure metabolites in sufficient quantities to utilize their potential as promising cosmeceutical ingredients.
Figure 1. Structures of catechins and their metabolite DHPVs.
Herein, we report a highly concise and scalable synthetic route for optically pure DHPVs. Moreover,
inspired by evidence that the bioavailability of (epi)catechin depends on their stereochemistry [19],
we envisioned that (R)- and (S)-DHPV might exhibit different anti-photoaging activities. Thus, we
describe our unique synthesis of both DHPV enantiomers and their MMP-1 [20
which is a representative biomarker of UV-induced skin wrinkling [22].
,21] inhibitory activities,
2. Results and Discussion
The syntheses of racemic or optically active DHPV were reported by several groups for further
biological investigation or testing of their own catalytic system, as summarized in Figure 2. In 1959,
Watanabe et al. achieved the first total synthesis of racemic DHPV in 6% overall yield over four
steps [
vigorous refluxing conditions to obtain the
synthesis of racemic DHPV to secure the tea catechin metabolites for testing their anticancer and
anti-inflammatory activities [23]. The -valerolactone core was constructed in a stepwise manner,
9]. The key step included an epoxide opening/lactonization/decarboxylation cascade under
γ
-valerolactone core. Lambert et al. reported another
γ
including epoxidation, regioselective epoxide opening, hydrolysis, and dehydrative lactonization,
which result in low overall yield. The first synthesis of optically pure DHPVs was accomplished by
Nakajima et al. in 2008 [24] and improved in 2010 [25], using (R)- and (S)-benzyl glycidyl ether as
chiral sources. Although the γ-valerolactone formation still included protection/deprotection and
homologation steps, overall yield was substantially increased up to 36%. Most recently, Curti et al.
reported an enantioselective method [26]. Their elegant catalytic approach exploiting vinylogous

Molecules 2020, 25, 1970
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Mukaiyama aldol reaction was able to introduce
γ
-lactone moiety onto benzylic position in a single
step with moderate to high stereoselectivity, although global steps was not successfully reduced.
Figure 2. Previous synthetic approaches toward DHPV.
Collectively, these synthetic routes feature the addition of requisite carbons onto the aromatic part,
followed by the construction of the γ-valerolactone structure. However, despite excellent previous
syntheses, the desire for efficient DHPV preparation in terms of number of steps, overall yield,
scalability, mild conditions, and stereocontrol still remains. In particular, the previous methods were
hampered by multiple steps or harsh conditions to construct the γ-valerolactone core.
To overcome these drawbacks, we employed an oxidative lactonization [27] of a hexenol rac-
4
generated by a regioselective epoxide opening reaction of rac- 24 25 28], which is able to conveniently
3
[
,
,
provide both enantiomers of DHPV with excellent enantiomeric excess and in a practical and
efficient manner.
We initially examined our synthetic strategy by synthesizing racemic DHPV (Scheme 1). Aryl
bromide
hexenol
2
4
, which is commercially available, was lithiated and coupled with racemic epoxide
in 81% yield with perfect regioselectivity. To obtain the -valerolactone core in a concise way,
, as summarized in Table 1. A classical
via a sequence of dihydroxylation,
3 to afford
γ
we investigated several conditions of oxidative lactonization of
three-step protocol successfully afforded the desired lactone
4
5
oxidative cleavage, and corresponding lactol oxidation (71%; entry 1). Confirming synthetic efficiency
and practicality, two-step protocols were examined by combining dihydroxylation-oxidative cleavage
sequence in a single operation (entry 2–4). Although the yield was slightly decreased, the use of NaIO4
and a catalytic amount of OsO4 gave moderate yield of desired product
5 (51%, entry 2). As expected,
the addition of base additives [29], such as pyridine or 2,6-luthidine, improved yield up to 73% (entry
3 and 4). Hexenol
4 did not undergo the one-step oxidative lactonization using catalytic OsO4 and
Oxone® as a co-oxidant [27].
With optimized condition in hand for the synthesis of lactone 5 (entry 4), demethylation of 5 using
boron tribromide was successfully conducted to provide 1.2 g of the desired phenolic metabolite 1 in
69% yield.
We next turned our attention to the syntheses of optically pure DHPVs (Scheme 2). Optically
active (S)-
route. For the synthesis of (R)-
the C4-stereocenter in the secondary alcohol (S)-
to yield alcohol (R)- . We obtained (R)- via the same procedure applied for the synthesis of (S)-4.
1
was conveniently obtained from optically pure epoxide (S)-
, as an antipode of (S)- , we initially inversed the stereochemistry of
by a Mitsunobu reaction, followed by methanolysis
3 via our established synthetic
1
1
4
4
1

Molecules 2020, 25, 1970
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NMR spectra (800MHz for 1H-NMR) and chiral HPLC analysis of the synthetic DHPVs (Supporting
Information, Figures S5–S7) confirmed almost perfect purity for biological experiments including
skin-protection against UV-irradiation.
Scheme 1. Synthesis of racemic DHPV (rac-1).
Table 1. Optimization of oxidative lactonization conditions.
Entry
Condition (Equivalent)
Result
(i)
OsO4 (0.02), NMO (4.0) in acetone/water
(ii) NaIO4 (2.0) in THF/water
(iii) NaOAc (2.0), PCC (2.0) in CH2Cl2
1
2
71% (3 steps)
(i)
(ii) NaOAc (2.0), PCC (4.0) in CH2Cl2
(i) OsO4 (0.01), NaIO4 (4.0), pyridine (2.0) in dioxane/water
(ii) NaOAc (2.0), PCC (4.0) in CH2Cl2
(i) OsO4 (0.01), NaIO4 (4.0), 2,6-lutidine (2.0) in dioxane/water
(ii) NaOAc (2.0), PCC (2.0) in CH2Cl2
OsO4(0.01), Oxone® (4.0) in DMF
No reaction. The bold is lactonization condition after optimization.
OsO4 (0.01), NaIO4 (4.0) in dioxane/water
51% (2 steps)
64% (2 steps)
3
4
5
73% (2 steps)
N.R. a (1 step)
a
Scheme 2. Synthesis of optically pure DHPVs ((S)-1 and (R)-1).
We finally examined the stereochemical effects on the skin wrinkle-reducing activity of the
DHPVs, which was previously reported by us [18]. The ROS in skin tissue is frequently increased
by UV radiation, which consequently causes an increase in expression of MMP-1 and transcription
activity of AP-1. AP-1 is a transcription factor of MMP-1, which breaks down collagen fiber, and is a
composition of skin dermis extracellular matrix. The degradation of collagen causes structural collapse
of skin dermis. Therefore, the increase of AP-1 and MMP-1 induces skin wrinkles as a phenotype of
photo-aging. For these reasons, we envisioned that MMP-1 could be an appropriate biomarker to
evaluate the skin-protecting activities of the synthetic DHPVs from UV-irradiation.

Molecules 2020, 25, 1970
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After the human dermal fibroblasts (HDF) was incubated with the tested metabolites at 1
µM
concentration under UV light for a day, the suppressive activities of the racemic, (S), and (R)-DHPVs (
1)
against the photoinduced MMP-1 protein expression [22] was evaluated. Interestingly, the (S)-DHPV
isomer revealed inhibitory effects two-folds greater than the (R)-DHPV isomer in quantitative real-time
PCR and western blot assay, as summarized in Figure 3. Currently, we do not fully understand
the stereochemical effects of DHPVs on their suppressive activities against UV-induced MMP-1
expression. However, these results confirmed that the metabolites of the flavan-3-ols possess different
suppressive effects in the photoinduced MMP-1 protein expression, which are quite dependent on the
C4-stereocenter of the DHPV.
Figure 3. Suppressive activities of the racemic and optically pure DHPVs against UV-induced MMP-1
expression assessed by quantitative real-time PCR (top) and western blot analysis (bottom).
3. Materials and Methods
3.1. General Experiment
Unless noted otherwise, all starting materials, reagents, and solvents were obtained from
commercial suppliers and used without further purification. (S)-( )-1,2-Epoxy-5-hexene was purchased
−
from BOC Sciences. Flash column chromatography was performed using silica gel 60 (230–400 mesh,
Merck, Kenilworth, NJ, USA) with the indicated solvents. Thin-layer chromatography was performed
using 0.25 mm silica gel plates (Merck, Kenilworth, NJ, USA). Optical rotations were measured with
JASCO P-2000 digital polarimeter (JASCO, Easton, MD, USA) at ambient temperature using 10 mm cell
of 0.1 mL capacity. Infrared spectra were recorded on a JASCO FT-IR-4200 spectrometer (JASCO, Easton,
MD, USA). High resolution mass spectra were obtained with Agilent Q TOF 6530 (Agilent, Santa
Clara, CA, USA). High performance liquid chromatography (HPLC) experiments were performed
with Waters 1525 binary pump (Waters, Milford, MA, USA) and Waters 2489 UV/Visible detector
(Waters, Milford, MA, USA) at 35 ◦C. The 1H- and 13C-NMR spectra were recorded using BRUKER
AVANCE-800 (Bruker, Billerica, MA, USA). Chemical shifts are expressed in parts per million (ppm,
δ
)
1
downfield from tetramethylsilane and are referenced to the deuterated solvent (CHCl3). H-NMR data
were reported in the order of chemical shift, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet;
ABq, AB quartet; quint, quintet; m, multiplet and/or multiple resonances), number of protons, and
coupling constant in hertz (Hz).

Molecules 2020, 25, 1970
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3.2. Experimental Part
3.2.1. Preparation of 1-(3,4-dimethoxyphenyl)hex-5-en-2-ol (rac-4)
To a solution of 4-bromo-1,2-dimethoxybenzene (3.0 g, 13.8 mmol) in THF (15 mL) was added
n-butyllithium (2.5 M in hexane, 6.1 mL, 15.2 mmol) dropwise at
78 ◦C. The mixture was stirred for
−
40 min at the same temperature and BF3OEt2 (1.9 mL, 15.2 mmol) and 2-(but-3-en-1-yl)oxirane (1.4 g,
13.8 mmol) was consecutively added. After stirring for 24 h at room temperature, the reaction mixture
was quenched with water and extracted with EtOAc. The combined organic layer was washed with
aqueous NaHCO3, dried over MgSO4, and concentrated in vacuo. The residue was purified by flash
column chromatography (EtOAc/Hexane = 1:5) to give 2.6 g (81%) of rac-4 as a colorless oil.
1H-NMR (800 MHz, CDCl3)
δ 6.80 (d, J = 8.1 Hz, 1H), 6.73 (dd, J = 8.1, 1.9 Hz, 1H), 6.71 (d, J = 1.9
Hz, 1H), 5.82 (ddt, J = 16.9, 10.2, 6.7 Hz, 1H), 5.03 (ddd, J = 17.1, 3.4, 1.6 Hz, 1H), 4.96 (ddd, J = 10.3, 2.9,
1.2 Hz, 1H), 3.85 (s, 3H), 3.84 (s, 3H), 3.79 (ddt, J = 12.5, 8.4, 4.3 Hz, 1H), 2.76 (dd, J = 13.7, 4.2 Hz, 1H),
2.57 (dd, J = 13.7, 8.6 Hz, 1H), 2.28 – 2.21 (m, 1H), 2.15 (ddt, J = 6.8, 5.6, 4.0 Hz, 1H), 1.66 – 1.53 (m, 4H).;
13C-NMR (200 MHz, CDCl3)
δ
148.9, 147.7, 138.4, 130.9, 121.3, 114.8, 112.5, 111.3, 72.1, 55.9, 55.8, 43.6,
35.8, 30.1.; HRMS (FAB+) calcd for C14H20O3 (M+) 236.1412, found 236.1417; IR (thin film, neat) νmax
3493, 2935, 1517, 1262, 1236, 1156, 1141, 1029 cm−1
.
3.2.2. Preparation of 5-(3,4-dimethoxybenzyl)dihydrofuran-2(3H)-one (rac-5)
To a solution of 1-(3,4-dimethoxyphenyl)hex-5-en-2-ol (2.6 g, 10.9 mmol) in 1,4-dioxane (10 mL)
and water (3 mL) was added 2,6-lutidine (2.6 mL, 21.8 mmol), OsO4 (0.1 M in toluene, 1.1 mL, 0.1 mmol),
and NaIO4 (9.4 g, 43.7 mmol) at 0 ◦C. After stirring for 17 h at room temperature, the reaction mixture
was quenched with water and extracted with CH2Cl2. The combined organic layer was washed with
brine, dried over MgSO4, and concentrated in vacuo to afford the corresponding lactol. The lactol was
used in the next step without further purification.
To a solution of the resulting lactol in CH2Cl2 (13 mL) was added NaOAc (1.8 g, 21.8 mmol) and
PCC (4.7 g, 21.8 mmol) at room temperature. After stirring for 1 h at the same temperature, the reaction
mixture was concentrated and the residue was directly purified by flash column chromatography
(EtOAc/Hexane = 1:2) to give 1.9 g (73%) of rac-5 δ 6.78 (d, J
as white solid.1H-NMR (800 MHz, CDCl3)
= 8.0 Hz, 1H), 6.73 (dd, J = 8.0, 2.0 Hz, 1H), 6.72 (d, J = 1.9 Hz, 1H), 4.72–4.67 (m, 1H), 3.84 (s, 3H), 3.83
(s, 3H), 2.94 (dd, J = 14.2, 5.9 Hz, 1H), 2.87 (dd, J = 14.2, 6.0 Hz, 1H), 2.41 (ddd, J = 17.8, 9.7, 8.9 Hz, 1H),
2.29 (ddd, J = 17.7, 9.5, 4.8 Hz, 1H), 2.22 (dddd, J = 12.8, 9.8, 6.9, 4.8 Hz, 1H), 1.91 (dtd, J = 12.9, 9.2, 7.3
Hz, 1H).; 13C-NMR (200 MHz, CDCl3)
δ
177.0, 148.9, 148.0, 128.3, 121.5, 112.6, 111.2, 80.8, 55.8, 55.8,
40.7, 28.6, 26.8.; HRMS (FAB+) calcd for C13H16O4 (M+) 236.1049, found 236.1045; IR (thin film, neat)
νmax 2938, 1770, 1517, 1465, 1262, 1238, 1178, 1159, 1144, 1027 cm−1
.
3.2.3. Preparation of 5-(3,4-dihydroxybenzyl)dihydrofuran-2(3H)-one (rac-1)
To a solution of 5-(3,4-dimethoxybenzyl)dihydrofuran-2(3H)-one (1.9 g, 8.0 mmol) in CH2Cl2
(8 mL) was added BBr3 (1.0 M in CH2Cl2, 33.6 mL, 33.6 mmol) at
78 ◦C. After stirring for 10 h at room
−
temperature, the reaction mixture was quenched with water and extracted with EtOAc. The combined
organic layer was washed with brine, dried over MgSO4, and concentrated in vacuo. The residue was
purified by flash column chromatography (EtOAc/Hexane = 2:1) to give 1.2 g (69%) of rac-
1
as white
1
solid. H-NMR (800 MHz, MeOD)
δ
6.69 (d, J = 8.0 Hz, 1H), 6.68 (d, J = 2.0 Hz, 1H), 6.55 (dd, J = 8.0,
2.1 Hz, 1H), 4.75–4.69 (m, 1H), 2.86 (dd, J = 14.1, 6.1 Hz, 1H), 2.78 (dd, J = 14.1, 6.1 Hz, 1H), 2.50–2.44
(m, 1H), 2.32 (ddd, J = 17.8, 9.5, 4.8 Hz, 1H), 2.23 (dddd, J = 12.7, 9.8, 6.9, 4.8 Hz, 1H), 1.97–1.92 (m,
1H).; 13C-NMR (200 MHz, MeOD)
δ
181.2, 147.1, 146.0, 129.9, 122.7, 118.5, 117.2, 84.1, 42.2, 30.3, 28.6.;
HRMS (FAB+) calcd for C11H13O4 [M + H] + 209.0814, found 209.0820; IR (thin film, neat) νmax 3377,
1753, 1520, 1446, 1285, 1230, 1191, 1116 cm−1
.

Molecules 2020, 25, 1970
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3.2.4. Preparation of (S)-1-(3,4-dimethoxyphenyl)hex-5-en-2-ol ((S)-4)
Alcohol (S)-
4
was prepared via the same procedure as that of rac-
4
using (S)-(
−
)-1,2-epoxy-5-hexene
as a starting material. [α]2D0 17.5 (c 0.15, CHCl3).
3.2.5. Preparation of (R)-1-(3,4-dimethoxyphenyl)hex-5-en-2-ol ((R)-4)
To a solution of (S)-1-(3,4-dimethoxyphenyl)hex-5-en-2-ol (297 mg, 1.3 mmol) in THF (13 mL) was
added p-nitrobenzoic acid (315 mL, 1.9 mmol), DIAD (0.4 mL, 1.9 mmol), and PPh3 (496 mg, 1.9 mmol)
at 0 ◦C. After stirring overnight at room temperature, the reaction mixture was concentrated and the
residue was directly purified by flash column chromatography (EtOAc/Hexane = 1:9) to give 496 mg
(100%) of (R)-1-(3,4-dimethoxyphenyl)hex-5-en-2-yl-4-nitrobenzoate as a yellow oil.
To a solution of above (R)-nitrobenzoate (496 mg, 1.3 mmol) in MeOH (13 mL) was added K2CO3
(267 mg, 1.9 mmol) at room temperature. After stirring for 1 h at the same temperature, the reaction
mixture was quenched with water and extracted with EtOAc. The combined organic layer was washed
with brine, dried over MgSO4, and concentrated in vacuo. The residue was purified by flash column
chromatography (EtOAc/Hexane = 1:5) to give 236 mg (77%) of (S)-
(c 1.0, CHCl3).
4
as a colorless oil. [α]D20
20.5
−
3.2.6. Preparation of (S)-5-(3,4-dimethoxybenzyl)dihydrofuran-2(3H)-one ((S)-5)
Lactone (S)- was prepared via the same procedure as that of rac- using (S)-
[α]2D0 21.4 (c 1.0, CHCl3).
5
5
4 as a starting material.
3.2.7. Preparation of (R)-5-(3,4-dimethoxybenzyl)dihydrofuran-2(3H)-one ((R)-5)
Lactone (R)-5 was prepared via the same procedure as that of rac-5 using (R)-4 as a starting
material. [α]2D0 −29.3 (c 1.0, CHCl3).
3.2.8. Preparation of (S)-5-(3,4-dihydroxybenzyl)dihydrofuran-2(3H)-one ((S)-1)
DHPV (S)- was prepared via the same procedure as that of rac- using (S)-
[α]2D0 8.1 (c 1.0, MeOH).
1
1
5
as a starting material.
3.2.9. Preparation of (R)-5-(3,4-dihydroxybenzyl)dihydrofuran-2(3H)-one ((R)-1)
DHPV (R)-5 was prepared via the same procedure as that of rac-1 using (R)-5 as a starting material.
[α]2D0 −10.5 (c 1.0, MeOH).
3.2.10. Cell Culture and Treatments
The HDFs (5
×
105 cells) were seeded onto a 100Φ culture dish. HDFs were cultured in DMEM
with 10% (v/v) feral bovine serum and 1% (v/v) penicillin/streptomycin at 37 ◦C and 5% CO2 for 48 h.
Monolayer cultures of the HDFs in the culture dish were washed with PBS and cultured in serum free
media for 24 h. HDFs were washed twice with PBS and exposed to UVB irradiation (5 mJ/cm2). After
removal of the PBS, the HDFs were treated with each DHPV isoform in serum-free culture media for
24 h. The HDFs were harvested to extract RNA or protein.
3.2.11. Quantitative Real-time Polymerase Chain Reaction
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The total RNA
was quantified via nanodrop, and equal amounts (1.5 µg) of total RNA was reverse-transcribed using
MMLV reverse transcriptase. To quantify the MMP-1 RNA expression revel, the cDNA was amplified
with specific primers. The sequences of the PCR primers used for the amplifications of GAPDH and
MMP1 were as follows. GAPDH forward: 50-ACCACAGTCCATGCCATCAC-30, GAPDH reverse:
50-TCCACCACCCTGTTGCTGTA-30; MMP1 forward: 50-CTGCTTACGAATTTGCCGACAGA-30,

Molecules 2020, 25, 1970
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MMP-1 reverse: 50-GTTGTAGGGAAGCCAAAGGAGCTG-30. Quantitative real time PCR analysis
was performed using the Power Green PCR Master Mix on a CFX ConnectTM Real-Time PCT Detection
System (Bio-Rad, Hercules, CA, USA). GAPDH was used as the normalization gene in these studies.
The relative expression levels of the target genes were◦given by 2∆∆Ct. GAPDH and MMP-1 were
◦
amplified for 40 cycles as follows: denaturation at 95 C for 30 s, annealing at 60 C for 30 s, and
extension at 72 ◦C for 30 s for 40 cycles. The experiments were performed in triplicate.
3.2.12. Western Blot Analysis
The HDFs were lysed with PRO-PREPTM protein extraction solution (iNtRON Biotechnology, MA,
USA). The cell lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
and transferred to 0.2 µm pore size PVDF membrane (Millipore, Burlington, MA, USA). The membrane
was blocked with 3% BSA (RMBIO, Missoula, MT, USA) in Tris-buffered saline Tween-20 for 1 h and
incubated with specific primary antibody at 4 ◦C overnight against MMP-1 (1:1000, Abcam, Cambridge,
MA, USA), β-actin (1:3000, Abcam, Cambridge, MA, USA), followed by incubation with IgG-HRP
(1:1000, GeneTex, Irvine, CA, USA). Protein bands were visualized by ClarityTM Western ECL Blotting
Substrates (Bio-Rad, Hercules, CA, USA).
4. Conclusions
In conclusion, we developed a scalable four-step synthetic route (41% overall yield) for optically
pure DHPVs. Our unique synthetic procedure, a key part of which involved efficient construction of the
γ
-valerolactone moiety via an oxidative lactonization, enabled us to elucidate the different biological
activities of each metabolite. In particular, we confirmed that the metabolites of the flavan-3-ols
possess different suppressive effects in the photo-induced MMP-1 protein expression, which are quite
dependent on the C4-stereocenter of the DHPV.
Based on these results, further studies, including the structure-activity relationship and mode of
action on DHPVs are in progress.
Supplementary Materials: The following are available online, Copies of NMR spectra (1H and 13C) and Chiral
HPLC chromatogram of relevant compounds.
Author Contributions: J.H. and J.S. performed the experiments and wrote the manuscript; A.-R.K., H.S.K., and
C.L. performed the experiments and analyzed the data; T.K. and T.-A.K. analyzed the data and reviewed the
manuscript; Y.-G.S. designed and supervised the study. All authors have read and agreed to the published version
of the manuscript.
Funding:
This research was funded by the National Research Foundation of Korea, grant
number NRF-2020R1A2C1010552.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
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