Design and efficient synthesis of novel ascorbyl conjugated peptide with high collagen biosynthesis stimulating effects

Article abstract of DOI:10.1016/j.bmcl.2008.10.112

Collagen is critical for skin strength and elasticity, and its degradation leads to wrinkles that accompany aging. Based emphasis on the aesthetics, we tried to make a new compound that can highly stimulate collagen biosynthesis and synthesized ascorbyl c

Full text of DOI:10.1016/j.bmcl.2008.10.112

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2079–2082
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and efficient synthesis of novel ascorbyl conjugated peptide
with high collagen biosynthesis stimulating effects
Ho-Il Choi a,b, Heung-Jae Kim a, Jong-Il Park a, Eun-Ho Shin a, Dong-Won Kim a,b, Soung-Soo Kim b,
*
a 385-19 Doryong-Dong, Yusong-Gu, Peptron, Inc., Daejeon, Republic of Korea
b Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, 134 Sinchon-Dong, Seodaemun-Gu, Seoul 120-749, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Collagen is critical for skin strength and elasticity, and its degradation leads to wrinkles that accompany
aging. Based emphasis on the aesthetics, we tried to make a new compound that can highly stimulate
collagen biosynthesis and synthesized ascorbyl conjugated peptide that is a complex form connected
by succinoyl linker. We conducted several in vitro and in vivo experiments to identify if the compound
has a potent activity, comparing to the ascorbic acid only for collagen biosynthesis. Our in vitro and
in vivo result identified that ascorbyl conjugated peptide can stimulate collagen biosynthesis in human
dermis and is assumably stable in the rat skin extracts. In conclusion, we strongly suggest that ascorbyl
conjugated peptide can be used as a main ingredient for cosmetic products as well as wound healing
agents.
Received 11 June 2008
Revised 1 October 2008
Accepted 27 October 2008
Available online 31 October 2008
Keywords:
Collagen
Ascorbyl conjugated peptide
Succinoyl linker
Ó 2008 Elsevier Ltd. All rights reserved.
Collagen is one of the long, fibrous proteins in structural con-
nective tissues. Bundles of collagen and collagen fibers are major
components of the extracellular matrix that serve many functions,
such as providing support and anchorage sites for cells and sepa-
rating tissues from one another. Therefore, the amount of collagen
plays a critical role in determining the natural aging and photo-
aging. It has been known that the amount of collagen decreases
by 10–50% from the age of 30 to 80.1–4 With the finding that acti-
vated collagen synthesis in the body increases the dermal matrix,
resulting in the effects of improving wound healing, skin elasticity,
wrinkle reduction, etc., collagen has been utilized in the nutrient
supplements and therapeutic agents. Some of oligopeptides having
less than 10 amino acids, which exist in collagen, can be the small-
est activation units which relate to functions as messenger, stimu-
lator, and neurotransmitter, and take part in such physiological
activities as growth control, nursing, immunity, digestion, blood
pressure, and healing. Particularly, effective peptides in skin regen-
eration are disclosed.5 For example, a tri-, tetra-, or hexa-peptide
selected from the group consisting Gly-Lys-His, Gly-His-Lys, Gly-
Pro-Hyp(hydroxyproline), and Glu-Glu-Met-Gln-Arg-Arg promotes
the synthesis of collagen and glycosaminoglycan in the dermis,
thereby increasing moisture retention ability and elasticity of the
dermis and improving wrinkle problems. In 1993, Katayama
et al. demonstrated that a penta-peptide from type I procollagen
promotes extracellular matrix production in the human lung fibro-
blasts.6 The group observed that 80% of an enhancement in the col-
lagen production was found with penta-peptide Lys-Thr-Thr-Lys-
Ser alone, which is the minimal size necessary for stimulating
ECM biosynthesis.7
However, typically peptides tend to be cleared from the blood-
stream from minutes to hours by enzymatic digestion, thereby
greatly reducing sufficient effects. Therefore, the development of
peptide with improved safety and stability and superior skin per-
meability are needed. Peptides can be modified in many ways to
prevent degradation by endopeptidases and exopeptidases. These
include acetylation and glycosylation at N-terminals or amidation
at C-terminals and the use of unnatural amino acids at specifically
labile sites within a complete peptide.8,9 Besides modifications
above, peptides can be also engineered with incorporation of
D-amino acids and cyclization, which reduce the conformational
flexibility of linear peptides and substantially increase the stability
against the proteolysis.8 Especially palmitated penta-peptide, a
major ingredient of MatrixylTM from Sederma, is one of the most
famous materials that succeeded in enhancing its stability and per-
meability by using lipid molecules. With the respect to the efficient
synthesis, solubility, and original activity, we selected the ascorbic
acid as a coupling agent to penta-peptide, which has been known
to have strong anti-oxidative activity, collagen biosynthesis, and
whitening effects. Moreover, we could expect that the activity of
collagen production would be improved by dual effects of ascorbic
acid and peptide in HDF (human dermal fibroblast) cells. In this let-
ter, regarding these facts, we have designed an ascorbyl conjugated
penta-peptide (Fig. 1) Lys-Thr-Thr-Lys-Ser identified by Katayama
et al. In order to attach ascorbic acid to penta-peptide, we intro-
duced a succinoyl linker between two molecules. We described
* Corresponding author. Tel.: +82 2 2123 2698; fax: +82 2 362 9897.
E-mail address: kimss518@yonsei.ac.kr (S.-S. Kim).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.10.112

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H.-I. Choi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2079–2082
The linear peptide KTTKS was synthesized on 2-chlorotrityl re-
NH2
HO
OH
OH
sin preloaded with Fmoc-serine using standard Fmoc chemistry
employing HBTU/HOBt as coupling reagents. To the peptide syn-
thesized, coupled up to a N-terminal amino acid, a 20% (piperi-
dine/N-methylpyrolidone) solution was added to remove the
Fmoc groups. Then, the peptide was washed with N-methylpyroli-
done and dichloromethane, and coupled with succinoyl ascorbate
8 derivative synthesized in Scheme 1. The resulting peptide-cou-
pled vitamin C derivative was incubated in a special cleavage cock-
tail to remove the peptide protection groups. Eliminating the
benzyl groups protecting the alcohol groups at carbon positions 2
and 3 of vitamin C affords a peptide-coupled vitamin C derivative
11 (Scheme 2).10
In addition to synthesis of ACP 11, it is also important to identify
the original activity of ECM production for the development of cos-
metic materials. We examined the effect of ACP 11 in collagen bio-
synthesis by using primary cultured neonatal human dermal
fibroblasts (HDFs). The quantity of collagen produced by HDFs
was measured by the modified method described by Martens,
Gut, 1992, 33, 1664–1670. In case of the cells not treated with
any test substance, their collagen levels were designed ‘100%’. This
assay will be described in detail below.
HDFs were seeded onto 96-well plates at the number of 3000
cells and grown in DMEM containing 5% fetal bovine serum
(Invitrogen, Maryland, USA) for 24 h. After changing with fresh
assay media including 0.1% FBS, test samples were treated into
HDFs to stimulate the collagen production. After incubation for
72 h, a bit of supernatant was added into the plate which is
coated with human collagen type I antibody. Then, the plate
was placed at room temperature for 2 h to allow the reaction of
antibody and antigen. The plate was washed with phosphate buf-
fered saline with 0.5% Tween 20 three times to remove unbound
collagen, and then biotin-labeled human collagen type I antibody
was added into each well. About an hour later at room tempera-
ture, streptavidin–horseradish peroxidase (St. Louis, Sigma) was
treated into each well to detect biotin-labeled antibodies bound
to type I collagen. The quantity of collagen in the plate was trans-
formed into OD value by treating tetramethylbenzidine (St. Louis,
Sigma), as a substrate of HRP. The reaction HRP and TMB were
stopped by 1 N HCl, and then OD value was measured at
450 nm. As shown in Figure 2, expectedly ACP 11 was found to
have the strong stimulatory activity on collagen synthesis than
ascorbic acid alone at every concentration. Actually, we could no-
tice that the cytotoxicity of Pal-KTTKS was found at more than
OH
H
O
O
O
O
O
H
O
H
N
N
O
N
O
N
N
OH
H
H
O
O
OH
OH
NH2
Figure 1. Structure of ascorbyl conjugated peptide 11(ACP 11).
the synthetic progress of ascorbyl conjugated peptide 11 (ACP 11)
and were able to recapitulate its biological activity in HDF cells and
stability in the rat skin extracts, comparing to the original activity
of ascorbic acid and penta-peptide alone.
There are three parts of synthesis in ACP 11. The first part is the
synthesis of succinoyl ascorbate 8. Second part is peptide synthesis
in solid phase chemistry. And third part is the conjugation of suc-
cinoyl ascorbate and peptide.
Scheme 1 shows the synthesis of succinoyl ascorbate in part I.
To begin with, original vitamin C must be blocked for linking be-
tween vitamin C and peptide at a carbon position 5 or 6. First
acetal 2 was synthesized by reaction with acetyl chloride, which
was alkylated to obtain the 2,3-di-O-protected derivative. After
cleavage of the isopropylidene-protecting group, tritylation was
carried out using trityl chloride to produce compound 5. Succi-
noyl ascorbate 8 was synthesized by protection and deprotecion
step of compound 5 followed by acylation of 6-OH using succinic
anhydride.
HO
O
O
i
ii
O
O
HO
O
O
HO OH
HO OH
2
1
O
O
HO
HO
O
O
iii
iv
O
O
BnO OBn
BnO OBn
3
4
100
inducing effects at 100
In this experiment, the ACP 11 was synthesized efficiently and
showed high activity in collagen biosynthesis from to
1000 M concentration, comparing others. According to the exper-
l
M of concentration, even if it had the highest collagen
TrtO
TrtO
HO
lM of concentration.
O
O
v
O
O
CBzO
a
1
BnO OBn
BnO OBn
l
6
5
imental results in vitro, the conjugation form with ascorbic acid
and penta-peptide has better abilities for stimulating collagen bio-
synthesis than any others. Although ACP 11 is potentially effective
in collagen biosynthesis, it does not clearly demonstrate whether
the linker is required to induce collagen biosynthesis in human
fibroblast cells. To further access about the linker effect of ACP
11, we treated each of ascorbic acid and penta-peptide(KTTKS) into
human fibroblast cells. As shown above the data, each of ascorbic
acid and penta-peptide, which is a loss of linker, is not so much sig-
nificant active as ACP 11. This result can indicate that the linker is
indispensible to make the strong collagen expression in human
fibroblast cells. However, we could not clarify how the chemical
works in stimulating collagen biosynthesis at the cell based system
still. One possibility that can explain why ACP 11 showed the high-
est collagen synthetic effect at every concentration is that the syn-
ergic effects of linkage between ascorbic acid and penta-peptide
would contribute to the collagen biosynthesis as well as the
HO
O
vii
vi
O
CBzO
BnO OBn
7
OBn
O
OCBz
OH
BnO
O
O
O
O
8
Scheme 1. Reagents and conditions: (i) L-ascorbic acid, acetone, acetyl chloride
(60%); (ii) K2CO3, DMF, BnBr, 50 °C (90%); (iii) 3 N HCl, THF, 50 °C (90%); (iv) trityl
chloride, CH2Cl2, TEA (90%); (v) carbobenzyloxy chloride, dimethylaminopyridine,
CH3CN (80%); (vi) 1.5 N HCl, CH3CN, 50 °C; (vii) succinic anhydride, DMF, K2CO3 (2
steps, 80%).

H.-I. Choi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2079–2082
2081
NHBoc
Ot-Bu
O
Fmoc SPPS
O
O
O
H
H
FmocHN
H2N
N
N
N
H
N
H
O
O
Ot-Bu
Ot-Bu
Ot-Bu
9
NHBoc
NHBoc
BnO
OBn
OCBz
Ot-Bu
H
O
i
O
O
H
O
N
O
H
O
N
O
N
O
N
O
N
H
H
O
Ot-Bu
Ot-Bu
NHBoc
10
NH2
HO
OH
OH
ii
OH
H
iii
O
O
O
O
N
O
H
O
H
N
N
O
N
O
N
OH
OH
H
H
O
O
OH
NH2
11
Scheme 2. Reagents: (i) 8, HBTU, HOBt, DIPEA, DMF; (ii) TFA/thioanisole/H2O/triisopropylsialne (92.5:2.5:2.5:2.5 (v/v)); (iii) 10% Pd/C, H2.
one more centrifuge step to get pure supernatant for 30 min at
20,000g at 4 °C. Then, the supernatant was collected and adjusted
with PBS to a protein concentration of 4 mg/ml determined by the
method of Lowry et al.12 Test samples at the indicated concentra-
tions were added into the 4 mg/ml of homogenate solution by 1–
1 v/v ratio. The mixture was preincubated at 37 °C for 10 min,
Collagen synthesis assay
800
700
600
500
400
300
200
100
0
Pal-KTTKS
Vc
KTTKS
ACP11
Vc+KTTKS
and then 50
ll of the sample was taken out of the whole mixture
at every 10 min. Each of samples was treated with 100
ll of meth-
anol to stop the reaction and centrifuged for 5 min at 10,000g at
4 °C for the removal of precipitated proteins and the analysis using
HPLC as well.
In Figure 3, we could observe ACP 11 was maintained for more
than 6 h, even if its content was less than 10%. The HPLC peak of
penta-peptide, however, was lost in the mixed sample and even
ascorbic acid could not be detected because of its instability (data
not shown). Based on the result, we are not a hundred percent sure,
but we can expect ACP 11 is more stable than others, because the
linkage of peptide and ascorbic acid could be protected from some
kinds of proteases in the rat skin extracts.
0
1
10
100
1000
Concentration (uM)
Figure 2. Collagen biosynthesis induced by ascorbyl conjugated peptide 11 (ACP
11). Equal amounts of human normal fibroblast cells were seeded in 96-well plates
including 0.1% FBS, and were treated with various concentrations of sample. After
72 h incubation, cells were collected and tested using ELISA (p < 0.01).
In conclusion, here we demonstrated that ACP 11 has the strong
effects in collagen biosynthesis and is significantly stable in the rat
increased stability of ACP 11 from peptidases. Thus, we addition-
ally examined the stability of ACP 11 using rat skin extracts indi-
rectly as an ex vivo test. In this stability test, we observed ACP
11 appear to be maintained for more than 6 h, comparing to the
penta-peptide. It is thus possible that ACP 11 has more stable
and highly effective in the collagen biosynthesis than each of pen-
ta-peptide and ascorbic acid. Since many peptides have had trou-
bles in solving the problem of skin permeation because of the
rapid degradation by proteases and the hydrophilic properties,
we thought ACP 11 was required to be tested for its stability. To
give an answer to this, we just followed the method from the
Yamamoto group.11 After preparing for the rat skin with hair-clip-
ping step, it was cut into small pieces. Ten percent homogenates
were made with PBS by homogenizing the skin, using a POLYTRON
homogenizer (Kinematica, GmbH, Switzerland). After centrifuged
for 5 min at 3000g at 4 °C, the supernatant was required to have
In vivo Stability test
100.00
penta
ACP 11
80.00
60.00
40.00
20.00
0.00
peptide
0hr
1hr
2hr
5hr
6hr
8hr
10hr
Figure 3. Stability test of ascorbyl conjugated peptide 11 (ACP 11) in rat skin
extracts. Each of samples was analyzed by a Shimadzu reversed phase HPLC in
Thermo Hypersil Gold C18 packed columns (250 Â 4.6 mm, 5
lm sized particle)
using a water (0.1% TFA) acetonitrile (0.1% TFA) gradient.

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H.-I. Choi et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2079–2082
2. Moragas, A.; Castells, C.; Sans, M. Anal. Quant. Cytol. Histol. 1993, 15, 75.
3. Lock-Andersen, J.; Therkildsen, P.; De Fine Olivarius, F.; Gniadecka, M.;
Dahlstrøm, K.; Poulsen, T.; Wulf, H. C. Photodermatol. Photoimmunol.
Photomed. 1997, 13, 153.
4. Makrantonaki, E.; Zouboulis, C. C. Dermatology 2007, 214, 352.
5. Loren, R. P. et al. U.S. Patent 4665,054, 1987.
6. Katayama, K.; Seyer, J. M.; Raghow, R.; Kang, A. H. Biochemistry 1991, 30,
7097.
7. Katayama, K.; Armendariz-Borunda, J.; Raghow, R.; Kang, A. H.; Seyer, J. M. J.
Biol. Chem. 1993, 268, 9941.
skin extracts. For the further study, ACP 11 was needed to check
the level of permeation in the in vivo model for the industrial field.
Besides ACP 11 was required to verify what the mechanism is or
how it works in collagen biosynthesis for the scientific filed. Final-
ly, we hopefully want to suggest that ACP 11 can be used as a ther-
apeutic agent for the wound healing as well as functional materials
for cosmetics, when the intracellular mechanism of this chemical is
defined.
8. Landon, L. A.; Zou, J.; Deutscher, S. L. Curr. Drug Discov. Technol. 2004, 1, 113.
9. Werle, M.; Bernkop-Schnurch, A. Amino Acids 2006, 30, 351.
10. Synthesis of compound 11: The dried product 10 (100 mmol) was incubated in a
mixture of trifluoroacetic acid: thioanisole/water/triisopropylsilane at a ratio
of 92.5:2.5:2.5:2.5 (v/v) for 3 h to remove the peptide protection groups and
resin. Removed resin by filtration and the reaction mixture was precipitated
with ether, was collected by filtration, washed with ether and dried in vacuo.
The precipitated peptide was treated with 10% Pd/C (20 g) in methanol with
stirring for 4 h at room temperature under a hydrogen atmosphere. Thereafter,
the solution was filtrated over Celite to remove the Pd/C, concentrated under
pressure, and purified by passage through reverse phase high performance
liquid chromatography using a isocratic of 0.1% TFA in 5% acetonitrile, freezing
dried to give 11 (41 g, 50% 2 step yield) 1H NMR (CD3OD): 4.78 (m, 3H), 4.31
(m, 10H), 3.90 (m, 2H), 2.96 (q, 4H), 2.65 (m, 4H), 1.92 (m, 2H), 1.74 (m, 6H),
1.52 (m, 4H), 1.23 (q, 6H). MS m/z 822.3 (M+).
Acknowledgment
Support to this work by Ministry of Knowledge & Economy
(A25-06-14).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2008.10.112.
References and notes
11. Yamamoto, A. et al Int. J. Pharm. 2003, 250, 119.
12. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. Biol. Chem. 1951, 193,
265.
1. Shuster, S. Br. J. Dermatol. 1975, 93, 639.