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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%).