In this work, we used the linear RGD hexapeptide or
nonapeptide as the templates to introduce two kinds of cyclic
RGD peptides, cyclo(RGDfK) and cylco(CRGDRGDC), onto the
C-terminus and side chains respectively. These linear RGD
peptide templates were expected to partially contribute to integrin
interaction and enhance the affinity of the full structure. For
cyclic RGD peptides, we sought to assemble cyclo(RGDfK) at
amino group of cyclo(RGDfK). After cleavage, the mono-cyclic
RGD peptide comprising the Asp side chain benzyl ester was
obtained, and benzyl ester was hydrazinolyzed by a 5%
hydrazine hydrate in DMF solution to obtain the corresponding
peptide hydrazide. Successively, it was treated with NaNO at a
2
pH 2-3 to give the carbonyl azide, then the solution was adjusted
to pH 5.5 and MPAA(4-Mercaptophenylacetic acid) was added
the
C-terminus
and
introduce
cyclo(RGDfK)
or
to form a thioester. The side-chain thioester reacted with peptide
cyclo(CRGDRGDC) onto the side-chain Asp of the RGD
CRGDRGDC via native chemical ligation and the resulted
bran
ched
pepti
de
was
oxidi
zed
with
air to
give
the
dual-
2 2 2
Scheme 2: Synthesis of compound P3. Reaction conditions: (a) 5% NH NH in DMF, r.t., 30 min,95%; (b) NaNO , pH 2, –10 °C, 15 min,
9
5%; (c)MPAA, pH 5.5, –10 °C, 15 min, 95%; (d) CRGDRGDC (5 equiv), pH 6.0, 4 °C, 12 h, 80%; (e) 20% DMSO, air, r.t., 4 h, 95%; (f)
(CH2) COOSu (2-5 equiv), pH 7.5, r.t., 15 min, 95%.
N
3
3
templates to investigate their activities. For late-stage conjugation
with small-molecule drugs or fluorescent dyes, we assembled an
azido group at the N-terminus of these dual-cyclic RGD peptides.
As shown in Scheme 1, a series of novel dual-cyclic peptide
derivatives were designed and synthesized to understand the SAR
of different branch sites and cyclic RGD motif combination.
cyclic RGD peptide. The N-terminus of the peptide was modified
with the active ester N (CH ) CO Su (Su=succinimidyl) to carry
3
2 3
2
an azido tag for conjugation with a small-molecule drug or a
fluorescent dye (SI, Fig. S9.). In order to facilitate the
comparison of integrin binding activity, we also synthesized an
azide-modified cyclic peptide P6 and a monocyclic peptide P5
comprising a linear RGD hexapeptide (SI. Fig. S1).
The synthesis of the target peptides followed our previously
2
0,21
published method for branched cyclic peptides
with
In this new procedure, on-resin synthesis of the C-terminal
cyclic peptide cyclo(RGDfK) simplified the late-stage processes
by avoiding the introduction of two different cyclic peptides at
the C-terminus and the side chain via an orthogonal design on
both reactive sites. Meanwhile, consumption of multiple
equivalents of cyclic peptide motif is also avoided, which is more
economical. In addition, we also optimized the process to reduce
the cyclization side reaction on side-chain Asp. During the
deprotection of the Fmoc with 20% piperidine for 10 minutes, the
side-chain benzyl ester may partially be removed under this basic
condition that lead to the cyclization of the side-chain Asp
carboxyl group with the adjacent amide nitrogen atom (Fig. 1,
Panel a). In an optimized condition, we conducted the de-Fmoc
by 3-minutes treatment of 20% piperidine, effectively reducing
the formation of cyclized by-products to a limit level (Fig. 1,
Panel a').
significant optimization. Side-chain benzyl ester on Asp was the
precursor that was readily converted to a hydrazide and an acyl
azide successively. Then, the corresponding cyclic peptide
moieties were assembled by direct amidation or side-chain native
chemical ligation. Peptides P1 and P2 were synthesized
2
1
according to our previous method (SI, Figs. S3, S11.). The
synthesis of P3 and P4 was dramatically improved as shown in
Scheme 2 and Figure 1. In the new procedure, we synthesized the
cyclo(fKRGD) on the solid support that avoided the twice
amidation with acyl azide or thioester in the previous method.
Thereafter, the RGD linear peptide was elongated on side-chain
With the target RGD dual-ring peptides in hands, we sought to
investigate their integrin binding properties. An integrin-
mediated cell adhesion assay model was utilized to evaluate the
integrin-targeting activity of RGD peptides, using the tumor cell
line SKOV-3 which indicated over-expression of integrin α β .
v
3
Fibrinogen is the natural ligand of α β integrin and is involved in
v
3
cell adhesion. Firstly, we coated fibrinogen on a plate overnight,
and then human ovary cancer cells (SKOV-3) and our synthetic
RGD peptide compound was added successively after washing
(No RGD peptide as a control). Since the RGD peptides
competed with fibrinogen for binding to the integrin receptor αvβ3
on SKOV-3 cells, tumor cells that bound to fibrinogen can
remain on the plate, while bound to RGD peptides that inhibited
adhesion would be eluted. Then stained it by MTT method, the
OD value was measured, and finally the statistical data
(Inhibition rate = (Control group - Experimental group) /
Control group). For comparison purpose, Cilengitide was used as
Figure 1: HPLC profiles of procedures in the synthesis of P3. (a) In situ RGD
peptide 1 by SPPS before optimization; (a’) In situ RGD peptide 1 by SPPS
after optimization; (b) in situ carbonyl hydrazide peptide 2; (c) in situ
carbonyl azide peptide 3; (d) in situ thioester peptide 4; (e) side-chain ligation
of thioester 4 with CRGDRGDC for 12 h; (f) in situ dual-ring branched cyclic
RGD peptide 4a after disulfide-bond formation; (g) azide labeling of dual-ring
3 2 3
branched peptide 4b with N (CH ) COOSu.