Synthesis, nano-scale assembly, and in vivo anti-thrombotic activity of novel short peptides containing l-Arg and l-Asp or l-Glu

Article abstract of DOI:10.1016/j.bmc.2008.04.064

Two tripeptides H-Asp(Arg)-Arg (3a) and H-Glu(Arg)-Arg (3b), four pentapeptides H-Asp(Arg-Asp)-Arg-Asp (6a), H-Glu(Arg-Asp)-Arg-Asp (6b), H-Asp(Asp- Arg)-Asp-Arg (10a), and H-Glu(Asp-Arg)-Asp-Arg (10b), and their Cu(II)-peptide complexes Cu(II)-Asp(Arg)-Arg [3a-Cu(II)], Cu(II)-Glu(Arg)-Arg [3b-Cu(II)], Cu(II)-Asp(Arg-Asp)-Arg-Asp [6a-Cu(II)], Cu(II)-Glu(Arg-Asp)-Arg-Asp [6b-Cu(II)], Cu(II)-Asp(Asp-Arg)-Asp-Arg [10a-Cu(II)], and Cu(II)-Glu(Asp-Arg)-Asp-Arg [10b-Cu(II)] were designed and synthesized. Their self-assembling properties and in vivo anti-thrombotic activities were investigated. In normal saline (NS), the Cu(II)-peptide complexes assembled into stable nano-particles surrounded by negative charges (-4.102 to -9.825 mV), with diameters ranging from 212.1 ± 4.0 to 632.4 ± 36.7 nm. TEM analysis exhibited that the compounds remained as nano-globes in the solid state, with diameters ranging from 15 to 20 nm. In an in vivo anti-thrombotic assay, peptides (3,6,10)a,b at 5 μmol/kg reduced the thrombus weights of a rat model by 15-40%. Aspirin, a widely used anti-thrombotic drug, achieved comparable activity in this model system at a dosage of ca. 110 μmol/kg. The required dosage of Cu(II)-peptide complexes [(3,6,10)a,b]-Cu(II), which assemble into stable nano-particles, was significantly reduced to 0.05 μmol/kg. Therefore, the anti-thrombotic activity of the nano-particles [(3,6,10)a,b]-Cu(II) increased dramatically by 100-fold over that of the corresponding peptides.

Full text of DOI:10.1016/j.bmc.2008.04.064

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 5914–5925
Synthesis, nano-scale assembly, and in vivo anti-thrombotic
activity of novel short peptides containing
L-Arg and L-Asp or L-Glu
Yu Chen,a Guohui Cui,b Ming Zhao,b,* Chao Wang,a Keduo Qian,c
Susan Morris-Natschke,c Kuo-Hsiung Leec,* and Shiqi Penga,b,
*
aCollege of Pharmaceutical Sciences, Peking University, Beijing 100083, PR China
bCollege of Pharmaceutical Sciences, Capital Medical University, Beijing 100069, PR China
cNatural Products Research Laboratories, School of Pharmacy, University of North Carolina, Chapel Hill, NC 27599, USA
Received 16 November 2007; revised 24 April 2008; accepted 24 April 2008
Available online 29 April 2008
Abstract—Two tripeptides H-Asp(Arg)-Arg (3a) and H-Glu(Arg)-Arg (3b), four pentapeptides H-Asp(Arg-Asp)-Arg-Asp (6a),
H-Glu(Arg-Asp)-Arg-Asp (6b), H-Asp(Asp- Arg)-Asp-Arg (10a), and H-Glu(Asp-Arg)-Asp-Arg (10b), and their Cu(II)–peptide
complexes Cu(II)-Asp(Arg)-Arg [3a–Cu(II)], Cu(II)–Glu(Arg)-Arg [3b–Cu(II)], Cu(II)-Asp(Arg-Asp)-Arg-Asp [6a–Cu(II)],
Cu(II)–Glu(Arg-Asp)-Arg-Asp [6b–Cu(II)], Cu(II)-Asp(Asp-Arg)-Asp-Arg [10a–Cu(II)], and Cu(II)–Glu(Asp-Arg)-Asp-Arg [10b–
Cu(II)] were designed and synthesized. Their self-assembling properties and in vivo anti-thrombotic activities were investigated.
In normal saline (NS), the Cu(II)–peptide complexes assembled into stable nano-particles surrounded by negative charges
(ꢀ4.102 to ꢀ9.825 mV), with diameters ranging from 212.1 4.0 to 632.4 36.7 nm. TEM analysis exhibited that the compounds
remained as nano-globes in the solid state, with diameters ranging from 15 to 20 nm. In an in vivo anti-thrombotic assay, peptides
(3,6,10)a,b at 5 lmol/kg reduced the thrombus weights of a rat model by 15–40%. Aspirin, a widely used anti-thrombotic drug,
achieved comparable activity in this model system at a dosage of ca. 110 lmol/kg. The required dosage of Cu(II)–peptide complexes
[( 3,6,10)a,b]–Cu(II), which assemble into stable nano-particles, was significantly reduced to 0.05 lmol/kg. Therefore, the anti-
thrombotic activity of the nano-particles [(3,6,10)a,b]–Cu(II) increased dramatically by 100-fold over that of the corresponding
peptides.
ꢀ 2008 Elsevier Ltd. All rights reserved.
1. Introduction
in preventing cerebrovascular thrombus formation.
Thus, the discovery of novel anti-thrombotic agents
has attracted much interest.5
Platelet activation and aggregation are principal pro-
cesses in thromboembolic disorders.1 The formation of
an occlusive thrombus in the lumen of an injured vessel
depends on a successive process of activation, adher-
ence, and aggregation of platelets.2,3 A thrombus can re-
sult in arteriosclerosis, heart attack, stroke, and
peripheral vascular disease.4 Although thrombosis is sig-
nificant in the development of cardiovascular diseases,
the currently available anti-thrombotic drugs are lim-
ited, and anti-platelet drugs are only partially effective
Optimizing the ability of a drug to inhibit platelet acti-
vation is essential in the development of anti-thrombotic
drugs.6 The L-arginine pathway in blood vessel endothe-
lial cells is among the numerous platelet activation path-
ways that have been extensively studied. It is responsible
for generation of the endogenous nitrovasodilator
(NO).7 NO plays a fundamental role in keeping blood
vessels in a dilated state and maintaining the surface
of the endothelium nonthrombogenic.8 By bonding the
ferroheme center of the enzyme and consequently gener-
ating cGMP, NO stimulates the release of soluble guan-
ylate cyclase,9 and by increasing cGMP concentration,
NO induces vasodilation and anti-platelet aggrega-
tion.10 Both the vasodilation and anti-platelet properties
of NO are considered as pharmacologic foundations of
Keywords: Asp-Arg-peptides; Glu-Arg-peptides; Cu(II)-complex; Syn-
thesis; Self-assembling; Anti-thrombotic activity.
*
Corresponding authors. Tel.:+86 10 82802482; fax: +86 10 82802482
(M.Z.); tel.: +1 919 962 0066; fax: +1 919 966 3893 (K.-H.L.); tel.:
+86 10 83911528; fax: +86 10 83911528 (S.P.); e-mail addresses:
khlee@unc.edu; sqpeng@email.bjmu.edu.cn
0968-0896/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.04.064

Y. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5914–5925
5915
nitrovasodilators.11 Thus, it is well established that L-
arginine pathway modulators, such as NO donors and
assembling peptides usually exhibit a tendency to form
nanosystems, which can have distinctive properties.21–
24
Thus, Cu(II) complexes of the designed peptides (3,
6,10)a,b were also prepared in our study. The self-assem-
bly and diameters of the complexes [(3,6,10)a,b]–Cu(II)
were investigated in both NS solution and solid state,
and compared with those of (3,6,10)a,b. Their in vivo
anti-thrombotic activities were also evaluated in our
rat model and compared with those of (3,6,10)a,b.
L-arginine,
are
beneficial
in
thromboembolic
complications.12
In our previous study, a polymer containing L-arginine
and L-aspartic acid was prepared as an anti-platelet
aggregation peptide (PDR).13,14 Although PDR (MW
more than 30,000) exhibited anti-thrombotic activity in
an in vivo assay, it had shortcomings that are inherent
to polymers, including inexact molecule weight and
uncertain proportion of L-arginine to L-aspartic acid
residues. Thus, structural simplification of PDR was
merited. In addition, we also explored the physico-
chemical properties of PDR, which exhibited low water
solubility and an amphipathic tendency. Thus, we pos-
tulated that structural simplification of PDR could re-
sult in proper regulation of the amphipathic nature
consistent with self-assembling property.
2. Results and discussion
2.1. Synthesis
The short peptides were synthesized as shown in Scheme
1. Solution methodology and stepwise synthesis from C-
terminus to N-terminus were applied. In the presence of
dicyclohexylcarbodiimide (DCC), separate reactions of
p-nitrophenol with Boc-Asp-OH and Boc-Glu-OH
yielded Boc-Asp(ONp)-ONp (1a) and Boc-Glu(ONp)-
ONp (1b) in 83% and 84% yields, respectively. At pH
8.5–9.0, amidation of 1a and 1b with H-Arg provided
Boc-Asp(Arg)-Arg (2a, 80% yield) and Boc-Glu(Arg)-
Arg (2b, 85% yield). Removal of the Boc group con-
verted 2a and 2b to 3a (88% yield) and 3b (78% yield).
In the presence of 1-hydroxybenzotriazole (HOBt),
DCC, and N-methylmorpholine (NMM), 2a and 2b
were coupled with H-Asp(OMe)-OMe to give Boc-As-
p[Arg-Asp(OMe)-OMe]-Arg-Asp(OMe)-OMe (4a, 58%
yield) and Boc-Glu[Arg-Asp(OMe)-OMe]-Arg-As-
p(OMe)-OMe (4b, 65% yield), respectively. Saponifica-
tion of 4a and 4b provided Boc-Asp(Arg-Asp)-Arg-
Asp (5a) and Boc-Glu(Arg-Asp)-Arg-Asp (5b) in 75%
In this study, two tripeptides H-Asp(Arg)-Arg (3a) and
H-Glu(Arg)-Arg (3b) as well as four pentapeptides H-
Asp(Arg-Asp)-Arg-Asp (6a), H-Glu(Arg-Asp)-Arg-Asp
(6b), H-Asp(Asp-Arg)-Asp-Arg (10a), and H-Glu(Asp-
Arg)-Asp-Arg (10b) were designed and synthesized to
mitigate the polymeric drawbacks of PDR, while retain-
ing its anti-thrombotic activity. The self-assembling
properties of these short peptides were investigated
and their in vivo anti-thrombotic activities were evalu-
ated in a rat model. Because peptides can have tremen-
dous potential for self-assembly by defining secondary
and tertiary structures and increasing various non-cova-
lent interactions, they are considered to be naturally
occurring self-assembling species.15–20 Moreover, self-
iv
H-AA(Asp-Arg)-Asp-Arg
Cu(II)-AA(Asp-Arg)-Asp-Arg
(10a,b)-Cu
10a,b
iii
vi
Boc-AA(Asp-Arg)-Asp-Arg
Boc-AA[Asp(OMe)-Arg]-Asp(OMe)-Arg-OMe
8a,b
9a,b
v, H-Arg-OMe
i
ii, H-Asp(OMe)
Boc-AA
Boc-AA[Asp(OMe)]-Asp(OMe)
Boc-AA(ONp)-ONp
7a,b
1a,b
ii, H-Arg-OH
iii
iv
Boc-AA(Arg)-Arg
H-AA(Arg)-Arg
Cu(II)-AA(Arg)-Arg
2a,b
(3a,b)-Cu
3a,b
v, H-Asp-(OMe)2
vi
Boc-AA[Arg-Asp(OMe)-OMe]-Arg-Asp(OMe)-OMe
Boc-AA(Arg-Asp)-Arg-Asp
5a,b
4a,b
iii
iv
Cu(II)-AA(Arg-Asp)-Arg-Asp
H-AA(Arg-Asp)-Arg-Asp
6a,b
(6a,b)-Cu
Scheme 1. Synthetic route of ( 3,6,10)a,b and [( 3,6,10)a,b]–Cu(II). In 1a–10a AA = L-Asp, in 1b–10b AA = L-Glu. Reagents: (i) DCC, HONp
anhydrous THF and Boc-Asp-OH or Boc-Glu-OH; (ii) NaHCO3 and NMM; (iii) CF3CO2H; (iv) CuCl2ÆH2O and Na2CO3; (v) HOBt, DCC and
NMM; (vi) NaOH and KHSO4.

5916
Y. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5914–5925
and 68% yields, respectively. Treating 5a and 5b with tri-
fluoroacetic acid (TFA) removed the Boc group and
generated 6a (63% yield) and 6b (69% yield). Similarly,
by using H-Asp(OMe) rather than H-Arg, amidation
of 1a and 1b gave Boc-Asp[Asp(OMe)]-Asp(OMe) (7a,
81% yield) and Boc-Glu[Asp(OMe)]-Asp(OMe) (7b,
67% yield), respectively. The same reaction sequences
described above were used to synthesize Boc-Asp[As-
p(OMe)-Arg-OMe]-Asp(OMe)-Arg-OMe (8a, 80%
yield) and Boc-Glu-[Asp(OMe)-Arg-OMe]-Asp(OMe)-
Arg-OMe (8b, 70% yield), followed by Boc-
Asp(Asp-Arg)-Asp-Arg (9a, 75% yield) and Boc-Glu-
(Asp-Arg)-Asp-Arg (9b, 90% yield), and finally 10a
(75% yield) and 10b (86% yield). At pH 8, reaction of
(3, 6, 10)a,b with CuCl2 provided Cu(II)–peptide com-
plexes 3a–Cu(II), 3b–Cu(II), 6a–Cu(II), 6b–Cu(II),
10a–Cu(II) and 10b–Cu(II) in 58%, 78%, 63%, 69%,
75% and 90% yields, respectively (Scheme 1).
Figure 2. UV spectra of aqueous solutions of (3,6,10)a,b and [(3,6,10)
a,b]–Cu(II). Maximum absorbance over 560–660 nm relates to the d–d
transition of Cu(II)–peptide complexes. Temperature 25 ꢁC; concen-
tration 1 mM; a1, a2, a3, a4, a5 and a6 represent10b, 10a, 6b, 6b, 6a, 3b
and 3a, respectively; b, c, d, e, f and g represent 10b–Cu(II), 10a–
Cu(II), 6b–Cu(II), 6a–Cu(II), 3b–Cu(II) and 3a–Cu(II), respectively.
2.2. Nano-scale assembling of (3,6,10)a,b in NS
Nano-scale assembling of (3,6,10)a,b in NS solution was
analyzed using a Malvern’s Zeta Sizer (Nano-ZS90)
with DTS (Nano) Program. However, (3,6,10)a,b were
only able to self-assemble to unstable nano-particles in
NS. On Day 1, the diameters of the nano-particles ran-
ged from 473.9 50.5 to 983.2 48.9 nm, while on Day
2, diameters increased to 666.1 23.3 to 1443.8
49.7 nm. These diameters are indicative of unstable
nano-particles. On Day 3, no nano-particles were de-
tected in the NS solutions. The recorded diameters are
shown in Figure 1.
Except for caudal absorbance from 190 to 240 nm, the
free peptides ( 3,6,10)a,b displayed no UV absorbance
at any other wavelength. After Cu(II) complexation,
the spectra changed dramatically and strong bands were
observed in the region from 560 to 660 nm for all six
complexes. These significant spectral changes, attributed
to Cu (II) d–d transition, indicate changes in the envi-
ronment of the carbonyl group in the peptide bond
and possible coordination of the peptide bonds with
the metal cation.25 A similar absorbance was also re-
2.3. Nano-scale assembling of [(3,6,10)a,b]–Cu(II)
complexes in NS
ported to occur in
nanostructure.26
a related Cu(II)-coordinated
As previously mentioned, peptides can have tremendous
potential for self-assembly through increased non-cova-
lent interactions, and can form nanosystems with dis-
tinctive properties. To increase the potential for our
newly synthesized peptides to form stable nano-parti-
cles, we selected Cu(II) as a complexing metal ion. Since
the d–d transition in UV–vis and CD spectra is generally
used to identify Cu(II) coordination, the UV–vis and
CD spectra of the free peptides ( 3,6,10)a,b and their
Cu(II) complexes [( 3,6,10) a,b]–Cu(II) were measured
and are shown in Figures 2 and 3.
The CD spectra of the free peptides (3,6,10)a,b and their
Cu(II) complexes [(3,6,10)a,b]–Cu(II) confirmed the UV
results and conclusions. The free peptides had positive
Cotton effects from 217 to 225 nm, suggesting they had
unordered structures. Stereoviews of the free peptides’
lowest energy conformations were drawn using Discovery
Studio Program (Fig. 4). These conformations showed
that the free peptides’ carbonyl groups were exposed
and unable to take part in repetitive H bonds. On the
other hand, in the spectral region from 600 to 660 nm,
1600
1400
1200
1000
800
600
400
200
0
Day 1
Day 2
3a
3b
6a
6b
Compd.
10a
10b
Figure 1. Particle diameters of (3,6,10)a,b assembled in NS. Test temperature was 25 ꢁC, solution concentration of (3,6,10)a,b in NS was 1 mg/mL,
particle diameter is expressed by X ꢁ SD nm, n = 10.

Y. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5914–5925
5917
Figure 3. CD spectra of (3,6,10)a,b and [(3,6,10)a,b]–Cu(II) in water. CD spectra of [(3,6,10)a,b]–Cu(II) displayed an absorbance around 600–660 nm,
which is considered to result from the d–d transition of Cu(II) coordination. Temperature 25 ꢁC; concentration 1 mM; a1, a2, a3, a4, a5 and a6
represent 10b, 10a, 6b, 6b, 6a 3b and 3a, respectively; b, c, d, e, f and g represent 10b–Cu(II), 10a–Cu(II), 6b–Cu(II), 6a–Cu(II), 3b–Cu(II) and 3a–
Cu(II), respectively.
Figure 4. Discovery Studio Program drawing lowest energy conformations of (3,6,10)a,b.
the free peptides displayed no absorbance, while their
Cu(II) complexes did, which is likely a result of the d–d
transition of Cu(II) coordination.27
averaged, and the difference between the average and
norm values was used to represent the diameter change.
The data are listed in Table 1.
A nano-particle size analysis of [(3,6,10)a,b]–Cu(II) com-
plexes in NS demonstrated that these complexes did
assemble more easily into stable nano-particles com-
pared with (3,6,10)a, b (Fig. 1). On Day 1, the diameters
of the nano-particles ranged from 212.1 4.0 to
632.4 36.7 nm, on Day 4, the diameter size increased
to 382.4 16.0 to 755.0 204.4 nm, and on Day 7, the
nano-particles were still stable with detectable diame-
ters. The recorded diameters over 4 days are shown in
Figure 5.
Comparison of the diameter change demonstrated the
following facts. The diameter increase of [(3,6)b]–Cu(II),
which have Glu as the central linker, was less than that
of [(3,6)a]–Cu(II), with Asp as the central linker, (2.2%
and 20.6% versus 58.4% and 102%, respectively). Corre-
spondingly, the diameter decrease of 10b–Cu(II), central
residue is Glu, was more than that of 10a–Cu(II), central
residue is Asp, (27.1% vs 0.5%). These results indicate
that the former Glu-linked complexes were more stable
as nano-particles than the latter Asp-linked complexes.
In the calculated lowest conformations, the distances be-
tween the guanidiniums in peptides with a Glu residue as
the central linker are larger than those of the peptides
with an Asp residue as the central linker, which implies
that the former are more flexible and may limit particle
growth.
To gain insights into the relationship between nano-par-
ticle stability and peptide structure, the diameter
changes were compared. For this purpose, the particle
diameter of the assembled Cu(II) complexes on Day 1
was taken as the norm, the diameters of Days 2–4 were

5918
Y. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5914–5925
1000
800
600
400
200
0
Day1 Day2 Day3 Day4
3a-Cu(II) 3b-Cu(II) 6a-Cu(II) 6b-Cu(II) 10a-Cu(II) 10b-Cu(II)
Compd.
Figure 5. Particle diameters of [(3,6,10)a,b]–Cu(II) assembled in NS. Test temperature was 25 ꢁC, solution concentration of [(3,6,10)a,b]–Cu(II) in NS
was 1 mg/mL, diameter is expressed by X ꢁ SD nm, and on the 7th day the nano-systems were still stable, n = 10.
2.4. Zeta-potentials of (3,6,10)a,b and [(3,6,10)a,b]–
Cu(II) nano-particles in NS
its nano-particles’ surface would be negatively, rather
than positively, charged and surrounded by positive
charges. The surfaces of [(3,6)a,b,10a]–Cu(II) nano-par-
ticles were also surrounded by negative charges, demon-
strating that, even after Cu(II) complexation, the
hydrophilic guanidiniums remained exposed. In addi-
tion, Cu(II) complexation of 10b apparently led to a
changed mode of self-assembly and consequently to ex-
posed hydrophilic guanidiniums.
To examine the electrical stabilities of (3,6,10) a,b and
[(3,6,10)a,b]–Cu(II) nano-particles in solution state, the
Zeta potentials in NS were measured. Table 2 lists the
Zeta potentials on the nano-particle surfaces of
(3,6,10) a,b at Hour 1 and of [(3,6,10) a,b]–Cu(II) on
Day 4 after their preparation in NS solution.
The results indicated that, except for 10b, the surfaces of
(3, 6)a,b and 10a nano-particles were surrounded by neg-
ative charges. It is reasonable to propose that the self-
assembly of (3, 6)a,b and 10a in NS is driven by hydro-
phobic interactions between residues and results in
hydrophilic guanidiniums being exposed. The confor-
mational stereoviews (Fig. 4) of (3,6)a,b and 10a support
such an arrangement of the protonated guanidiniums.
Therefore, the nano-particles’ surfaces will be positively
charged and surrounded by negative charges. However,
looking at the stereoview of 10b, controlled self-assem-
bly could instead expose negative charges. Therefore,
Surface zeta potential is generally considered as a mea-
surement of the charge density per unit surface, which
depends on both the total of charged groups and the size
of the nano-particle. Although 3a and 3b have equal
numbers of protonated guanidiniums, the particle size
of the former is significantly smaller than that of the lat-
ter; thus, the zeta potential of the former is significantly
higher than that of the latter. In contrast, although the
particle size of 6a is significantly smaller than that of
6b, the two compounds have comparable zeta poten-
tials. An explanation is found from the stereoviews of
the lowest energy conformations shown in Figure 4.
When 6a assembles, two guanidiniums will likely be ex-
posed; however, when 6b assembles, only one guanidi-
nium will be exposed. These opposing effects (size and
total charge) lead to the two compounds having equal
zeta potentials. Similarly, although 10a is smaller than
10b, each exposed guanidinium of 10a has at least two
protonated nitrogens, while each exposed carboxyl can
ionize giving only one acid group. Therefore the abso-
lute Zeta potential of 10a is significantly higher than that
of 10b.
Table 1. Dependence of particle diameter on peptide–Cu(II) structure
Compound
Diameter
Mean of
Days 2-4
Central
residue
Day 1
Increase in
respect to
Day 1 (%)
3a–Cu(II)
3b–Cu(II)
6a–Cu(II)
6b–Cu(II)
10a–Cu(II)
10b–Cu(II)
Asp
Glu
Asp
Glu
Asp
Glu
212.1
424.4
311.6
465.1
553.1
632.4
335.9
433.8
629.4
560.8
550.3
459.9
58.4
2.2
102
20.6
ꢀ0.5
ꢀ27.3
Table 2 also indicates, except for 3b and 10b, the Zeta
potentials of the remaining peptides (3a, 6a, 6b, and
10a) at Hour 1 generally were equal to those of their cor-
Table 2. Zeta potential and size of (3,6,10)a,b and [(3,6,10)a,b]–Cu(II) nano-particles
Compound
Hour 1
Compound
Day 4
Zeta potential
Diameter
Zeta potential
Diameter
3a
3b
ꢀ6.405
ꢀ1.901
ꢀ5.578
ꢀ4.592
ꢀ9.159
2.101
304.9 10.3
431.4 32.1
749.8 17.8
536.4 21.1
550.8 27.2
503.5 16.5
3a–Cu(II)
3b–Cu(II)
6a–Cu(II)
6b–Cu(II)
10a–Cu(II)
10b–Cu(II)
ꢀ6.284
ꢀ6.472
ꢀ5.262
ꢀ4.102
ꢀ9.825
ꢀ8.959
382.4 16.0
416.0 33.1
755.0 20.4
579.6 17.8
518.3 50.2
460.1 15.7
6a
6b
10a
10b

Y. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5914–5925
5919
responding Cu(II) complexes on Day 4. This observa-
tion is consistent with the requirement that zeta poten-
tial depends on the particle size, as the nano-particle
diameters at Hour 1 and on Day 4 were also close as
shown in Table 2. However, the particle size of 3b was
significantly larger than that of its Cu(II) complex and
consequently the Zeta potential of 3b was significantly
lower than that of its Cu(II) complex. The distinct Zeta
potentials for 10b and 10b–Cu(II) should be the result of
their separate assembly models, as discussed above.
ing Cu complexes at 0.05 lmol/kg in NS were assayed in
our rat model. The thrombus weight was used as the
measure of activity. The data indicated that the throm-
bus weights of rats receiving only NS (negative control)
averaged 29.44 5.35 mg. Intravenous dosing with (
3,6,10)a,b at 5 lmol/kg reduced thrombus weights signif-
icantly by 15–40%, with weights ranging from
17.51 3.71 to 25.12 3.52 mg. Dosage with
110 lmol/kg of aspirin, a current widely used anti-
thrombotic agent, was needed to achieve similar anti-
thrombotic activity. These results suggested that our no-
vel, designed tripeptides and pentapeptides are active
against thrombosis (Fig. 7).
2.5. Nano-particle powders of (3,6,10)a,b and
[(3,6,10)a,b]–Cu(II)
Generally nano-solutions can be converted into nano-
solids. To confirm this possibility with our compounds,
solutions (1 mg/mL) of (3, 6,10)a,b and [(3,6,10)a,b]–
Cu(II) in triple-distilled water were evaporated at room
temperature to form powders. The particle sizes of the
powders were analyzed by using transmission electron
microscopy (TEM). The obtained TEM photographs
of (3,6,10)a,b and [(3,6,10)a,b]–Cu(II) powders showed
that they were present as nano-globes in the solid state,
and their diameters ranged from 15 to 30 nm. Represen-
tative photographs of 3a, 10a, 3a–Cu(II), 3b–Cu(II),
10a–Cu(II), and 10b–Cu(II) powders are shown in
Figure 6. The particle sizes shown in the photographs
indicate that both peptides (3,6,10)a,b and the complexes
[(3,6,10)a,b]–Cu(II) are stable nano-globes in the solid
state.
Because the peptides do not assemble to stable nano-
particles, while the [(3,6,10) a,b]–Cu(II) complexes do
form stable nano-systems, the anti-thrombotic activities
of the nano-particle solutions of [(3,6,10) a,b]–Cu(II)
complexes were also tested using the same general proce-
dure. Only 0.05 lmol/kg of [(3,6,10) a,b]–Cu(II) com-
plexes were needed to achieve similar anti-thrombotic
activity as 5 lmol/kg of (3,6,10)a,b. By dosing [(3,6,10)
a,b]–Cu(II) at 0.05 lmol/kg intravenously to the rats,
their thrombus weights reduced significantly by 21–
31%, with thrombus weights ranging from 20.31 3.79
to 23.27 3.32 mg. The results indicated that the forma-
tion of stable nano-particles increased the anti-throm-
botic activity of the newly designed peptides by 100-
fold (Fig. 7).
2.7. Dose-dependent activities of 6b, 10a, 10b, and 10a–
Cu(II) in NS
2.6. In vivo anti-thrombotic activity of intravenously
injected (3,6,10)a,b and [(3,6,10)a,b]-Cu(II) in NS
Three peptides (6b, 10a, and 10b) and one Cu complex
(10a–Cu(II)) were selected for a dose-dependent activity
study. The evaluation was performed in a rat model
The in vivo anti-thrombotic activities of 3a, 3b, 6a, 6b,
10a, and 10b at a dose of 5 lmol/kg and the correspond-
Figure 6. TEM (Model JEM-1230, JEOL, Japan) photographs of 3a, 10a, 3a–Cu(II), 3b–Cu(II), 10a–Cu (II) and 10b–Cu(II) powders.
35
a
30
25
a
b
b
b
b
b
b
b
b
c
d
20
b
15
10
5
0
Compd.
Figure 7. Thrombus weights of (3,6,10)a,b or [(3,6,10)a,b]–Cu(II) treated rats. NS, vehicle, n = 12; A, Aspirin; (3,6,10)a,b/dose = 5 lmol/kg, [(3,6,10)
a,b]–Cu(II)/dose = 0.05 lmol/kg, Aspirin/dose = 20 mg/kg. (a) Compared to NS p < 0.05; (b) Compared to NS p < 0.01; (c) Compared to NS and
6b–Cu(II) p < 0.01; (d) Compared to NS and 10a–Cu(II) p < 0.01.

5920
Y. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5914–5925
according to standard procedures. The data obtained
indicated significant differences in the in vivo anti-
thrombotic activities of the intravenously injected pep-
tides at concentrations of 5, 0.5, 0.05, and 0.005 lmol/
kg, as well as for intravenously dosed 10a–Cu(II) at
0.5, 0.05, 0.005, and 0.0005 lmol/kg. These observations
indicated that the in vivo anti-thrombotic activities of
6b, 10a, 10b, and 10a–Cu(II) in NS are dose-dependent
(Fig. 8).
bus weights of rats receiving 5 lmol/kg ( 3,6,10)a,b or
0.05 lmol/kg [(3,6,10)a,b]–Cu(II) and the corresponding
nano-particle diameters were compared in Table 4. Gen-
erally, a decrease in the nano-particle diameter led to an
increase in the anti-thrombotic activity of (3,6,10)a,b and
[(3,6,10)a,b]–Cu(II) complexes.
3. Conclusions
2.8. In vivo activities of orally administrated 10a (5 lmol/
kg) and 10a–Cu(II) (0.05 lmol/kg) in NS
In conclusion, we designed and synthesize novel tripep-
tides and pentapeptides containing L-arginine and L-
aspartic acid or L-glutamic acid in order to increase
the anti-thrombotic activity of polyaspartyl-L-arginine
(PDR), as well as alleviate its polymer shortcomings.
When the residue proportions of L-arginine to L-aspartic
acid or L-glutamic acid are less than 60% or more than
40%, the peptides may self-assemble. To form stable
nano-particles in NS solution, Cu(II) complexes of the
new peptides (3,6,10) a,b were prepared, and their
nano-particle properties were measured. The in vivo
anti-thrombotic activities of both (3,6,10) a,b and the
Cu(II)–peptide complexes [(3,6,10) a,b]–Cu(II) were
tested. Five micromoles per kilograms dose of (3,6,10)
a,b resulted in similar anti-thrombotic activity as
110 lmol/kg of aspirin. By assembling into stable
nano-particles, [(3,6,10)a,b]-Cu(II) complexes were 100-
fold more potent than ( 3,6,10)a,b against thrombosis,
with comparable activity at the 0.05 lmol/kg level. Po-
tency was retained when the administration method
was changed from intravenous to oral, which should
facilitate potential clinical application.
Compounds 10a and 10a–Cu(II) were selected to test
their oral administration profiles. The oral and intrave-
nous data are listed in Table 3. The data indicated that
orally administered 10a (5 lmol/kg) can effectively block
the
thrombosis
process
(thrombus
weight
23.18 3.75 mg; compared to that of NS receiving rats,
29.44 5.35 mg, p < 0.01). Orally dosed 10a–Cu(II)
complexes (0.05 lmol/kg) also effectively blocked the
thrombosis process (thrombus weight 25.22 3.79 mg;
p < 0.05). Comparable thrombus weights were found
after oral and intravenous dosing of 10a–Cu(II). These
results suggested that both 10a and its Cu complex
[10a–Cu(II)] can be sufficiently absorbed in the stomach
or intestine and can be administrated orally.
2.9. Correlation of the activity and nano-particle size of
(3,6,10)a,b and [(3,6,10)a,b]–Cu(II)
To evaluate a possible relationship between nano-parti-
cle size and in vivo anti-thrombotic activity, the throm-
Table 3. Effect of oral administration of 10a and 10a–Cu(II) on
thrombus weighta
4. Experimental
4.1. General procedures
Compound
Thrombus
weight
Compound
Thrombus
weight
8.50 0.70b
17.506 3.52b
23.19 3.71b
Amino acids with L-configuration used were purchased
from Sigma Chemical Co. Chromatography was per-
formed on Qingdao silica gel H. The purity of interme-
diates and products was confirmed by TLC (Merck
silica gel plates of type 60 F254, 0.25 mm layer thickness)
IR spectroscopy was performed on a 330 FT-IR spec-
trometer (Nicolet Avatar Thermo Electron Corpora-
tion). NMR spectroscopy was determined on 500 and
300 MHz spectrometers (AVANCE II, BRUKER
NS (oral)
10a(oral)
10a–Cu(II)
(oral)
29.44 3.35
23.18 3.75b
25.22 3.79c
Aspirin (iv)
10a (iv)
10a–Cu(II) (iv)
a Thrombus weight is expressed by X ꢁ SD mg, dose of 10a = 5 lmol/
kg and of 10a–Cu(II) = 50 nmol/kg and of Aspirin = 20 mg/kg;
NS,vehicle; n = 12.
b Compare to NS p < 0.01.
c Compare to NS p < 0.05.
35
c
30
5µmol/kg
f
c
b
c
b
b
25
20
15
10
5
0
a
a
a
0.5µmol/kg
e
d
0.05µmol/kg
0.005µmol/kg
0.0005µmol/kg
10a-Cu
6b
10b
10a
Compd. & Dose
Figure 8. Dose-dependent action of 6a, b and 10a and 10a–Cu. NS, vehicle, n = 10; (a) compare to NS p < 0.001, to 0.5 lmol/kg group p < 0.01; (b)
compare to NS p < 0.001, to 0.05 lmol/kg group p < 0.05; (c) compare to NS p < 0.01, to 0.005 lmol/kg group p < 0.05; (d) compare to NS
p < 0.001, to 0.05 lmol/kg group p < 0.01; (e) compare to NS p < 0.01, to 0.005 lmol/kg group p < 0.01; (f) compare to NS p < 0.01, to 0.0005
lmol/kg group p < 0.01.

Y. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5914–5925
5921
Table 4. Interrelation of thrombus weight and particle size
Compound Thrombus weight Nano-particle diameter on Day 1 Compound Thrombus weight Nano-particle diameter on Day 4
3a
3b
20.03 3.07c
24.59 2.89
25.12 3.52
18.68 3.61c
17.51 3.71b
19.19 3.61c
575.3 32.9e
983.2 48.9
904.0 20.9
507.7 12.8d
473.9 50.5d
653.0 52.0f
3a–Cu(II)
3b–Cu(II)
6a–Cu(II)
6b–Cu(II)
10a-Cu(II)
10b–Cu(II)
20.31 3.79
21.90 3.31
22.86 3.43
23.27 3.32
23.19 3.71
21.14 3.33b
382.4 16.0g
416.0 33.1h
755.0 20.4
579.6 17.8k
518.3 50.2j
460.1 15.7i
6a
6b
10a
10b
aThrombus weight is expressed by X ꢁ SD mg and n = 12, diameter is expressed by X ꢁ SD nm and n = 6; (3,6,10)a,b/Dose = 5 lmol/kg, [(3,6,10)
a,b]–Cu(II)/Dose = 50 nmol/kg.
b Compare to 3b and 6a p < 0.001.
c Compare to 3b and 6a p < 0.01.
d Compare to 3a p < 0.05, to 3b, 6a and 10b p < 0.001.
e Compare to 10b p < 0.01, to 3b and 6a p < 0.001.
f Compare to 3b and 6a p < 0.01.
g Compare to 3b p < 0.05, to 6a, 6b, 10a, and 10b p < 0.001.
h Compare to 10b p < 0.05, to 6a, 6b, and 10a p < 0.001.
i Compare to 10a p < 0.05, to 6a, b p < 0.001.
j Compare to 6b p < 0.05, to 6a p < 0.001.
k Compare to 6a p < 0.001.
UltraShiel). ESI-MS spectroscopy was obtained with a
Waters HPLC/MS (Quattro microTM API). Optical rota-
tions were measured on a P-1020 Jasco polarimeter
(Japan).
1H NMR (BHSC-500, D2O) d 4.36 (t, J = 5.0 Hz, 1H),
4.18 (dd, J = 5.0, 8.5 Hz, 2H), 3.10 (dd, J = 7.0,
14.0 Hz, 4H), 2.68 (dd, J = 9.5, 16.0 Hz, 2H), 1.79 (m,
4H), 1.64 (m, 4H), 1.32 (s, 9H); ESI-MS (m/z) 546
20
[M+H]+; ½aꢂ ꢀ 15:5 (c 0.01, H2O). Anal. Calcd for
D
4.2. Compounds
C21H39N9O8: C 46.23, H 7.20, N 23.11; found: C
46.40, H 7.05, N 23.30.
4.2.1. Boc-Asp(ONp)-ONP (1a). At 0 ꢁC, 7.82 g
(0.038 mol) of dicyclohexylcarbodiimide (DCC) were
added portionwise to a solution of 4.36 g (0.019 mol)
of Boc-Asp-OH and 5.28 g (0.038 mol) of p-nitrophenol
(HONp) in 50 mL of anhydrous tetrahydrofuran
(THF). The reaction mixture was stirred at 0 ꢁC for
30 min, and then at room temperature for 12 h until
TLC (CHCl3/MeOH, 10:1) indicated the complete dis-
appearance of Boc-Asp-OH. The precipitated dic-
ylohexylcarbourea (DCU) was removed by filtration
and the filtrate was evaporated under reduced pressure.
The residue was crystallized from the mixture of EtOAc
and petroleum ether to provide 7.5 g (83%) of the title
compound.
4.2.3. H-Asp(Arg)-Arg (3a). A solution of 500 mg
(0.92 mmol) of 2a, 1 mL of TFA, and 3 mL of CH2Cl2
was stirred at 0 ꢁC for 1 h. Upon removal of TFA and
CH2Cl2 in vacuum, the residue was triturated with
diethyl ether, and the obtained powder was purified on
a C18 column (CH3CN/1% HOAc, 2.5:100) to provide
351 mg (86%) of the title compound as a colorless pow-
der. Mp 141–145 ꢁC; 1H NMR (BHSC-500, D2O) d 4.23
(t, J = 5.0 Hz, 1H), 4.07 (dd, J = 5.0, 8.0 Hz, 1H), 4.03
(dd, J = 5.0, 8.5 Hz, 1H), 3.09 (dd, J = 7.0, 14.0 Hz,
4H), 2.91 (dd, J = 5.0, 16.0 Hz, 1H), 2.82 (dd, J = 6.5,
16.0 Hz, 1H), 1.73 (m, 4H), 1.48 (m, J = 7.5 Hz, 4H);
20
ESI-MS (m/z) 446 [M+H]+; ½aꢂ ꢀ 16:6 (c 0.01, H2O).
D
Anal. Calcd for C16H31N9O6: C 43.14, H 7.01, N
28.30; found: C 43.00, H 7.14, N 28.11.
4.2.2. Boc-Asp(Arg)-Arg (2a). A solution of 1.85 g
(8.82 mmol) of HClÆH-Arg-OH and 1.48 g (17.6 mmol)
of NaHCO3 in 25 mL of distilled water was stirred at
room temperature, and then a solution of 2.0 g
(4.2 mmol) of 1a in 5 mL of dioxane was added portion-
wise. The suspension was stirred and maintained at pH
8.5–9.0 by adding N-methylmorpholine (NMM), during
which 1a gradually dissolved and TLC (CHCl3/MeOH,
20:1) indicated complete disappearance of 1a. The reac-
tion mixture was evaporated under reduced pressure.
The residue was dissolved in 30 mL of distilled water,
and the solution was extracted with diethyl ether (3·
30 mL). The aqueous phase was acidified with a satu-
rated aqueous solution of KHSO4 to pH 2 and extracted
with EtOAc (3· 30 mL). The aqueous phase was evapo-
rated under reduced pressure and the residue was dis-
solved in 10 mL of anhydrous EtOH. After filtration
and evaporation under reduced pressure, the residue
was chromatographed (CHCl3/MeOH, 1:1) to give
1.83 g (80%) of the title compound. Mp 160–162 ꢁC;
4.2.4. Cu(II)-Asp(Arg)-Arg [3a–Cu(II)]. To a solution of
100 mg (0.225 mmol) of 3a in 2 mL of distilled water,
37.0 mg (0.220 mmol) of CuCl2ÆH2O was added. The
solution was adjusted to pH 8 with an aqueous solution
of Na2CO3 (1 mol/L) and stirred at room temperature
for 4 h. After filtration, the filtrate was evaporated un-
der reduced pressure and the residue was purified on a
Sephadex G10 column to provided 66 mg (58%) of the
title compound as blue powder. IR (KBr) 3342, 2966,
2360, 1589, 1409, 1180, 699 cmꢀ1; ESI-MS (m/z) 507
20
D
[Mꢀ1]+; ½aꢂ 62:5 (c 0.01, H2O). Anal. Calcd for
C16H30CuN9O6Æ2H2O: C 35.32, H 6.30, N 23.17; found:
C 35.50, H 6.17, N 23.36.
4.2.5. Boc-Asp[Arg-Asp(OMe)-OMe]-Arg-Asp(OMe)-
OMe (4a). At 0 ꢁC, 0.238 g (1.76 mmol) of HOBt and
0.363 g (1.76 mmol) of DCC were added to a solution
of 0.458 g (0.84 mmol) of 2a in 5 mL of anhydrous

5922
Y. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5914–5925
DMF. The reaction mixture was stirred at 0 ꢁC for
10 min and then 0.346 g (1.76 mmol) of HClÆH-As-
p(OMe)-OMe in 5 mL of anhydrous THF was added.
The reaction mixture was adjusted to pH 8–9 with
NMM, and stirred at 0 ꢁC for 3 h and at room temper-
ature for 12 h. The precipitated DCU was removed by
filtration and the filtrate was evaporated under reduced
pressure. The residue was dissolved in 10 mL of distilled
water and the solution was extracted with EtOAc (3·
30 mL). The aqueous phase was acidified with saturated
aqueous KHSO4 to pH 2 and extracted with EtOAc (3·
30 mL). The aqueous phase was evaporated under re-
duced pressure and the residue was dissolved in 10 mL
of anhydrous EtOH. After filtration and evaporation
under reduced pressure, the residue was chromato-
graphed (CHCl3/MeOH, 10:1) to give 0.558 g (80%) of
at pH 8.5–9.0 by adding NMM, during which 1a grad-
ually dissolved and TLC (CHCl3/MeOH, 20:1) indicated
complete disappearance of 1a. The reaction mixture was
evaporated under reduced pressure. The residue was dis-
solved in 30 mL of distilled water, and the solution was
extracted with diethyl ether (3· 30 mL). The aqueous
phase was acidified with saturated aqueous solution of
KHSO4 to pH 2 and extracted with EtOAc (3·
30 mL). The EtOAc phase was dried over anhydrous so-
dium sulfate, filtered and evaporated under reduced
pressure to give 5.3 g (71%) of the title compound. Mp
1
151–152 ꢁC; H NMR (BHSC-300, DMSO-d6) d 12.85
(s, 2H), 8.29 (d, J = 6.9 Hz, 1H), 8.20 (d, J = 8.1 Hz,
1H), 6.90 (d, J = 8.1 Hz, 1H), 4.55 (t, J = 5.5 Hz, 2H),
4.24 (t, J = 4.5 Hz, 1H), 3.60 (s, 6H), 2.67 (m, 3H),
2.47 (m, 3H), 1.43 (s, 9H); ESI-MS (m/z) 492 [M+H]+;
20
D
1
the title compound. Mp 99–100 ꢁC; H NMR (BHSC-
½aꢂ 15:1 (c 0.01, CH3OH). Anal. Calcd for
300, D2O)
d
4.26 (t, J = 5.2 Hz, 2H), 4.06 (t,
C19H29N3O12: C 46.44, H 5.95, N 8.55; found: C
46.63, H 5.83, N 8.75.
J = 7.35 Hz, 2H), 3.79 (t, J = 5.2 Hz, 1H), 3.67 (d,
J = 7.35 Hz, 12H), 3.15 (t, J = 6.9 Hz, 4H), 2.91 (m,
3H), 2.85 (m, 3H), 1.72 (m, 4H), 1.58 (m, 4H), 1.31 (s,
4.2.10. Boc-Asp[Asp(OMe)-Arg-OMe]-Asp(OMe)-Arg-
OMe (8a). At 0 ꢁC, 1-hydroxybenzotriazole (HOBT)
(0.334 g, 2.48 mmol) and DCC (0.510 g, 2.48 mmol)
were added to a solution of 0.578 g (1.18 mmol) of 7a
in 5 mL of anhydrous THF. The reaction mixture was
stirred at 0 ꢁC for 10 min and then 0.553 g (2.47 mmol)
of HClÆH-Arg-OMe in 5 mL of anhydrous DMF was
added. The reaction mixture was adjusted to pH 8–9
with NMM, stirred at 0 ꢁC for 3 h and at room temper-
ature for 12 h. The precipitated dicyclohexylurine
(DCU) was removed by filtration and the filtrate was
evaporated under reduced pressure. The residue was dis-
solved in 10 mL of distilled water and the solution was
extracted with EtOAc (3· 30 mL). The aqueous phase
was acidified with saturated aqueous solution of KHSO4
to pH 2 and extracted with EtOAc (3· 30 mL). The
aqueous phase was evaporated under reduced pressure
and the residue was dissolved in 10 mL of anhydrous
EtOH. After filtration and evaporation under reduced
pressure the residue was purified by chromatography
(CHCl3/MeOH, 10:1) to give 0.784 g (80%) of the title
compound. Mp 117–118 ꢁC; 1H NMR (BHSC-300,
D2O) d 4.36 (t, J = 4.5 Hz, 2H), 4.34 (t, J = 3.6 Hz,
2H), 3.89 (t, J = 3.9 Hz, 1H), 3.68 (d, J = 7.8 Hz,
12H), 3.12 (t, J = 3.6 Hz, 4H), 2.80 (m, 3H), 2.71 (m,
20
9H); ESI-MS (m/z) 832 [M+H]+; ½aꢂ ꢀ 72:8 (c 0.01,
D
CH3OH). Anal. Calcd for C33H57N11O14: C 47.65, H
6.91, N 18.52; found: C 47.67, H 6.80, N 18.71.
4.2.6. Boc-Asp(Arg-Asp)-Arg-Asp (5a). A solution of
300 mg (0.361 mmol) of 4a, 2 mL of MeOH, and 3 mL
of aqueous NaOH (2 N) was stirred at 0 ꢁC for 4 h.
The reaction mixture was adjusted to pH 6 with satu-
rated aqueous KHSO4 and evaporated in vacuum. The
residue was dissolved in 10 mL of MeOH. After filtra-
tion and evaporation under reduced pressure, 210 mg
(75%) of the title compound was obtained as a colorless
powder, which was used directly in the next reaction.
4.2.7. H-Asp(Arg-Asp)-Arg-Asp (6a). Using the same
procedure as for 3a and starting from 200 mg
(0.258 mmol) of 5a, 110 mg (63%) of the title compound
was obtained. Mp 129–130 ꢁC; IR (KBr) 3340, 2950,
1
2360, 1662, 1558, 1386, 1270, 750, 640 cmꢀ1; H NMR
(BHSC-300, D2O) d 4.52 (dd, J = 6.0, 12.3 Hz, 2 H),
4.26 (t, J = 5.7 Hz, 2H), 4.20 (t, J = 6.9 Hz, 1H), 3.09
(t, J = 6.3 Hz, 4H), 2.94 (dd, J = 5.1, 16.8 Hz, 4H),
2.86 (t, J = 6.9 Hz, 2H), 1.70 (m, 4H), 1.54 (t,
20
J = 6.6 Hz, 4H); ESI-MS (m/z) 676 [M+H]+; ½aꢂ ꢀ 9:8
D
(c 0.01, H2O). Anal. Calcd for C24H41N11O12: C 42.66,
H 6.12, N 22.80; found: C 42.50, H 6.00, N 22.62.
3H), 1.78 (m, 4H), 1.50 (m, 4H), 1.31 (s, 9H); ESI-MS
20
(m/z) 831[M+H]+; ½aꢂ ꢀ 50:9 (c 0.01, CH3OH). Anal.
D
Calcd for C33H57N11O14: C 47.65, H 6.91, N 18.52;
found: C 47.82, H 6.80, N 18.34.
4.2.8. Cu(II)-Asp(Arg-Asp)-Arg-Asp [6a–Cu(II)]. Using
the same procedure as that for 3a-Cu(II), starting from
100 mg (0.148 mmol) of 6a, 35 mg (32%) of the title
compound was obtained as blue powder. IR (KBr)
4.2.11. Boc-Asp(Asp-Arg)-Asp-Arg (9a). Using the same
procedure as that for 5a, starting from 700 mg
(0.842 mmol) of 8a, 87 mg (90%) of the title compound
was obtained as a colorless powder, which was used di-
rectly in the next reaction.
3347, 2968, 2358, 1575, 1403, 1123, 669 cmꢀ1; ESI-MS
20
(m/z) 737[M-1]+; ½aꢂ ꢀ 18:8 (c 0.01, H2O). Anal. Calcd
D
for C24H40CuN11O12Æ2H2O: C 37.23, H 5.73, N 19.90;
found: C 37.05, H 5.60, N 19.71.
4.2.12. H-Asp(Asp-Arg)-Asp-Arg (10a). Using the same
procedure as that for 3a, starting from 587 mg
(0.75 mmol) of 9a, 358 mg (71%) of the title compound
was obtained as a colorless powder. Mp 213–214 ꢁC; IR
(KBr) 3352, 2953, 2360, 1655, 1560, 1397, 1259, 752,
4.2.9. Boc-Asp[Asp-(OMe)]-Asp-(OMe) (7a). A solution
of 5.86 g (32.0 mmol) of HClÆH-Asp(OCH3) and 5.37 g
(64.0 mmol) of NaHCO3 in 30 mL of distilled water
was stirred at room temperature, and a solution of
7.23 g (15.2 mmol) of 1a in 10 mL of dioxane was added
portionwise. The suspension was stirred and maintained
653 cmꢀ1
;
1H NMR (BHSC-300, D2O) d 4.48 (t,
J = 4.5 Hz, 2H), 4.26 (t, J = 3.9 Hz, 1H), 4.12 (t,

Y. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5914–5925
5923
J = 5.1 Hz, 2H), 3.09 (t, J = 6.6 Hz, 4H), 2.80 (m, 6H),
1.74 (t, J = 6.6 Hz, 4H), 1.64 (m, 4H); ESI-MS (m/z)
4.2.19. Boc-Glu(Arg-Asp)-Arg-Asp (5b). Using the same
procedure as that for 5a, starting from 500 mg
(0.592 mmol) of 4b, 319 mg (68%) of the title compound
was obtained, which was used in the next reaction
directly.
20
676 [M+H]+; ½aꢂ ꢀ 14:0 (c 0.01, H2O). Anal. Calcd
D
for C24H41N11O12: C 42.66, H 6.12, N 22.80; found: C
42.49, H 6.01, N 22.99.
4.2.13. Cu(II)-Asp(Asp-Arg)-Asp-Arg
[10a–Cu(II)].
4.2.20. H-Glu(Arg-Asp)-Arg-Asp (6b). Using the same
procedure as that for 3a, starting from 300 mg
(0.380 mmol) of 5b, 180 mg (69%) of the title compound
was obtained. Mp 161–163 ꢁC; IR (KBr) 3346, 2879,
Using the same procedure as that of 3a–Cu(II), starting
from 124 mg (0.15 mmol) of 10a, 34 mg (31%) of the ti-
tle compound was obtained as blue powder. IR (KBr)
3351, 2964, 2401, 1635, 1396, 1193, 640 cmꢀ1; ESI-MS
2360, 1652, 1589, 1386, 1259, 748, 669 cmꢀ1; H NMR
1
20
(m/z) 737[M-1]+; ½aꢂ ꢀ 29:4 (c 0.01, H2O). Anal. Calcd
(BHSC-500, D2O) d 4.65 (dd, J = 5.0, 10.0 Hz, 2H),
4.35 (t, J = 7.5 Hz, 1H), 4.23 (dd, J = 6, 8 Hz, 1H),
3.85 (dd, J = 5.0, 8.5 Hz, 1H), 3.11 (t, J = 6.5 Hz, 4H),
2.86 (m, J = 5.0 Hz, 4H), 2.37(t, J = 7.0 Hz, 1H), 2.28
(dd, J = 8.0, 16.0 Hz, 1H), 2.12 (t, J = 8.0 Hz, 1H) 2.00
(t, J = 6.5 Hz, 1H), 1.76 (m, J = 8.0, 14.5 Hz, 2H),
D
for C24H40CuN11O12Æ2H2O: C 37.23, H 5.73, N 19.90;
found: C 37.06, H 5.61, N 19.73.
4.2.14. Boc-Glu(ONp)-ONp (1b). Using the same proce-
dure as that for 1a, starting from 5.0 g (0.02 mol) of
Boc-Glu, 8.2 g (84%) of the title compound was ob-
tained, which was used directly in the next reaction.
1.70–1.62 (m, 2H), 1.57 (m, J = 6.5, 16.5 Hz, 4H); ESI-
20
MS (m/z) 690 [M+H]+; ½aꢂ ꢀ 22:6 (c 0.01, H2O). Anal.
D
Calcd for C25H43N11O12: C 43.54, H 6.28, N 22.34;
found: C 43.36, H 6.16, N 22.50.
4.2.15. Boc-Glu(Arg)-Arg (2b). Using the same procedure
as that for 2a, starting from 1.8 g (3.68 mmol) of 1b and
1.6 g (7.7 mmol) of HClÆH-Arg, 1.75 g (85%) of the title
compound was obtained. Mp 89–91 ꢁC; 1H NMR
(BHSC-300, D2O) d 4.15 (t, J = 4.2 Hz, 2H), 3.80 (t,
J = 5.2 Hz, 1H), 3.08 (t, J = 7.35 Hz, 4H), 2.28 (t, J =
4.2.21. Cu(II)-Glu(Arg-Asp)-Arg-Asp [6b-Cu(II)]. Using
the same procedure as that for 3a–Cu(II), starting from
0.100 g (0.145 mmol) of 6b, 40 mg (37%) of the title
compound was obtained as blue powder. ESI-MS (m/
20
D
7.2 Hz, 2H), 1.98 (m, 2H), 1.80 (m, 4H), 1.64 (m, 4H),
z) 750 [Mꢀ1]+; ½aꢂ ꢀ 8:8 (c 0.01, H2O). Anal. Calcd
20
1.32 (s, 9H); ESI-MS (m/z) 560 [M+H]+; ½aꢂ ꢀ 22:7 (c
for C25H42CuN11O12Æ2H2O: C 38.09, H 5.88, N 19.55;
found: C 38.26, H 5.75, N 19.70.
D
0.01, H2O). Anal. Calcd for C22H41N9O8: C 47.22, H
7.38, N 22.53; found: C 47.03, H 7.27, N 22.33.
4.2.22. Boc-Glu[Asp(OMe)]-Asp(OMe) (7b). Using the
same procedure as that for 7a, starting from 5.0 g
(0.01 mol) of 1b and 3.84 g (0.02 mmol) of HClÆH-As-
p(OCH3), 4.1 g (81%) of the title compound was ob-
tained. Mp 83–85 ꢁC; H NMR (BHSC-300, DMSO-
d6) d 12.85 (s, 2H), 8.16 (d, J = 8.1 Hz, 1H), 8.12 (d,
J = 8.1 Hz, 1H), 6.92(d, J = 8.1 Hz, 1H), 4.55 (t,
J = 5.2 Hz, 2H), 3.88 (t, J = 7.0 Hz, 1H), 3.59 (s, 6H),
4.2.16. H-Glu(Arg)-Arg (3b). Using a similar procedure
as that for 3a, starting from 0.5 g (0.89 mmol) of 2b,
319 mg (78%) of the title compound was obtained. Mp
140–142 ꢁC; 1H NMR (BHSC-300, D2O) d 4.22 (dd,
J = 4.5, 9.0 Hz, 1H), 4.11 (dd, J = 3.9, 9.0 Hz, 1H),
3.74 (dd, J = 4.5, 11.1 Hz, 1H), 3.11 (dd, J = 5.7,
19 Hz, 4H), 2.37 (dd, J = 4.8, 14.7 Hz, 2H), 2.22 (m,
1
2H), 1.73 (m, 4H), 1.57 (m, 4H); ESI-MS (m/z) 460
2.70 (m, 4H), 2.19 (m, 2H), 1.72 (m, J = 6.5 Hz, 2H),
20
D
20
D
[M+H]+; ½aꢂ ꢀ 11:8 (c 0.01, H2O). Anal. Calcd for
1.43 (s, 9H); ESI-MS (m/z) 506 [M+H]+; ½aꢂ 3:2 (c
C17H33N9O6: C 44.44, H 7.24, N 27.43; found: C
44.61, H 7.11, N 27.25.
0.01, CH3OH). Anal. Calcd for C20H31N3O12: C 47.52,
H 6.18, N 8.31; found: C 47.67, H 6.06, N 8.50.
4.2.17. Cu(II)-Glu(Arg)-Arg [3b–Cu(II)]. Using the same
procedure as that for 3a–Cu(II), starting from 100 mg
(0.218 mmol) of 3b, 48 mg (42%) of the title compound
4.2.23. Boc-Glu[Asp(OMe)-Arg-OMe]-Asp(OMe)-Arg-
OMe (8b). Using the same procedure as that for 8a,
starting from 4.0 g (0.008 mol) of 7b and 3.8 g
(0.017 mmol) of HClÆH-Arg-OCH3, 4.68 g (70%) of the
title compound was obtained. Mp 118–119 ꢁC; 1H
NMR (BHSC-500, D2O) d 4.38 (t, J = 5.2 Hz, 2H),
4.14 (t, J = 5.2 Hz, 2H), 3.91 (t, J = 7.35 Hz, 1H), 3.62
(s, J = 7.2 Hz, 12H), 3.10 (t, J = 6.5 Hz, 4H), 2.85 (m,
J = 6.5 Hz, 2H), 2.78 (m, J = 6.4 Hz, 2H), 2.29 (d,
J = 6.4 Hz, 2H), 2.04 (m, 2H), 1.78 (m, 4H), 1.52 (m,
was obtained. IR (KBr) 3367, 2966, 2360, 1596, 1411,
20
1207, 732 cmꢀ1; ESI-MS (m/z) 521 [Mꢀ1]+; ½aꢂ þ 43:4
D
(c 0.01, H2O). Anal. Calcd for C17H32CuN9O6Æ2H2O: C
36.59, H 6.50, N 22.59; found: C 36.41, H 6.37, N 22.78.
4.2.18. Boc-Glu[Arg-Asp(OMe)-OMe]-Arg-Asp(OMe)-
OMe (4b). Using the same procedure as that for 4a,
starting from 1.22 g (2.18 mmol) of 2b and 902 mg
(4.58 mmol) of H-Asp(OMe)-OMe, 1.2 g (65%) of the ti-
4H), 1.31 (s, 9H); ESI-MS (m/z) 846 [M+H]+;
20
D
½aꢂ ꢀ 30:3 (c 0.01, CH3OH). Anal. Calcd for
1
tle compound was obtained. Mp 116–118 ꢁC; H NMR
C34H59N11O14: C 48.28, H 7.03, N 18.21; found: C
48.11, H 7.13, N 18.40.
(BHSC-500, D2O) d 4.25 (t, J = 7.0 Hz, 2H), 4.01 (t,
J = 5.2 Hz, 2H), 3.87(t, J = 7.35 Hz, 1H), 3.65 (d,
J = 7.2 Hz, 12H), 3.11 (t, J = 4.5 Hz, 4H), 2.29 (m,
4.2.24. Boc-Glu(Asp-Arg)-Asp-Arg (9b). Using the same
procedure as that for 5a, starting from 0.239 g
(0.282 mmol) of 8b, 200 mg (90%) of the title compound
was obtained, which was used directly in the next
reaction.
4H), 2.01 (m, 4H), 1.80 (m, 4H), 1.55 (m, 4H), 1.33 (s,
20
9H); ESI-MS (m/z) 846 [M+H]+; ½aꢂ ꢀ 97:3 (c 0.01,
D
CH3OH). Anal. Calcd for: C34H59N11O14: C 48.28, H
7.03, N 18.21; found: C 48.11, H 7.16, N 18.38.

5924
Y. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5914–5925
4.2.25. H-Glu(Asp-Arg)-Asp-Arg (10b). Using the same
procedure as that for 3a, starting from 200 mg
(0.253 mmol) of 9b, 150 mg (86%) of the title compound
was obtained. Mp 147–149 ꢁC; IR (KBr) 3349, 2952,
sures that the welfare of the animals was maintained in
accordance to the requirements of the animal welfare act
and according to the guide for care and use of labora-
tory animals.
1
2360, 1652, 1558, 1394, 1253, 752, 611 cmꢀ1; H NMR
(BHSC-500, D2O) d 4.49 (t, J = 4.0 Hz, 2H), 4.14 (t,
J = 4.5 Hz, 2H), 4.11 (t, J = 4.5 Hz, 1H), 3.07 (t,
J = 6.5 Hz, 4H), 2.68 (dd, J = 9.0 Hz, J = 14.5 Hz,
4.4.1. Intravenous administration of (3,6,10)a,b and
[(3,6,10)a,b]–Cu(II). Aspirin, (3,6,10) a,b, and [(3,6,10)
a,b]–Cu(II) were dissolved in NS just before use and
kept in an ice bath. Male Wistar rats weighing 250–
300 g (purchased from Animal Center of Peking Univer-
sity) were used, and the method of Ji et al.28 was fol-
lowed. Venom-derived fractions were dissolved in NS
just before use and kept in an ice bath. The rats were
anesthetized with pentobarbital sodium (80.0 mg/kg,
ip), and the right carotid artery and left jugular vein
were separated. A weighed 6 cm thread was inserted into
the middle of a polyethylene tube. The polyethylene tube
was filled with heparin sodium (50 IU/mL in NS) and
one end was inserted into the left jugular vein. From
the other end of the polyethylene tube, heparin sodium
was injected as anticoagulant, then NS or ( 3,6,10)a,b
or [(3, 6, 10)a,b]–Cu(II) were injected, and this end was
inserted into the right carotid artery. Blood flowed from
the right carotid artery to the left jugular vein through
the polyethylene tube for 15 min. The thread was recov-
ered to obtain the thrombus weight.
4H), 2.08 (m, 4H), 1.75 (m, 4H), 1.50 (m, J = 4.5 Hz,
20
4H); ESI-MS (m/z) 690 [M+H]+; ½aꢂ ꢀ 11:5 (c 0.01,
D
H2O). Anal. Calcd for C25H43N11O12: C 43.54, H 6.28,
N 22.34; found: C 43.40, H 6.21, N 22.52.
4.2.26.
Cu(II)-Glu(Asp-Arg)-Asp-Arg
[10b–Cu(II)].
Using the same procedure as that for 3a–Cu(II), starting
from 100 mg (0.145 mmol) of 10b, 26 mg (23%) of the ti-
tle compound was obtained. IR (KBr) 3350, 2965, 2340,
1589, 1397, 1186, 779 cmꢀ1; ESI-MS (m/z) 750 [Mꢀ1]+;
20
½aꢂ ꢀ 40:7 (c 0.01, H2O). Anal. Calcd for C25H42Cu-
D
N11O12Æ2H2O: C 38.09, H 5.88, N 19.55; found: C
38.25, H 5.74, N 19.37.
4.3. In vitro anti-platelet aggregation assay
An H-10 cell counter was used to determine the platelet
count. A two-channel Chronolog aggregometer was
used to evaluate platelet aggregation. After collection,
pig blood was centrifuged at 100g for 10 min and the
platelet-rich plasma (PRP) was removed. The remaining
blood was centrifuged for a further 10 min at 1500g to
prepare platelet-poor plasma (PPP). The final platelet
count of the citrated plasma samples was adjusted to
2·108 platelets/mL with autologous PPP. In the assays
0.5 mL of the adjusted plasma sample and 5 lL of NS
or a solution of (3,6,10)a,b (in a range of concentrations
from 10ꢀ2 to 10ꢀ4 M) or [(3,6,10)a,b]–Cu(II) in NS (10ꢀ2
M to 10ꢀ4 M) was added to the testing tube. After
adjustment of the baseline, 5 lL of a solution of plate-
let-activating factor in NS (PAF, final concentration
10ꢀ7 M), or 5 lL of a solution of adenosine diphosphate
in NS (ADP, final concentration 10ꢀ5 M), or 5 lL of a
solution of arachidonic acid in NS (AA, final concentra-
tion 3.5·10ꢀ4 M), or 5 lL of a solution of thrombin in
NS (TH, final concentration 0.2 U/mL) was then added
to the testing tube. The testing tube was stirred at 37 ꢁC
for 5 min allowing the platelets having a maximal aggre-
gation, while the aggregometer automatically recorded
the aggregation curve. The peak height of this aggrega-
tion curve was defined as the maximal aggregation rate
(Am). The inhibition effect of (3,6,10)a,b and [(3,6,10)
a,b]–Cu on PAF-, ADP-, AA-, or TH-induced platelet
aggregation was represented by the difference between
the Am of NS and that of (3,6,10)a,bor [(3,6,10)a, b]–
Cu, the aggregation inhibition rate was calculated by
Inhibition = [(Am of NS) ꢀ (Am of ( 3,6,10) a,bor [(
3,6,10)a,b]–Cu(II)) ꢃ (Am of NS), and the IC50 value
was determined via the program GWBASIC.EXE.
4.4.2. Oral administration of 10a. Solutions of aspirin or
10a in NS were given orally to male Wistar rats (weigh-
ing 250–300 g, purchased from Animal Center of Peking
University). Then the rats were anesthetized with pento-
barbital sodium (80.0 mg/kg, ip). Thirty min later, the
right carotid artery and left jugular vein of the rat were
separated. A weighed 6 cm thread was inserted into the
middle of a polyethylene tube. The polyethylene tube
was filled with heparin sodium (50 IU/mL in NS) and
one end was inserted into the left jugular vein and the
other end was inserted into the right carotid artery.
Blood flowed from the right carotid artery to the left
jugular vein through the polyethylene tube for 15 min.
The thread was removed and weighed, and the weight
of the wet thrombus was recorded.
4.5. Particle size and zeta-potential tests of (3,6,10)a,b
and [(3,6,10)a,b]–Cu(II)
The size of the nano-particles of (3,6,10)a,b and [(3,6,10)
a,b]–Cu(II) assembled in NS was analyzed using a Mal-
vern’s Zeta Sizer (Nano-ZS90) with DTS (Nano) Pro-
gram. In the determination, 1 mg each of ( 3,6,10)a,b
or [(3,6,10)a,b]–Cu(II) was dissolved in 1 mL of normal
saline (NS, 0.9% NaCl aqueous solution, pH 7.3–7.4) to
prepare the testing solution (1 mg/mL, pH 6.8–7.1). This
stock solution was kept in a 4 ꢁC refrigerator before use.
In the test, the system temperature was 25 ꢁC, the data
collection time was 50 s and total collection 5 min. The
Sizer automatically read the mean size, half-peak width
and % intensity of the peak.
4.4. In vivo anti-thrombosis activity in rat model
Zeta-potentials of the nano-particles assembled from
(3,6, 10)a, b and [(3,6,10)a,b]–Cu(II) in NS were analyzed
using a ZetaPlus Potential Analyzer (ZetaPlus S/N
21394, Brookhaven Instruments Coorporation) with
The assessments described herein were performed based
on a protocol reviewed and approved by the ethics com-
mittee of Capital Medical University. The committee as-

Y. Chen et al. / Bioorg. Med. Chem. 16 (2008) 5914–5925
5925
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illustrated by photographs, and particle sizes repre-
sented by diameters.
Acknowledgments
This work was supported by Beijing Area Major Labo-
ratory of Peptide and Small Molecular Drugs, the 973
Project of China (2006CB708501) and Natural Scientific
Foundation of China (30472071).
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