Coordination of Thrombolytic Pro-Ala-Lys peptides with Cu (II

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J. Phys. Chem. B 2008, 112, 8174–8180

Coordination of Thrombolytic Pro-Ala-Lys peptides with Cu (II): Leading to Nanoscale Self-assembly, Increase of Thrombolytic Activity and Additional Vasodilation Xue Ren,† Guohui Cui,‡ Ming Zhao,*,‡ Chao Wang,† and Shiqi Peng*,†,‡ College of Pharmaceutical Sciences, Peking UniVersity, Beijing 100083, P.R. China, and College of Pharmaceutical Sciences, Capital Medical UniVersity, Beijing 100069, P.R. China ReceiVed: January 23, 2008; ReVised Manuscript ReceiVed: April 5, 2008

Vasodilation is one of the biologically important properties for thrombolytic agents because of it may help thrombolysis via dilating blood vessels. Aimed at discovering agents with the dual-action of vasodilative and thrombolytic activities, H-Pro-Ala-Lys (PAK, 3a) and five novel analogs H-Pro-Ala-AA (2b-f, AA ) Val, Phe, Ser, Glu, and His) were coordinated with Cu(II) to form Cu(II)-Pro-Ala-AA [(3a-f)-Cu(II)]. The coordination chemistry was confirmed by the d-d transition occurred in their UV and circular dichroism (CD) spectra and the molecular ion in their electrospray ionization mass spectrometry (ESI-MS) spectra. The particle size tests of their solution and powders revealed that the coordination generally resulted in nanoscale self-assembly. Zeta potential and half-peak width tests indicated that the formed nanoparticles were sufficiently stable during the monitored 8 days. The bioassays implied that comparing to the PAK peptides themselves and CuCl2 the coordination led to a 3000-fold increase of the in vitro thrombolytic activity, a 10-fold increase of the in vivo thrombolytic activity, and especially an additional vasodilation. Thus Cu(II)-peptide coordination indeed is a way for thrombolytic peptide design. I. Introduction It is well-known that in the atherosclerotic coronary artery occlusive thrombi cause a variety of heart diseases such as heart attack, stroke, and other peripheral vascular diseases. However, currently used protein thrombolytic agents, such as tissue-type plasminogen activator (t-PA), urokinase (UK) and streptokinase (SK), are known to have severe side effects, including hemorrhagic tendency and immunogenic reactions.1–5 Discovering small peptides has been considered an approach toward making the side-effect-benefit ratio favorable for clinical therapy and has attracted a lot of effort. In such an effort, fibrin(ogen) Bβ chain was incubated with plasmin and led to finding the thrombolytic product H-Ala-Arg-Pro-Ala-Lys (P6A).6–11 Afterward, from P6A metabolites, H-Pro-Ala-Lys (PAK, 3a) was identified as a thrombolytic compound with free solution conformation.12,13 Thus rigidifying the conformation of PAKlike small peptides has become one of our resent interests. It is known that, when the secondary and tertiary structures of peptides are defined, various noncovalent interaction propensities will generally be increased and may gain tremendous self-assembling potential. As naturally occurring motifs, the selfassembling peptides are able to gain rapid access to an array of three-dimensional structures, thus to a rigid conformation 14–19 and a nanosystem.20–23 Having various conformational rigidities means that Cu(II)-peptide complexes are known to be excellent scaffolds.24–27 In this context, Cu(II)-PAK-like peptide complexes entered our vision field. In this paper, 3a was first altered at its C-terminal residue to form five analogues of H-Pro-AlaAA (3b-f, AA ) Val, Phe, Ser, Glu, and His). Subsequently, * To whom correspondence should be addressed. S.P.: College of Pharmaceutical Sciences, Capital University of Medical Sciences, Beijing 100069, P.R. China; tel: 86-10-8391-1528; fax: 86-10-8391-1528; e-mail: [email protected]. M.Z.: Tel.: +86-10-8280-2482; fax: +86-108280-2482. † Peking University. ‡ Capital Medical University.

Cu(II)-Pro-Ala-AA complexes (3a-f)-Cu(II) were prepared. Finally the generated nanoscale self-assembling ability and vasodilation activity, and the enhanced in vitro and in vivo thrombolytic activity via coordination of 3a-f and Cu(II) were defined by a series of tests and evaluations. II. Results and Discussion II-1. UV and Circular Dichroism (CD) Spectra Confirming the Coordination of 3a-f and Cu(II). UV and CD spectra are widely used as the tools judging the coordination status of Cu(II) and a peptide. To confirm the coordination of 3a-f and Cu(II), the UV spectra of 3a-f (Figure 1) and the complexes (3a-f)-Cu(II) (Figure 2) were first analyzed. It is well established that the d-d transition of Cu(II) resulted in the absorbance from 520 nm to 650 nm.28–32 As seen, in this area, the UV spectra of 3a-f gave no absorbance, and the UV spectra of (3a-f)-Cu (II) gave typical peaks, suggested that Cu(II) successfully coordinated with 3a-f. On the other hand, the fact that the UV spectra of (3a-f)-Cu(II) are distinct from that of 3a-f also implies that the coordination may be generally used to judge the coordination of Cu(II) and PAK-like peptides. To confirm the coordination of 3a-f and Cu(II), the CD spectra of 3a-f (Figure 3) and (3a-f)-Cu(II) (Figure 4) were also analyzed. Figure 3 indicates that the CD spectra of 3a-f give two sets of peaks at ca. 200 nm and 225 nm. The peaks at ca. 200 nm and 225 nm usually reflect the flexibility of the side chain and the peptide backbone, respectively. Thus the observed two sets of peaks supported the idea that 3a-f were free peptides. Figure 4 explores that, upon the effect of Cu (II) coordination, the peaks at ca. 200 nm shift to about 210 nm, while the peaks at ca. 225 nm shift to about 250 nm and become very weak, suggesting that Cu(II) coordination decreased the flexibility of the peptide backbone. Figure 4 also gave two sets of additional peaks at ca. 310 nm and 550 nm, respectively. These additional peaks were clearly due to the charge transfer between N amide and Cu(II) and Cu(II) d-d transition. 28–33 It

10.1021/jp800645g CCC: $40.75  2008 American Chemical Society Published on Web 06/12/2008

Coordination of Pro-Ala-Lys Peptides

Figure 1. UV spectra of 3a-f in water. No absorbance from 520 to 650 nm was observed. Test temperature: 25 °C; concentration 0.1 mM. Here a, b, c, d, e, and f represent 3a, 3b, 3c, 3d, 3e, and 3f, respectively.

Figure 2. UV spectra of (3a-f)-Cu(II) in water. The d-d transition that occurred at 520-560 nm in the spectra is shown. Test temperature: 25 °C; concentration 0.1 mM. Here g, h, i, j, k, and l represent 3a-Cu, 3b-Cu, 3c-Cu, 3d-Cu, 3e-Cu, and 3f-Cu, respectively.

was also found that the CD spectra of (3b,c)-Cu(II) themselves exhibited distinct intensity. For instance, the positive peaks at ca. 210 nm of (3b,c)-Cu(II) with hydrophobic side chains had higher intensity, while (3a,f)-Cu(II) with hydrophilic side chains had lower intensity. The different solvolytic ability of hydrophobic and hydrophilic side chains is likely responsible for these intensity differences. Because of the charge transfer between N and Cu(II) occurring at a similar amide around 310 nm (3a,f)-Cu(II) gave positive peaks with same intensity. The negative peaks at ca. 550 nm could be the result of {NH, N-, 2CO} coordination of (3a-f)-Cu (II). The intensity of these negative peaks likely correlated with the side chain or the amino acid residue at C-terminus. For instance, with C-terminal basic and polar side chains, (3a,f)-Cu(II) had lower peak intensity, with C-terminal hydrophobic side chain (3b,c)-Cu(II) had modest peak intensity, and with C-terminal neutral and polar

J. Phys. Chem. B, Vol. 112, No. 27, 2008 8175 side chains (3d,e)-Cu (II) had higher peak intensity. The exact reason of these correlations remains to be investigated. In spite of these the comparison of the CD spectra of (3a-f)-Cu(II) and 3a-f supports that Cu(II) successfully coordinated with 3a-f, and implies that CD spectrum may be generally used to judge the coordination of Cu(II) and PAK-like peptide. The binding mode of Cu(II) and peptide can also be identified by d-d transition. The coordination of Cu(II) was reported starting usually at the peptide’s N-terminal amino nitrogen, following a coordination model of {NH2, 2N-, CO} and having the N/Cu(II) charge transfer transition at ca. 310 nm and the d-d transitions at ca. 540 nm. 33 Accordingly, N-terminal Pro residue of a peptide is also capable of taking part in Cu(II) complexes following a coordination model of {NH, 2N-, CO}, which is supported by the N/Cu(II) charge transfer transition at 310 nm and the d-d transitions at ca. 550 nm. 34,35 Taking the d-d transitions at ca. 540 nm in the UV spectra (Figure 2), the N/Cu(II) charge transfer transition at 310 nm and the d-d transitions at ca. 550 nm in the CD spectra (Figure 4), the [M-H]+ of MS, and the Pro residue at the N-terminus into account, the binding mode of Cu(II) and 3a-f was postulated as {NH, N-, 2CO}. 33,36 The structures of (3a-f)-Cu(II) corresponding to this coordination model were optimized using the QSAR module of Cerius2 and are summarized in Figure 5. II-2. Nanoscale Self-Assembly of (3a-f)-Cu(II) in Its Normal Saline (NS) Solution. The self-assembly of (3a-f)-Cu(II) in NS was explored by testing the formed particle size. The mean size and half-peak width of the particles are listed in Table 1. The data demonstrate that, in NS, (3a-f)-Cu(II) spontaneously assemble to nanoparticles. During the first 30 min, the particle diameters ranged from 253.39 nm to 276.16 nm, and the half-peak width ranged from 16.01 nm to 50.20 nm. After 8 consecutive days, the particle diameters ranged from 210.03 nm to 345.79 nm, and the half-peak width ranged from 8.72 nm to 35.18 nm. The fact that the particle diameters of 30 min are close to the mean value of 8 days suggests that the self-assembly of (3a-f)-Cu(II) in NS is a fast process. The half-peak widths of 8 days were significantly smaller than those of 30 min suggesting that, with lengthening the time, the nanoparticles were able to reassemble, evening their size. II-3. Morphology and Particle Size of (3a-f)-Cu(II) Powders. To explore the self-assembling property of (3a-f)-Cu(II) in solid state, their powders’ transmission electron microscopy (TEM) photographs were made and are shown in Figure 6. TEM measurements reveal that all the powders consisted of even nanoparticles, and the diameters of the powders ranged from 15 nm to 50 nm. The results not only demonstrate that the nanoscale self-assembling property of (3a-f)-Cu(II) in aqueous solution can be kept during the evaporation of the water, but also indicate that, via limiting water, the originally spherical particles lost internal volume and became smaller.37 II-4. Zeta-Potential of the Nanosystems of (3a-f)-Cu(II) Assembled in NS. The electrical property of the nanosystems was defined by zeta potential measurements, and the data are listed in Table 2. It was found that, for the first 1 h, the zeta potential of the nanosystems ranged from -12.89 mV to -18.86 mV, and on the eighth day ranged from -8.86 mV to -13.10 mV. Surface zeta potential is generally considered as a measurement of the charge density per unit of surface, which depends on both the amount of charging groups and the size of the nanoparticle. The moderate negative value of the zeta potential of the nanosystems of (3a-f)-Cu(II) assembled in NS demonstrated that the nanoparticles had positive charges, and

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Figure 3. CD spectra of 3a-f in normal saline (NS), temperature 25 °C, concentration 1 mM. There is no any peak of dichroic effect transition between 250 and 750 nm. Here, a, b, c, d, e, and f represent 3a, 3b, 3c, 3d, 3e, and 3f, respectively.

Figure 4. CD spectra of (3a-f)-Cu in NS, testing temperature 25 °C, and concentration 1 mM. In the CD spectrum of 3f-Cu(II), a positive peak occurs around 500 nm, and a negative peak occurs around 570 nm; in the CD spectra of (3a-e)-Cu(II), the negative peaks occur around 540 nm to 570 nm. Here, g, h, i, j, k, and l represent 3a-Cu, 3b-Cu, 3c-Cu, 3d-Cu, 3e-Cu, and 3f-Cu, respectively.

their surfaces were surrounded by comparatively sufficient negative charges and therefore the nanoparticles had proper size. Since during the monitored 8 days the zeta potential only exhibited a temperate decrease, the nanosystems should be stable for more than 8 days. II-5. Biological Benefits Resulting from the Coordination and Self-Assembly of 3a-f with Cu(II). To explore the biological benefits resulting from the coordination and the selfassembly of 3a-f with Cu(II), a series of bioassays, including in vitro vascular relaxation assay, in vitro thrombolytic assay, and in vivo thrombolytic assay for 3a-f and(3a-f)-Cu(II) were performed, and the corresponding activities of both the peptides and the Cu(II)-peptide complexes were compared. II-5.1. Coordination of 3a-f with Cu(II) Leading to a More than 3000-Fold Increase for the In Vitro Thrombolytic ActiWity. To evaluate the fibrillolytic potency of 3a-f and (3a-f)-Cu(II), the in vitro thrombolytic experiments were performed with NS and UK (final concentration, 100 000 IU/ L) as the negative and positive controls, respectively.38,39 The reduction of the thrombus weight was used to represent the in vitro thrombolytic activity. The data are listed in Table 3. It is clear that the reduced weights of the thrombi incubated in 3.75 mM of CuCl2 (22.31 mg) and NS (21.08 mg) are equal, and those in 375 µM of 3a-f (ranging from 17.48 mg to 33.3 mg) and 0.1 µM of (3a-f)-Cu(II) (ranging from 25.28 mg to 29.17 mg) are substantially equal. These data suggest that CuCl2 possesses little in vitro thrombolytic activity, and the in vitro thrombolytic activities of (3a-f)-Cu(II) are 3000-fold higher

than those of 3a-f. These comparisons suggest that the potent in vitro thrombolytic activity of (3a-f)-Cu(II) obviously results from coordination and self-assembly, and is independent of the contribution from free peptide and the Cu(II) ion. On the other hand, the in vitro thrombolytic activity of (3a-f)-Cu(II) likely correlated with their structure (Figure 5) with minimal energy and particle size (Table 1). According to the structures of the most potent (3a,f)-Cu(II) in Figure 5, their side chain amino group of Lys residue or imidazole group of Val residue is capable of forming a strong hydrogen bond with the carbonyl group of Ala residue. According to the particle diameters of the most potent (3a,f)-Cu (II) in Table 1, their self-assembling nanoparticles have smaller size. However, 3c-Cu(II), the compound with lower activity, is not capable of forming a similar hydrogen bond, and its nanoparticle has larger size. II-5.2. Coordination of 3a-f with Cu(II) Leading to an Additional Vasodilation. To explore the vasodilation gained from the coordination, the vasodilative activities of 3a-f and (3a-f)-Cu(II) were assayed using a rat aortic strip experiment of a standard procedure with noradrenaline (NE) as the aortic strip constriction agent.40 The dilation of 3a-f and (3a-f)-Cu(II) (in a series of final concentrations ranging from 150 µM to 0.1 µM) to NE-induced vasoconstriction was recorded, and the determined EC50 values are listed in Table 4. The results showed that the EC50 of CuCl2-inhibited NE-induced vasoconstriction was more than 10.0 mM, and those of 3a-f ranged from 84.6 µM to 116.5 µM, while those of (3a-f)-Cu(II) ranged from 1.0 µM to 5.7 µM. These data

Coordination of Pro-Ala-Lys Peptides

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Figure 5. Structures of (3a-f)-Cu(II) corresponding to {NH, N-, 2CO} coordination.

TABLE 1: Nanoparticle Diameters of (3a-f)-Cu(II) in NSa after the first 30 min

after 8 consecutive days

compound

diameter

half-peak width

diameter

half-peak widthb

3a-Cu 3b-Cu 3c-Cu 3d-Cu 3e-Cu 3f-Cu

235.35 ( 12.27 263.42 ( 12.00 331.57 ( 14.33 276.16 ( 6.09 253.39 ( 5.92 253.62 ( 6.58

16.01 ( 3.62 40.32 ( 5.28 37.83 ( 5.06 42.05 ( 2.02 33.72 ( 1.62 50.20 ( 3.49

210.03 ( 286.04 ( 9.78c 345.79 ( 16.08d 248.24 ( 9.06c 251.01 ( 6.88d 249.53 ( 4.28d

8.72 ( 1.43 31.46 ( 2.84 24.66 ( 3.32 13.78 ( 2.13 25.44 ( 1.81 35.18 ( 2.79

12.05c

a Diameter and half-peak width are expressed by jx ( SD nm, n ) 10. b Compared to the corresponding half-peak width after the first 30 min, p < 0.01. c Compared to the corresponding diameter after the first 30 min, p < 0.01. d Compared to the diameter after the first 30 min, p > 0.05.

suggest, in contrast with (3a-f)-Cu(II), CuCl2, PAK, and PAK-like peptides possess little in vitro vasodilation activity. These comparisons suggest that the potent vasodilation of (3a-f)-Cu(II) obviously results from coordination and assembly, and is independent of the contribution from free peptide and Cu(II) ion. II-5.3. Coordination of 3a-f with Cu(II) Leading to a 10Fold Increase of the In ViWo Thrombolytic ActiWity. To compare the in vivo thrombolytic activity of 3a-f and (3a-f)-Cu(II), the rat thrombolytic experiment of a standard procedure was performed with NS as the negative control and UK as the positive control. 41 The reduction weight of the thrombi inside the blood circulation was used to represent the in vivo thrombolytic activity, and the data are listed in Table 5. The data explore that the reduced weights of NS (10.43 mg) and 10.0 µM/kg of CuCl2 (11.69 mg) receiving rats are equal, and those of 1.0 µM/kg of 3a-f (ranging from 13.09 mg to 16.18 mg) and 0.1 µM/kg of (3a-f)-Cu(II) (ranging from 13.82 mg to 16.47 mg) receiving rats are substantially equal. These data suggest that CuCl2 possesses no in vivo thrombolytic activity, and that the activity of (3a-f)-Cu(II) is 10 times higher than that of 3a-f. These comparisons suggest that the 10-fold increase of the in vivo thrombolytic activity of (3a-f)-Cu(II) obviously results from coordination and selfassembly, and is independent of the contribution from free peptide and Cu(II) ion. On the other hand, the in vivo thrombolytic activity of (3a-f)-Cu(II) likely correlated with

their structure (Figure 5) with minimal energy and particle size (Table 1). According to the structures of the most potent (3a,f)-Cu(II) in Figure 5, their side chain amino group of Lys residue or imidazole group of Val residue is capable of forming a strong hydrogen bond with the carbonyl group of Ala residue. According to the particle diameters of the most potent (3a,f)-Cu(II) in Table 1, their self-assembling nanoparticles have smaller size. However, 3c-Cu(II), the compound with lower activity, is not capable of forming a similar hydrogen bond, and its nanoparticle has larger size. III. Conclusion In conclusion, using general procedures, Cu(II)-PAK-like peptide complexes (3a-f)-Cu(II) can be successfully prepared with {NH, N-, 2CO} coordination. In water and NS, these complexes quickly self-assembled to form stable nanoparticles, and this nanoscale self-assembling property was also gained in their powders. The diameters of their nanoparticles in both the solution and powders range from 15 to 346 nm, which are the diameters of the stable nanoparticles. In solution, the moderate negative charge surrounded the surfaces of their particles, and this was one of the factors stabilizing the nanosystems. The bioassays emphasized that the free Cu(II) ion had neither vasodilative activity nor thrombolytic activity, PAK peptides had little vasodilative activity and modest thrombolytic activity, and this self-assembly property of Cu(II)-PAK peptide com-

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Figure 6. TEM photographs of the powders of (3a-f)-Cu(II). The diameters of the powders ranged from 15 nm to 50 nm.

TABLE 2: Zeta Potential of (3a-f)-Cu zeta potential (mV) compound 3a-Cu 3b-Cu 3c-Cu 3d-Cu 3e-Cu 3f-Cu

after the first 1 h -14.34 -17.44 -18.86 -12.89 -14.16 -16.30

on the 8th day

compound

EC50 (µM)

compound

EC50 (µM)

-8.86 -9.85 -12.50 -12.76 -13.10 -11.93

3a 3b 3c 3d 3e 3f CuCl2

84.59 ( 10.86 100.54 ( 12.38 112.74 ( 13.11 90.78 ( 10.16 116.48 ( 14.12 98.67 ( 11.70 >10 mM

3a-Cu 3b-Cu 3c-Cu 3d-Cu 3e-Cu 3f-Cu

1.03 ( 0.08b 4.93 ( 0.10b 2.57 ( 0.09b 5.74 ( 0.20b 3.89 ( 0.18b 5.06 ( 0.23b

TABLE 3: In Vitro Trombolytic Activities of 3a-f and (3a-f)-Cu(II)a compound NS 3a 3b 3c 3d 3e 3f UK

reduced thrombus weight 21.08 ( 2.28 30.30 ( 4.47b 30.19 ( 5.17b 30.03 ( 2.13b 26.29 ( 3.88b 25.81 ( 2.20b 17.48 ( 2.32 63.74 (10.23

compound CuCl2 3a-Cu 3b-Cu 3c-Cu 3d-Cu 3e-Cu 3f-Cu

TABLE 4: EC50 of 3a-f and (3a-f)-Cu against NE-Induced Vasoconstrictiona

reduced thrombus weight 22.31 ( 2.33 28.10 ( 4.95c 28.82 ( 2.88c 28.24 ( 3.70c 27.02 ( 2.79d 27.32 ( 1.42d 28.25 ( 2.69e

a Reduction of the thrombus weight is used to represent the in vitro thrombolytic activity and is expressed by jx ( SD mg; NS (normal saline) ) vehicle; concentration of UK (urokinase): 100 000 IU/L; concentration of 3a-f: 375 µM; concentration of (3a-f)-Cu: 0.1 µM; concentration of CuCl2: 3.75 mM; n ) 6. b Compared to NS and CuCl2, p < 0.01. c Compared to NS and CuCl2, p < 0.01, to 3a-c, p > 0.05. d Compared to NS and CuCl2, p < 0.01, to 3d,e, p > 0.05. e Compared to NS, CuCl2, and 3f, p < 0.01.

plexes led to gaining additional vasodilation and improving thrombolytic activity. In a previous paper we pointed out that, as one of the essential metals, the content of Cu in the key organs of mouse, such as the kidney, liver, and brain, ranging from 2.30 to 3.41 µg/g tissue,42 the 0.1 µM/kg dose of the present Cu(II)-complexes means that, in this therapy, the mouse receives a total of 0.122 µg of Cu, which is far less than the level of its individual organ. Therefore, there is a very large safe index for (3a-f)-Cu(II)’s thrombolytic therapy.

a EC50 is expressed by jx ( SD µM, n ) 6. b Compared to 3a-f, p < 0.001.

TABLE 5: In Vivo Thrombolytic Activities of 3a-f and (3a-f)-Cu(II)a compound

reduced thrombus weight

NS 3a 3b 3c 3d 3e 3f UK

10.43 ( 2.26 15.41 ( 3.45b 15.15 ( 3.99b 14.50 ( 2.13b 14.53 ( 2.47b 13.09 ( 2.56c 15.36 ( 2.41b 19.64 ( 3.98

compound

reduced thrombus weight

CuCl2 3a-Cu 3b-Cu 3c-Cu 3d-Cu 3e-Cu 3f-Cu

11.69 ( 2.31 16.09 ( 2.14d 14.88 ( 3.06d 13.87 ( 2.82d 14.99 ( 3.27d 15.65 ( 3.16d 16.47 ( 2.80d

a Reduction of the thrombus weight is used to represent the in vivo thrombolytic activity and is expressed by jx ( SD mg; NS (normal saline) ) vehicle; dose of UK (urokinase): 25 000 IU/kg; dose of 3a-f: 1 µM/kg; dose of (3a-f)-Cu(II): 0.1 µM/kg; dose of CuCl2: 10.0 µM/kg; n ) 10. b Compared to NS and CuCl2, p < 0.01. c Compared to NS and CuCl2, p < 0.05. d Compared to NS and CuCl2, p < 0.01, to 3a-f, p > 0.05.

IV. Experimental IV-1. General. The protected amino acids with L-configuration used were purchased from Sigma Chemical Co. All coupling and deprotective reactions were carried out under anhydrous conditions. Chromatography was performed on Qingdao silica gel H. The purities of the intermediates and the products were determined by thin-layer chromatography (TLC,

Coordination of Pro-Ala-Lys Peptides SCHEME 1: Synthetic Route of H-Pro-Ala-AA (3a-f) and Cu(II) Complexes Cu(II)-Pro-Ala-AA [(3a-f)-Cu(II)]a

a (i) Dicyclohexylcarbodiimide (DCC); (ii) NaHCO3; (iii) DCC, 1-hydroxybenzotriazole (HOBt), and N-methylmorpholine (NMM); (iv) NaOH/MeOH; (v) Pd/C, H2, MeOH/H2O; (vi) HCl/EtOAc (4N); (vii) CuCl2. In 1a, 2a AA ) Lys(Z); in3a, AA ) Lys; in 1b-3b, AA ) Val; in 1c-3c, AA ) Phe; in 1d-3d, AA ) Ser; in 1e-3e, AA ) Glu; and in 1f-3f, AA ) His.

Merck silica gel plates of type 60 F254, 0.25 mm layer thickness) and high-performance liquid chromatography (HPLC, Waters, C18 column 4.6 × 150 mm) analysis. Electrospray ionization mass spectrometry (ESI-MS) was determined on a Waters Quattro Micro ZQ2000. Optical rotations were determined on a P-1020 Jasco instrument. The statistical analysis of all the biological data was carried out by use of an ANOVA test with p < 0.05 as the significant cutoff. IV-2. Preparing Compounds and (3a-f)-Cu(II). As depicted in Scheme 1, the preparations of 3a-f were performed by use of the solution method and a (2 + 1) strategy. At first, Boc-Pro was converted into its N-hydroxysuccinimide (HOSu) active ester and then coupled with H-Ala to form Boc-Pro-Ala (80% yield). Subsequently, Boc-Pro-Ala was converted into its HOSu active ester and then coupled with H-Lys-OBzl to give Boc-Pro-Ala-Lys(Z)-OBzl (1a, 90% yield), or with H-AA-OMe to give Boc-Pro-Ala-AA-OMe (1b-f in 82-98% yields). After saponification, the methylesters 1b-f were converted into acids 2b-f in 63-96% yields. Upon removing the Boc groups in 2b-f and 1a the H-Pro-Ala-AA 3b-f and 2a were obtained, the yields ranged from 72% to 100%. After hydrogenolysis, 2a was converted into 3a in 95% yield. In an aqueous solution (pH 9) the coordination of 3a-f and CuCl2 provided (3a-f)-Cu(II) in 61-87% yields. All of the data indicate that using the present synthetic strategy, 3a-f and (3a-f)-Cu(II) can be prepared in acceptable yields. The MS data of (3a-f)-Cu(II) indicated that the ratio of Cu(II) to peptide was 1:1. The detailed procedure is described in the Supporting Information, which also contains physical chemical data. IV-3. Bioassay. IV-3.1. Thrombolytic ActiWity In Vitro Assay. The assessments described herein were performed based on a protocol reviewed and approved by the ethics committee of Capital Medical University. The committee assures 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 laboratory animals. IV-3.1.1. Preparing Cylindrical Thrombus. A male SpragueDawley (SD) rat was anesthetized with pentobarbital sodium (80 mg/kg, ip), and its right carotid artery was separated. A plastic tube was inserted into the artery, and the blood was discharged into a plastic bottle; a syringe was used to inject the blood into a cylindrical thrombus-forming glass tube mounted vertically on a bowl-like plastic bottom, and a cylindrical-thrombus supporting helix was put into its center immediately. The blood was left for another 40 min to form the cylindrical thrombus. IV-3.1.2. Simulating In ViWo EnWironment for the Cylindrical Thrombus. According to the average weight of the rats, it was postulated that each rat had a total of about 13 mL of blood;

J. Phys. Chem. B, Vol. 112, No. 27, 2008 8179 during the experiment there was a total of about 8 mL of blood that could attach to the prepared thrombi clot, and, accordingly, 8 mL of water or solution of tested compound was used instead of blood to fill the incubation bottle. In order to simulate the temperature and flow of rat blood, the incubation was carried out at 37 °C and 70 rpm on a rocking bed. IV-3.1.3. In Vitro Thrombolytic Assay. In order to establish the thrombus, at first the prepared cylindrical thrombus was carefully taken out of cylindrical thrombus-forming glass tube and hung into the incubation bottle filled with 8 mL of distilled water for 1 h. Then the thrombus was taken out, weighed precisely to record its initial weight, and hung into another incubation bottle filled with 8 mL of NS or the solution of UK (100 000 IU/L), 3a-f (375 µM), and (3a-f)-Cu(II) (0.1 µM). The bottle was incubated at 37 °C and 70 rpm on a rocking bed for 3 h, and the thrombus was precisely weighed to record its final weight. From the initial and final weights of the thrombus, the reduced weight of the thrombus was obtained and used to represent the in vitro thrombolytic potency of the tested compounds. IV-3.2. In Vitro Vasodilation Assay. A constant temperature trough (CS501, Chongqing YinHe Experimental Apparatus Ltd. of China) was used to ensure the buffer warmed; a tension transducer (Hang JZ101, Beidian Xinghang Machine and Equipment Ltd.) and a two-channel physiological recorder (LMS-2B, Chengdu apparatus manufacturer) were used to evaluate the vasodilative effect. Male Wistar rats weighing 250-300 g (purchased from the Animal Center of Peking University) were used. Immediately after decapitation, rat aortic strips were taken and put in a perfusion bath with 15 mL warmed (37 C°), oxygenated (95% O2/5% CO2) Kreb’s solution (pH 7.4). The aortic strip was connected to a tension transducer, and the relaxation contraction curve of muscles was registered. Administration of 59 µM NE induced hypertonic contraction of the vessel strip. As the contraction reaches its maximum, NE was washed out, and the vessel strip was stabilized for 30 min. After renewal of the solution, 59 µM NE was added. When the hypertonic contraction value of the aortic strip reached the peak, CuCl2, PAK peptides (3a-f), and their Cu(II) coordinates (3a-f)-Cu(II) (in a series of final concentrations of a range from 150 µM to 0.1 µM) were administrated to observe their vasodilation, and the IC50 values of 3a-t and (3a-t)-Cu to NE-induced vasoconstriction were determined by use of the program GWBASIC EXE.39 IV-3.3. In ViWo Thrombolytic Assay. A male SD rat was anesthetized with 20% urethane (6 mL/kg, ip). The right carotid artery and left jugular vein of the animal were separated. The cylindrical thrombus-supporting helix (15 circles, pitch 1.2 mm, diameter 1.0 mm) was put into the cylindrical thrombus-forming glass tube, which was then filled with artery blood (0.2 mL) from the right carotid artery of the animal immediately. After 15 min, the cylindrical thrombus was carefully taken out of the cylindrical thrombus-forming glass tube, weighed precisely to record its initial weight, and then put into the middle polyethylene tube. The polyethylene tube was filled with heparin sodium (50 IU/mL of NS), and one of the ends was inserted into the left jugular vein. Heparin sodium was injected via the other end of the polyethylene tube as the anticoagulant, after which a solution of 3a-f (1 µM/kg) or (3a-f)-Cu(II) (100 nmol/kg) or UK (20 000 IU/kg) or CuCl2 (10.0 µMol/kg) was injected. The blood was circulated through the polyethylene tube for 90 min, after which the cylindrical thrombus was carefully taken out of the middle polyethylene tube, and weighed precisely to record its final weight. From the initial and final weights of

8180 J. Phys. Chem. B, Vol. 112, No. 27, 2008 the thrombus, the reduced weight of the thrombus was obtained and used to represent the in vivo thrombolytic potency of the tested compounds. IV-4. Spectral and TEM Tests. IV-4.1. Particle Size and Zeta-Potential Tests of (3a-f)-Cu(II) in NS. The size of the nanoparticles of (3a-f)-Cu(II) assembled in NS was analyzed using a Malvern Zeta Sizer Nano Series (Nano-ZS90) with DTS (Nano) Program. The concentration of the solution of (3a-f)-Cu(II) in NS was 1 mg/mL, and the testing temperature was 25 °C. After the solution was filtered using a 0.45 µM membrane filter, the test was begun, the time interval used was 3 min, and the mean size and half-peak width for 10 times were recorded. The zeta-potential of the nanoparticles assembled by (3a-f)-Cu(II) in NS was analyzed using a Malvern Zeta Sizer Nano Series (Nano- ZS90) with DTS (Zeta) Program. The concentration of the solution of (3a-f)-Cu(II) in NS was 1 mg/mL, and the testing temperature was 25 °C. IV-4.2. UV and CD Spectral Tests of 3a-f and of (3a-f)-Cu(II). UV spectra of 3a-f (pH 3.5-4.0, aqueous solution, 1 × 10-4 mol/L, 25 °C) and (3a-f)-Cu(II) (pH 7.0-7.5, aqueous solution, 1 × 10-4 mol/L, 25 °C) were recorded with a Shimadzu UV-2550 UV-visible spectrophotometer over the range of 200-800 nm. CD spectra of 3a-f (pH 3.5-4.0, aqueous solution, 1 × 10-3 mol/L, 25 °C) and (3a-f)-Cu(II) (pH 7.0-7.5, aqueous solution, 1 × 10-3 mol/ L, 25 °C) were tested on a JASCO J-810 spectropolarimeter with the JASCO Canvas Program (model J-810, Jasco, Japan) over the range of 200-750 nm. IV-4.3. Nanosize Particle Tests of (3a-f)-Cu(II) Powders. The solution of (3a-f)-Cu(II) in double-distilled water (1 mg/ mL, pH 7.0-7.5) was evaporated at room temperature to provide dry powder. The particle size of (3a-f)-Cu(II) powders was observed by TEM (model JEM-1230, JEOL, Japan). Their morphologies were illustrated by photographs, and particle sizes were represented by diameters. Acknowledgment. This work was supported by the Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, the 973 Project of China (2006CB708501), Natural Scientific Foundation of China (20772082). Supporting Information Available: Preparation procedures, and physical, analytical, and spectrometric data of all compounds. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Banerjee, A.; Chisti, Y.; Banerjee, U. C. Biotechnol. AdV. 2004, 22, 287. (2) Khan, I. A.; Gowda, R. M. Int. J. Cardiol. 2003, 91, 115. (3) Wardlaw, J. M.; Warlow, C. P.; Counsell, C. Lancet 1997, 350, 607. (4) Rouf, S. A.; Young, M. M.; Chisti, Y. Biotechnol. AdV. 1996, 14, 239. (5) Hellebrekers, B.W. J.; Trimbos-Kemper, T. C. M.; Trimbos, J. B. M. Z.; Emeis, J. J.; Kooistra, T. Fertil. Steril. 2000, 74, 203.

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