Synthesis of a Stable Helical Peptide and Grafting on Gold

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Notes Synthesis of a Stable Helical Peptide and Grafting on Gold Nanoparticles Paolo Pengo,† Quirinus B. Broxterman,‡ Bernard Kaptein,‡ Lucia Pasquato,*,§ and Paolo Scrimin*,† CNR-ITM Sezione di Padova and Universita` di Padova, Dipartimento di Chimica Organica, via Marzolo 1, I-35131 Padova, Italy, and DSM Research, DFC Advanced Synthesis & Catalysis, 6160 MD Geleen, The Netherlands Received May 23, 2002. In Final Form: November 4, 2002

Introduction Much effort is currently devoted to the molecular design and synthesis of de novo proteins taking advantage of specific folding motifs, with one of the final goals being the obtainment of new and selective catalysts.1,2 Protein tertiary structures can be considered as assemblies of secondary structural domains (R- and 310-helices, β-strands, reverse turns). Accordingly, by assembling on a proper template relatively short polypeptides, one may obtain complex structures with protein-like properties.3 Such an approach, introduced by Mutter4 (template assembled synthetic proteins, TASP) has been exploited also by others.5 The use of solid supports has been pursued, too.6,7 This may provide access to new materials with remarkable properties.8 Interesting is the possibility to obtain nanometer size systems suitable for catalytic applications. Such a strategy may take advantage of the use of a dendrimer template.9 We decided to exploit a selfassembling strategy for the preparation of a putative “artificial protein” by assembling short histidine-based * To whom correspondence should be addressed. LP: phone, (039) 049-8275255; fax, (039) 049-8275239; e-mail, lucia. [email protected]. PS: phone, (039) 049-82752765; fax, (039) 049-8275239; e-mail, [email protected]. † University of Padova. ‡ DSM Research. § CNR-IMT Padova Section. (1) (a) Venkatraman, J.; Shankaramma, S. C.; Balaram, P. Chem. Rev. 2001, 101, 3131-3152. (b) Baltzer, L.; Nilsson, H.; Nilsson. J. Chem. Rev. 2001, 101, 3153-3163. (2) (a) Hill, R. B.; Raleigh, D. P.; Lombardi, A.; DeGrado, W. F. Acc. Chem. Res. 2000, 33, 745-754. (b) Lombardi, A.; Summa, C. M.; Geremia, S.; Randaccio, C.; Pavone, V.; DeGrado, W. F. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 6298-6305. (c) DeGrado, W. F.; Summa, C. M.; Pavone, V.; Nastri, F.; Lombardi, A. Annu. Rev. Biochem. 1999, 68, 779-819. (3) Das, C.; Shankaramma, S. C.; Balaram, P. Chem. Eur. J. 2001, 7, 840-847. (4) Mutter, M.; Vuilleumier, S. Angew. Chem., Int. Ed. Engl. 1989, 28, 535-554. (5) (a) Mezo, A. R.; Sherman, J. C. J. Am. Chem. Soc. 1999, 121, 8983-8994. (b) Rau, H. K.; DeJonge, N.; Haehnel, W. Angew. Chem., Int. Ed. 2000, 39, 250-253. (6) (a) Niwa, M.; Morikawa, M.; Higashi, N. Angew. Chem., Int. Ed. 2000, 39, 960-963. (b) Whitesell, J. K.; Chang, H. K. Science 1993, 261, 73-76. (7) Whitesell, J. K.; Chang, H. K. Science 1993, 261, 73-76. (8) (a) Waley, S. R.; English, D. S.; Hu, E. L.; Barbara, P. F.; Belcher, A. M. Nature 2000, 405, 665-668. (b) Matsumura, S.; Sakamoto, S.; Ueno, A.; Mihara, H. Chem. Eur. J. 2000, 6, 1781-1788. (c) Pieroni, O.; Fissi, A.; Angelini, N.; Lenci, F. Acc. Chem. Res. 2001, 34, 9-17. (9) (a) Higashi, N.; Koga, T.; Niwa, N.; Niwa, M. Chem. Commun. 2000, 361-362. (b) Sakamoto, M.; Ueno, A.; Mihara, H. Chem. Eur. J. 2001, 7, 2449-2458.

peptides on gold nanoparticles. There is an obvious advantage in pursuing a self-assembling strategy7 instead of a full synthesis: the synthetic efforts (often quite demanding) are reduced to the minimum, as they are restricted only to the components of the resulting nanostructure. Peptide 7, shown in Scheme 1, comprising six CR-tetrasubstituted amino acids and a histidine residue, has been chosen as the monomeric unit for the preparation of the functional nanoparticles. Short peptides (7-8 residues) rich in CR-tetrasubstituted amino acids are usually rather structured, preferentially adopting a 310-helical conformation.10 Such peptides provide a conformationally robust element to be assembled on a proper template. However, there is no guarantee that such a conformation is maintained in the final material, a mandatory condition for any application requiring a precise disposition in the space of the structural elements. We show that this is indeed the case with the present system despite 7 being only a heptapeptide. The structural motifs of peptide 7 are the following: (i) the (Aib-(RMe)Val-Aib)2 sequence expected to guarantee a right-handed 310-helical conformation; (ii) a histidine residue suitable as a potential catalytic unit in transacylation reactions (This amino acid was introduced at the C-terminus to be confined on the surface of the nanoparticles, to make it accessible to a putative substrate. Several research groups have demonstrated that dendrimers bearing catalytic units covalently linked to the terminal sites can combine the best features of homogeneous and hetereogeneous catalysts.11 In addition, cooperativity between proximal groups may be exhibited.12); (iii) a hydrocarbon tether between the peptide and the sulfur atom in order to allow enough space for the helical peptide, once bound to the gold cluster, to maintain the folded conformation (more space is available as the distance from the surface increases because of the spherical geometry of the nanoparticles). Previous work by Whitesell and collaborators on the functionalization of planar gold surfaces with oligopeptides had shown that their helical conformation is lost in the absence of an adequate spacer between the oligomer and the supporting surface.9 To the best of our knowledge, this is the first report of the synthesis of a thiol-functionalized, highly structured peptide and of its utilization for the functionalization of monolayer-protected gold nanoparticles.13 (10) Polese, A.; Formaggio, F.; Crisma, M.; Valle, G.; Toniolo, C.; Bonora, G. M.; Broxterman, Q. B.; Kamphuis, J. Chem. Eur. J. 1996, 2, 1104-1111. (11) (a) Vogtle, F.; Schalley, C. A. Dendrimers IV: Metal Coordination, Self-assembly, Catalysis; Spring-Verlag: Berlin, 2001. (b) Oosterom, G. E.; Reek, J. N. H.; Kamer, P. C. J.; Leeuwen van P. W. N. M. Angew. Chem., Int. Ed. 2001, 40, 1828-1849. (c) Astruc, D.; Chardac, F. Chem. Rev. 2001, 101, 2991-3023. (12) This situation is similar to the one we have recently observed in surface-confined imidazoles in gold nanoparticles where cooperativity has been found: Pasquato, L.; Rancan, F.; Scrimin, P.; Mancin, F.; Frigeri, C. Chem. Commun. 2000, 2253-2254. It is noteworthy that dendrimers carrying [Co(salen)] complexes on the surface exhibit significantly enhanced catalytic activity in the hydrolytic kinetic resolution of terminal epoxides: Breinbauer, R.; Jacobsen, E. N. Angew. Chem., Int. Ed. 2000, 39, 3604-3607.

10.1021/la025982v CCC: $25.00 © 2003 American Chemical Society Published on Web 02/04/2003

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Langmuir, Vol. 19, No. 6, 2003 Scheme 1. Synthesis of 7

Experimental Section General. Melting points were determined with a Kofler Ernst Leitz apparatus and are uncorrected. NMR spectra were recorded on Bruker AC 200 or AC 250 or Avance 400 spectrometers at 25 °C. For 1H NMR, data are reported as follows: chemical shift in ppm, from the chemical shifts of the residual protons in the deuterated solvents, on the δ scale, integration, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; sp, septet; br, broad; dsp, doublet of septets), coupling constants (Hz), and assignment. For 13C NMR, the signals of the solvent were used as reference. FTIR absorption spectra were recorded using a Perkin-Elmer 1720X or a Perkin-Elmer 1600X Series FTIR spectrophotometer, nitrogen-flushed, equipped with a sample-shuttle device, at 2 cm-1 nominal resolution, averaging 100 scans. Solvent (baseline) spectra were obtained under the same conditions. Cells with path lengths of 0.1, 1.0, and 10.0 mm (with CaF2 windows) were used. CD spectra were recorded using a Jasco model J-715 spectropolarimeter equipped with a Haake thermostat. Cylindrical, fused cells of 0.2 mm path lengths were employed. UV spectra were recorded on a Perkin-Elmer Lambda 16 spectrophotometer equipped with a thermostated cell holder. Mass spectra were obtained by electrospray (ESI) ionization on a Navigator LC/MS Thermo Quest Finningan instrument. Transmission electron microscopy (TEM) images of the particles were obtained with a JEOL JEM 2010 electron microsope operating at 200 kV. Samples for TEM were prepared by spreading a mixture of nanoparticles/ SiO2 (∼1 mg/10 mg) onto standard carbon-coated copper grids (200 mesh). Commercial reagents and known compounds were purchased from standard chemical suppliers or prepared according to literature procedures and purified to match the reported physical and spectral data. Solvents were purified according to standard procedures. The synthesis and characterization of AcS-(CH2)7-Aib-L-(RMe)Val-Aib2-L-(RMe)Val-Aib-His-OMe are described in detail in the Supporting Information. MPC-C12 was prepared following the reported procedure14 using a Au/C12H25SH ratio of 1:3 and by addition of NaBH4 in 10 s at 0 °C. 1H NMR (250 MHz, C6D6) δ: 1.45 (br, CH2); 1.03 (br, CH3, fwhm 21.8 Hz). 13C NMR {1H} (62.90 MHz, CD2Cl2) δ: 32.11, 30.06, 29.07, 22.81, 13.96. IR (film) ν (cm-1): 3859, 3847, 3757, 3463, 2956, 2922, 2871, 2852, 2383, 2348, 1753, 1737, 1726, 1711, 1679, 1659, 1597, 1589, 1566, 1549, 1513, 1501, 1465, 1416, 1378, 1367, 1345, 1293, 1262, 1240, 1212, 1120, 1072, 1020, 1006, 983, 961, 935, 721. Anal. Calcd for Au225(C12H25S)90: C, 20.77; (13) Tripeptides have been anchored to gold nanoparticles: (a) Schaaff, T. G.; Knight, G.; Shafigullin, M. N.; Borkman, R. F.; Whetten, R. L. J. Phys. Chem. B 1998, 102, 10643-10646. (b) Templeton, A. C.; Hostetler, M. J.; Warmoth, E. K.; Chen, S.; Hartshorn, C. M.; Krishnamurthy, V. M.; Forbes, M. D. E.; Murray, R. W. J. Am. Chem. Soc. 1998, 120, 4845-4849. (14) Hostetler, M. J.; Wingate, J. E.; Zhong, C.-J.; Harris, J. E.; Vachet, R. W.; Clark, M. R.; Londono, J. D.; Green, S. J.; Stokes, J. J.; Wignall, G. D.; Glish, G. L.; Porter, M. D.; Evans, N. D.; Murray, R. W. Langmuir 1998, 14, 17-30.

Notes H, 3.63; S, 4.62. Found: C, 20.33; H, 3.59; S, 4.62. TEM: core diameter 2.2 ( 0.4 nm. MPC-C12/7. AcS-(CH2)7-Aib-L-(RMe)Val-Aib2-L-(RMe)ValAib-His-OMe (52 mg, 0.045 mmol) was thiol deprotected by reaction in methanol (3 mL), dichloromethane (10 mL), and acetyl chloride (2 mL) added at 0 °C. After 5 h of stirring at room temperature, the solvent was removed in vacuo and the residue dissolved in 10 mL of AcOEt. The solution was washed with 5% NaHCO3 (2 × 10 mL) and dried over Na2SO4. The reaction is quantitative, and 42 mg of thiol 7 were obtained. In a thermostated reactor a solution of MPC-C12 (21 mg) and thiol 7 (44 mg) in dichloromethane (10 mL) was left under stirring at 28 °C for 60 h. Then methanol was added (2 mL), and after an additional 12 h the solution was concentrated at reduced pressure. The exchanged gold nanoparticles were purified from the excess of thiol by chromatography on Sephadex LH60 (CH2Cl2/MeOH 8/2). The product, after removal of the solvent at reduced pressure, had a very low solubility (if any) in organic solvents or water. IR (KBr) ν (cm-1): 3981, 3925, 3852, 3750, 3442, 3435, 3311, 2980, 2957, 2922, 2852, 1883, 1743, 1655, 1536, 1465, 1458, 1444, 1384, 1363, 1293, 1374, 1223, 1201, 1169, 1118, 1019, 1056, 1042, 1019, 998, 970, 957, 937, 928, 874, 847, 828, 799, 775, 755, 720, 671, 655, 623, 581, 563, 525, 504, 480, 433. TEM: core diameter 2.3 ( 0.4 nm.

Results and Discussion The synthetic strategy toward peptide 7 is outlined in Scheme 1. Dipeptide Z-L-(RMe)Val-Aib-OtBu (2) was obtained15 by first converting Z-L-(RMe)Val into Z-L(RMe)Val-F by using TFFH in dry methylene chloride followed by coupling of the isolated fluoride with H-AibOtBu in the presence of an excess of DIEA in dry acetonitrile at reflux for 3 days (70% isolated yield).16 Tripeptide Z-τ-OtBu (3) was obtained by reacting H-L(RMe)Val-Aib-OtBu, 3 equiv of Z-Aib-F, and an excess of DIEA in dry acetonitrile for 3 days at room temperature in 77% yield after purification. For the preparation of hexapeptide Z-τ-τ-OtBu (4), we first converted the tripeptide into the oxazolone by treatment of Z-τ-OH with acetic anhydride at 120 °C for 20 min, and subsequently reacted it with H-τ-OtBu (1.1 equiv) in dry acetonitrile at reflux (61% yield). S-Acetyl-8-thiooctanoic acid was obtained in 86% yield from 8-bromooctanoic acid and thiolacetate, following a reported procedure.17 The acid was then converted into the corresponding acyl chloride by treatment with SOCl2. The coupling between the tether and the hexapeptide was accomplished by reacting H-τ-τ-OtBu with the acyl chloride in the presence of pyridine in methylene chloride (89% yield). The coupling between hexapeptide 5 and the tritylprotected histidine does not deserve any special comment but for the fact that the reaction is quite slow and after 4 days the product was isolated in a modest 40% yield. Removal of the trityl group let us obtain the target peptidefunctionalized thiol 7 in 90% yield. A solution conformational analysis of dipeptide 2, tripeptide 3, hexapeptides 4 and 5, and heptapeptide 6 was carried out by means of FT-IR and NMR techniques. Peptide 5 was also studied by CD spectroscopy. (15) L-(RMe)Val is prepared via a chemoenzymatic synthesis developed by DSM Research (see: Sonke, T.; Kaptein, B.; Boesten, W. H. J.; Broxterman, Q. B.; Schoemaker, H. E.; Kamphuis, J.; Formaggio, F.; Toniolo, C.; Rutjes, F. P. J. T. In Stereoselective Biocatalysis; Patel, R. N., Ed.; Marcel Dekker: New York, 1999; pp 23-58). (16) The monitoring of the reaction by proton NMR spectroscopy suggests that the fluoride is first converted into the corresponding oxazolone followed by the coupling with formation of the dipeptide. This is in accord with our recent findings: Fiammengo, R.; Licini, G.; Nicotra, A.; Modena, G.; Pasquato, L.; Scrimin, P.; Broxterman, Q. B.; Kaptein, B. J. Org. Chem. 2001, 66, 5905-5910. (17) Bader, M. M. Phosphorous, Sulfur and Silicon Relat. Elem. 1996, 116, 77-92.

Notes

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Figure 2. FT-IR absorption spectra (KBr) of MPC-C12, peptide 7 (partial), and petide-functionalized MPCs (MPC-C12/7). Figure 1. FT-IR absorption spectra (3500-3200 cm-1 region) in CDCl3 of Z-L-(RMe)Val-Aib-OtBu (2), Z-Aib-L-(RMe)Val-AibOtBu (3), Z-Aib-L-(RMe)Val-Aib2-L-(RMe)Val-Aib-OtBu (4), AcS-Oct-Aib-L-(RMe)Val-Aib2-L-(RMe)Val-Aib-OtBu (5), and AcS-Oct-Aib-L-(RMe)Val-Aib2-L-(RMe)Val-Aib-His(Trt)OMe (6). Peptide concentration: 1.0 mM.

The progressive increase of H-bond-driven conformational organization as the peptide sequence elongates is evident from the FT-IR absorption spectra in the 35003200 cm-1 region for the protected tri-, tetra-, penta-, and hexapeptides in CDCl3: the intensity of the free NH stretching band (∼3430 cm-1) relative to that of the H-bonded NH groups (∼3350 cm-1) decreases with increasing main chain length (Figure 1). From the analysis of the spectra in the CdO stretching region (see Supporting Information) we note that hexapeptide 4 and heptapeptide 6 absorb at 1662 and 1658 cm-1, respectively. Here the shift is less important, as expected for a carbonyl bond.18 The combined IR data suggest a helical conformation.19 The comparison of the spectra relative to those of the hexapeptides 4 and 5 and heptapeptide 6 indicates that the introduction of the spacer does not induce substantial changes in the secondary structure whereas the addition of the His residue increases the extent of the folding (free NH absorption decreases). The CD spectrum of hexapeptide 5 in methanol (see Supporting Information) is characterized by a negative Cotton band centered at 205 nm with a shoulder at a slightly higher wavelength than that expected for a 310-helical conformation (222 nm). This is probably due to the dichroic contribution of the thiolacetate present at the end of the tether. To avoid this problem, we independently synthesized the analogue of peptide 7 where the hydrocarbon tether (with the thiolate moiety) was replaced by an acetyl group. In this case the CD spectrum in methanol is fully consistent with a 310-helical conformation.20 The CD spectra do not change in the 2-20 mM concentration range, suggesting the absence of aggregation in this solvent and this concentration interval. Further support for a highly organized structure of the hexa- and heptapeptides is provided by the 1H and 13C NMR investigation carried out in CDCl3 solution at 2.0-2.5 mM peptide concentration. The participation of the NH groups in intramolecular H-bonding was assessed by examining the behavior of the NH resonances upon (18) Cantor, C. R.; Schimmel, P. R. Biophysical Chemistry, Part II; Freeman Ed.: San Francisco, CA, 1980; p 468. (19) Kennedy, D. F.; Crisma, M.; Toniolo, C.; Chapman, D. Biochemistry 1991, 30, 6541-6548. (20) Toniolo, C.; Polese, A.; Formaggio, F.; Crisma, M.; Kamphuis, J. J. Am. Chem. Soc. 1996, 118, 2744-2745.

Figure 3. TEM image of MPC-C12/7 dispersed on SiO2; the scale bar corresponds to 10 nm.

addition to the CDCl3 solution of increasing amounts of DMSO, a strong H-bonding acceptor solvent. For both peptides, only the chemical shifts of two NH protons are significantly shifted by the addition of the additive (likely N(1)H, ∆δ ∼ 2 ppm, and N(2)H, ∆δ ∼ 0.3 ppm, see Supporting Information) while all other NH protons are substantially unaffected, suggesting they are involved in intramolecular H-bonds. This is again consistent with a 310-helical conformation, as in this ordered secondary structure only the two N-terminal NHs do not participate in intramolecular H-bonding. In the case of heptapeptide 6, all NH proton resonances were unambiguously assigned by means of HMQC and 2D ROESY experiments (20 mM peptide concentration in CD2Cl2), starting from the histidine N(7)H proton, which is a doublet and shows crosspeaks with CR and Cβ of the His residue. The strong intensities of the NH(i) f NH(i+1) cross-peaks are consistent with the hypothesis that peptide 6 adopts a helical conformation in this solvent. Regrettably, the absence of CRH protons in the sequence (but for C-terminal histidine) does not allow us to determine long-range connectivities that could lend further support in favor of a specific helical conformation. Thiol 7 was then used for the preparation of peptidefunctionalized gold nanoparticles (MPCs) through place exchange21 with 2 nm size dodecanethiol-functionalized MPCs (MPC-C12).14 The reaction was carried out in methylene chloride using a 1:1.5 ratio between exiting (21) Hostetler, M. J.; Templeton, A. C.; Murray, R. W. Langmuir 1999, 15, 3782-3789.

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and entering thiols, at 28 °C for 72 h. The resulting material was purified by exclusion chromatography (Sephadex LH 60, CH2Cl2/MeOH 8:2). This procedure ensures the separation of unbound thiols and disulfides as well as of unfuntionalized gold particles from the MPCs.22 The FT-IR spectrum undoubtedly shows the presence of peptide 7 in the monolayer protecting the gold nanocluster (Figure 2). The spectrum is characterized by an intense broad band in the NH-stretching region and by two sharp bands in the CdO-stretching one. These latter match those of the parent, unbound peptide, suggesting that the folded conformation of the peptide is maintained when it is bound to the MPCs. Unfolding would have shifted the CdO stretching band to higher wavenumbers (1687-1677),19 which is not the case. This means that the packing of the peptide on the monolayer does not disturb the main chain intramolecular H-bonds of each peptide unit. These peptide-functionalized gold nanoclusters show a very peculiar solubility behavior. So, although they could be solubilized in the solvent used for their purification, they became almost insoluble once brought to dryness. TEM images (Figure 3) show that the nanoparticles containing the peptide in the monolayer have a core diameter of 2.3 ( 0.4 nm, very similar to that of the precursor MPC-C12. This suggests that the low (22) A reviewer suggested that the peptide could interact with the hydrocarbon monolayer without binding to the Au surface. We have ruled out this possibility by showing that a peptide devoid of the thiolate function does not bind to MPC-C12 under identical conditions of the exchange experiment.

Notes

solubility is due neither to aggregation or increased size of the nanoparticles nor to peptide depletion from the surface with formation of large gold colloids. We suggest that intermolecular hydrogen bonds23 (between NH and CdO not involved in the formation of the helix) may be formed, giving rise to a tightly packed monolayer and hence unsoluble aggregates. We plan to address this problem with a new generation of MPCs where we will change the length of the tether present in the thiol and the position of the histidine. We hope to report applications of these new nanoparticles as transacylation catalysts soon. Acknowledgment. We are indebted to Professors C. Toniolo and F. Formaggio for helpful discussion. This work was supported by the University of Padova (Progetti di Ricerca di Ateneo). Supporting Information Available: Experimental details with the characterization of all the described compounds. FT-IR spectra (1800-1600 cm-1 region) in CDCl3 of peptides 2-6; CD spectra (methanol) of peptide 5 and of the acetyl analogue of peptide 7; dependence of the NH chemical shifts of peptides 4-6 as a function of increasing percentages of DMSO added to the CDCl3 solution; 2D ROESY spectrum of heptapeptide 6 in CD2Cl2. ESI-MS of heptapeptide 6. This material is available free of charge via the Internet at http://pubs.acs.org. LA025982V (23) The formation of intramonolayer hydrogen bonds has been observed in MPCs protected with thiols containing amide moieties, and it depends on the distance of the amide from the nanoparticle core: Boal, A. K.; Rotello, V. M. Langmuir 2000, 16, 9527-9532.