Chinese Chemical Letters  2014, Vol.25 Issue (05):659-662   PDF    
Metalloporphyrin receptors for histidine-containing peptides
Hui Liua , Zhan-Ting Lib    
a Key Laboratory for Green Chemical Process of Ministry of Education, School of Chemical Engineering & Pharmacy, Wuhan Institute of Technology, Wuhan 430073, China;
b State Key Laboratory of Bio-Organic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
Abstract: Two new ditopic metalloporphyrin receptors constructed by combining metalloporphyrin with crown ethers have been prepared and characterized. 1H NMR and MS spectra confirmed the complexation of receptor with peptide driven by coordination interaction and hydrogen bonding. UV/vis experiments revealed that the receptors exhibited high binding affinity to histidine-containing peptides. These receptors could differentiate short peptides of C-terminal histidine and N-terminal histidine and formed the most stable complexes with tripeptide.
Key words: Metalloporphyrin     Crown ether     Synthetic receptor     Peptide    
1. Introduction

Selective recognition of short peptides by synthetic receptors has attracted a great deal of interest in past decade because of their important roles in nature [1, 2, 3, 4, 5]. The intrinsic properties of short peptides originated from flexible conformation and irregular topology placed a number of challenges in the design of receptors [6]. In the recently reported examples,one strategy has been proved to be effective for selective recognition of peptides,this is, the binding of peptide N terminus and side chain of peptide in ditopic fashion [7, 8, 9, 10].

Crown ethers are ideal binding units for ammonium ions in amino acids [11, 12, 13, 14]. On the other hand,metalloporphyrins have been documented to coordinate the nitrogen atom of imidazole [15, 16, 17, 18]. Porphyrin platform can further provide a large molecular surface for dispersive interaction with peptide backbone [19, 20, 21]. In this context,the aim of this letter is to develop new receptors by combining crown ethers with metalloporphyrin and to investigate their recognition behavior toward histidine-containing short peptides.

The strapped porphyrindeveloped by our group was served asa scaffold to align peptide backbone in one direction from the N to C terminus [22, 23]. The metal cation Zn(II) in the center of porphyrin could provide additional coordination site to bind nitrogen atom of histidine. Aza-crown ethers of varied size were incorporated to porphyrin via amide linkage as recognition site of ammonium ion, which allowed the receptor preorganized as the distance between N terminus and side chain of peptides (Scheme 1).

Scheme 1. Metalloporphyrin receptors and modified peptides.
2. Experimental

NMRspectra were recordedon a Varian spectrometer operating at 300 and 400 MHz for 1H and 13C respectively in the indicated solvents. Chemical shifts were expressed in parts per million (d) using residual solvent protons as internal standards. MALDI-TOF mass spectra were recorded on a Voyager-DE STR mass spectrom- eter (AB SCIEX,USA). UV/vis absorption spectra were measured with a Cary 100 UV/vis spectrophotometer (Varian,USA). Elemental analysis was carried out at the SIOC analytical center. Unless otherwise indicated,all starting materials were obtained from commercial suppliers and were used without further purification. All solvents were dried before use following standard procedures. All reactions were performed under an atmosphere of dry nitrogen. Compound 5 was prepared following our previous method [24, 25]. Column chromatography was carried out using silica gel (300-400 mesh). All of the modified peptides were prepared following standard procedures in the solution (see Supporting information).

The UV/vis titration experiments were performed according to the following procedure. A solution of receptor (2.0 mL, 1.1 × 10-6mol/L) in chloroform was titrated with 10mL of histidine-containing peptide (2.0 × 10-5mol/L to 5.0 × 10-5mol/ L in chloroform,adjusted with 10 fold HCl). After each addition the solution was allowed to equilibrate for 5 min and the absorption intensity was recorded in the wavelength region of 350-600 nm at 25 ℃.

2.1. Generalprocedureforthesynthesisofmetalloporphyrinreceptors

To a solution of porphyrin acid 5 in dichloromethane oxalyl chloride and several drops of DMF were added. The mixture was stirred at room temperature for 5 h. After evaporating the solvents under vacuum,the residue was dissolved in dichloromethane and then aza-crown ether and triethyl amine were added and stirred overnight at room temperature. After removal of solvent,the resulting residue was purified by column chromatography to afford aza-crownetherporphyrin.Thefreebaseporphyrinwasdissolvedin dichloromethane/methanol (3:1) and zinc acetate was added with stirring.Themixturewasstirredunderrefluxovernight.Thesolvent was removed in vacuo and the resulting residue was subjected to column chromatography to afford metalloporphyrin receptor as a purple solid in high yield (Scheme 2).

Scheme 2.Synthetic route of metalloporphyrin receptors 1 and 2 .

Compound 1 :Purplesolid(68%).Mp > 250 ℃.1H NMR(300 MHz, CDCl3):δ-1.39(m,2H),-1.21(m,2H),-1.00(m,2H),-0.80(m,2H), -0.60(br,4H),0.65(br,4H),0.90(t,6H),1.25-1.56(m,20H),1.89(p, 4H),3.35-3.86 (m,20H),4.49 (t,4H,J = 6.75 Hz),7.28 (m,2H),7.46 (d-t,1H,J = 0.9,7.5 Hz),7.76 (d-t,1H,J = 1.8,5.1 Hz),7.86 (d-d,1H, J = 2.25,8.55 Hz),8.19 (d-d,2H,J = 1.05,7.95 Hz),8.32 (d-d,1H, J = 7.5,1.8 Hz),8.37-8.48 (m,7H),8.81 (m,8H).13C NMR (100 MHz, CDCl3):δ 14.1,22.7,25.1,26.1,27.7,28.3,28.9,29.1,29.3,29.4,31.9, 65.5,70.1,112.9,113.8,116.4,117.8,119.5,120.0,127.7,128.0, 129.1,129.6,129.8,131.6,131.7,131.9,132.2,132.8,134.4,134.7, 147.7,149.5,150.4,150.7,159.8,160.6,167.0,172.0,172.3. MS (MALDI-TOF)(m/z):1407(M+H+).Anal.Calcd.forC83H97N5O11Zn:C, 70.90; H,6.95; N,4.98. Found: C,70.39; H,7.04; N,4.91.

Compound 2 : Purple solid (71%). Mp > 250 ℃. 1H NMR (300 MHz,CDCl3): δ -1.39 (br,2H),-1.26 (br,2H),-1.00 (br,2H),-0.85 (br,2H),-0.69 (br,4H),0.61 (br,4H),0.90 (t, 6H),1.25-1.55 (m,20H),1.88 (p,4H),2.29-3.06 (m,20H),3.67 (m, 4H),4.42 (t,4H,J = 6.75 Hz),7.09 (d,1H,J = 9.0 Hz),7.29 (s,1H), 7.46(t,1H,J = 7.5 Hz),7.56(d,1H,J = 5.4 Hz),7.76(t,1H,J = 5.1 Hz), 7.99 (s,1H),8.22-8.45 (m,9H),8.75-8.88 (m,8H). 13C-NMR (100 MHz,CDCl3): δ 14.1,22.7,25.1,26.2,29.1,29.3,29.4,29.7, 31.9,65.4,70.4,112.8,113.8,116.3,117.7,119.4,119.9,127.6, 128.9,129.5,129.8,131.4,131.6,131.8,132.1,134.3,134.7,147.8, 149.5,150.5,150.9,159.8,160.5,167.0,171.8,172.0. MS (MALDI- TOF) (m/z): 1451 (M+H+). Anal. Calcd. for C85H101N5O12Zn: C, 70.40; H,7.02; N,4.83. Found: C,70.61; H,7.02; N,4.64.

3. Results and discussion

To investigate the binding modes of the metalloporphyrin receptor and the histidine-containing peptides,1H NMR experi- ments were firstly carried out. When 5 equiv. of HisN8 was added to a solution of metalloporphyrin 1 or 2 in CD3OD/CDCl3(1/1),the signals of the protons of the imidazole group shifted upfield (ca. 0.17 ppm) as a result of the coordination interaction between the imidazole nitrogen atom and the zinc cation (Fig. 1). The strong upfield shifts were also observed when other histidine-containing peptides were used (Figs. S1-S3 in Supporting information), indicating the placement of the peptide within the ring current of metalloporphyrin.Duetotheoverlappingofsignalsintheregionof 3.0-4.0 ppm,the changes of the protons of the crown ether were hardly estimated. However,the binding strength between crown ether and ammonium ions should be much weaker than that of Zn2+and imidazole according to previous studies [26, 27, 28].

Fig. 1. Partial 1H NMR spectra (300 MHz) in CD3OD/CDCl3 (1/1) at 25 ℃: (a) Receptor 1; (b) Receptor 1 + 5 equiv. HisN8; (c) HisN8.

Following the NMR study,mass spectrometry was used to probe the non-covalent interactions between the metallopor- phyrin receptors and the histidine-containing peptides. The formation of a stable complex from receptor 1 and GlyHisN8 driven by coordination interaction and hydrogen bonding was confirmed by MALDI-TOF analysis (Fig. 2). It gave a peak at m/z 1785.1,which corresponded to 1:1 complex of 1 and GlyHisN8, although the intensity was weak.

Fig. 2.MALDI-TOF mass spectra of 1.GlyHisN8 complex.

The binding constants were determined by UV/vis titration experiments in chloroform. Both receptors 1 and 2 showed the maximum absorption at the wavelength of 421 nm. Upon the addition of a histidine-containing peptide to a solution of receptor 1 or 2,a pronounced decrease in the absorption for all those peptides was observed. No significant shift was observed for receptors 1 and 2 when a modified histidine (HisN8) was added to theirsolutions.Upon additionofdipeptide (HisGlyN8orGlyHisN8) two clear isosbestic points appeared at 432 and 559 nm, respectively. Furthermore,the addition of tripeptide (GlyGly- HisN8) also resulted in a significant bathochromic shift (ca. 12 nm and 10 nm) (Fig. 3 and Figs. S4 and S5 in Supporting information).

Fig. 3. Absorption spectral changes of 2 (1.1 × 10-6mol/L) upon addition of HisGlyN8 (1.0 × 10-7-7.0 × 10-6mol/L) in chloroform at 25 ℃.

On the basis of the absorption data obtained at 421 nm,binding constants were calculated using a 1:1 binding model [29]. The results are summarized in Table 1,which reveals that those histidine-containing peptides bind well to the metalloporphyrins. In contrast to receptor 1,receptor 2 shows slightly higher binding affinity to all the four peptides. It can be attributed to a higher binding ability of aza-18-crown-6 toward ammonium ion than that of aza-15-crown-5 [30].

Table 1
Binding constants (L/mol) and the associated free energy change (kcal/mol) of peptides with receptors 1 and 2.

Receptors 1 and 2 could differentiate short peptides with C- terminal histidine and N-terminal histidine. For example,recep- tors exhibited higher binding affinity toward GlyHisN8 than HisGlyN8. It might be attributed to a more matched spatial distance between ammonium ion and imidazole group in GlyHisN8. It was also noteworthy that the binding energy increased with the increasing length of the peptides,which was attributed to the dispersive interaction of the amide groups with the porphyrin unit. Receptors 1 and 2 bound best to tripeptide GlyGlyHisN8,which bears three amide oxygen atoms with negative charge of high polarizibility. Based on these results and the previous reports,it was proposed that complexes formed by the binding of receptors and peptides in the following modes (Fig. 4).

Fig. 4. Proposed structures of the complexes formed from receptors and peptides.
4. Conclusion

In summary,the combination of a strapped metalloporphyrin with aza-crown ethers resutled in two new synthetic receptors. They exihibited high binding affinity to histidine-containing short peptides and showed higher binding affinity to C-terminal histidine than to N-terminal histidine. As a consequece of additive dispersive interaction of the amide group with porphyrin,both receptors formed the most stable complex with tripeptide. In addition,the binding affinity of receptor 2 to the histidine- containing peptides was slightly higher than 1,which could be attributed to the stronger binding of ammonium ion by the aza crown ethers of larger ring size in receptor 2. Current efforts are focusing on exploring selective recognition of short peptide in aqueous media by modifying the metalloporphyrin receptors.


This research was financially supported by Basic Science Research Program through the National Natural Science Founda- tion of China (No. 21302149).

Appendix A. Supplementary data

Supplementarymaterialrelatedtothisarticlecanbefound,inthe online version,at

[1] M. Kruppa, C. Mandl, S. Miltschitzky, B. Konig, A luminescent receptor with affinity for N-terminal histidine in peptides in aqueous solution, J. Am. Chem. Soc. 127 (2005) 3362-3365.
[2] A.T. Wright, E.V. Anslyn, Cooperative metal-coordination and ion pairing in tripeptide recognition, Org. Lett. 6 (2004) 1341-1344.
[3] A. Buryak, K. Severin, An organometallic chemosensor for the sequence-selective detection of histidine- and methionine-containing peptides in water at neutral pH, Angew. Chem. Int. Ed. 43 (2004) 4771-4774.
[4] M. Sirish, V.A. Chertkov, H.J. Schneider, Porphyrin-based peptide receptors: syntheses and NMR analysis, Chem. Eur. J. 8 (2002) 1181-1188.
[5] R. Arienzo, J.D. Kilburn, Combinatorial libraries of diamidopyridine-derived ‘tweezer' receptors and sequence selective binding of peptides, Tetrahedron 58 (2002) 711-799.
[6] T. Schrader, S. Koch, Artificial protein sensors, Mol. Bio. Syst. 3 (2007) 241-248.
[7] C. Schmuck, L. Geiger, Dipeptide binding in water by a de novo designed guanidiniocarbonylpyrrole receptor, J. Am. Chem. Soc. 126 (2004) 8898-8899.
[8] T. Braxmeier, M. Demarcus, T. Fessmann, S. McAteer, J.D. Kilburn, Identification of sequence selective receptors for peptides with a carboxylic acid terminus, Chem. Eur. J. 7 (2001) 1889-1898.
[9] A. Ojida, Y. Mito-oka, K. Sada, I. Hamachi, Molecular recognition and fluorescence sensing of monophosphorylated peptides in aqueous solution by bis(zinc(Ⅱ)- dipicolylamine)-based artificial receptors, J. Am. Chem. Soc. 126 (2004) 2454- 2463.
[10] C.P. Mandl, B. Konig, Luminescent crown ether amino acids: selective binding to N-terminal lysine in peptides, J. Org. Chem. 70 (2005) 670-674.
[11] P. Gunning, A.C. Benniston, R.D. Peacock, A modular ditopic crown-shielded phosphate ion-pair receptor, Chem. Commun. (2004) 2226-2227.
[12] A. Spath, B. Konig, Ditopic crown ether-guanidinium ion receptors for the molecular recognition of amino acids and small peptides, Tetrahedron 66 (2010) 1859- 1873.
[13] S.I. Sasaki, A. Hashizume, D. Citterio, E. Fuji, K. Suzuki, Fluororeceptor for zwitterionic form amino acids in aqueous methanol solution, Tetrahedron Lett. 43 (2002) 7243-7245.
[14] G.W. Gokel, E. Abel, Comprehensive Supramolecular Chemistry, vol. 1, Pergamon Press, New York, 1996, pp. 511-535.
[15] J.L. Hou, H.P. Yi, X.B. Shao, et al., Helicity induction in hydrogen-bonding-driven zinc porphyrin foldamers by chiral C60-incorporating histidines, Angew. Chem. Int. Ed. 45 (2006) 796-800.
[16] D. Paul, F. Melin, C. Hirtz, et al., Induced fit process in the selective distal binding of imidazoles in zinc(Ⅱ) porphyrin receptors, Inorg. Chem. 42 (2003) 3779-3787.
[17] A. Satake, Y. Kobuke, Dynamic supramolecular porphyrin systems, Tetrahedron 61 (2005) 13.
[18] J.K.M. Sanders, in: K.M. Kadish, K.M. Smith, R. Guilard (Eds.), The Porphyrin Handbook, vol. 3, Academic Press, New York, 2000, pp. 347-368.
[19] H.J. Schneider, T.J. Liu, M. Sirish, V. Malinovski, Dispersive interactions in supramolecular porphyrin complexes, Tetrahedron 58 (2002) 779-786.
[20] M. Sirish, H.J. Schneider, Electrostatic interactions between positively charged porphyrins and nucleotides or amides: buffer-dependent dramatic changes of binding affinities and modes, Chem. Commun. (2000) 23-24.
[21] M. Sirish, H.J. Schneider, Porphyrin derivatives as water-soluble receptors for peptides, Chem. Commun. (1999) 907-908.
[22] X.B. Shao, X.K. Jiang, X. Zhao, et al., Recognition through self-assembly. A quadruply- hydrogen-bonded, strapped porphyrin cleft that binds dipyridyl molecules and a [2]rotaxane, J. Org. Chem. 69 (2004) 899-907.
[23] H. Liu, X.B. Shao, M.X. Jia, et al., Selective recognition of sodium cyanide and potassium cyanide by diaza-crown ether-capped Zn-porphyrin receptors in polar solvents, Tetrahedron 61 (2005) 8095-8100.
[24] X.B. Shao, X.Z. Wang, X.K. Jiang, Z.T. Li, S.Z. Zhu, A novel strapped porphyrin receptor for molecular recognition, Tetrahedron 59 (2003) 4881-4889.
[25] K. Senokuchi, H. Nakai, Y. Nakayama, et al., New orally active serine protease inhibitors: structural requirements for their good oral activity, J. Med. Chem. 38 (1995) 4508-4517.
[26] S. Belanger, M.H. Keefe, J.L. Welch, J.T. Hupp, Rapid derivatization of mesoporous thin-film materials based on Re(I) zinc-porphyrin ‘molecular squares': selective modification of mesopore size and shape by binding of aromatic nitrogen donor ligands, Coord. Chem. Rev. 190-192 (1999) 29-45.
[27] V. Rudiger, H.J. Schneider, V.P. Solov'ev, V.P. Kazachenko, O.A. Raevsky, Crown ether-ammonium complexes: binding mechanisms and solvent effects, Eur. J. Org. Chem. 8 (1999) 1847-1856.
[28] A. Spath, B. Konig, Modular synthesis of di- and tripeptides of luminescent crown ether aminocarboxylic acids, Tetrahedron 65 (2009) 690-695.
[29] K.A. Conners, Binding Constants: The Measurement of Molecular Complex Stability, Wiley, New York, 1987.
[30] J.W. Steed, J.L. Atwood, Supramol. Chem. (2000) 120-121.