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Complexation of arginine with a cyclopeptide in polar solvents and water
Journal of Supramolecular Chemistry 1 (2001) 293–297
Complexation of Arginine with a Cyclopeptide in Polar Solvents and Water Joachim Bitta and Stefan Kubik* Institut fu¨r Organische Chemie und Makromolekulare Chemie, Heinrich-Heine-Universita¨t, Universita¨tsstr. 1, D-40225 Du¨sseldorf, Germany
We dedicate this article to Professor J. L. Atwood on the occasion of his 60th birthday
Abstract—A cyclic hexapeptide containing free carboxylate groups in the periphery of a macrocyclic cavity binds guanidinium ions and arginine derivatives in polar solvents and even in water. Guest binding can be demonstrated by NMR spectroscopy and electrospray ionization mass spectrometry. The spectroscopic results indicate that in CD3OD, the complexes of enantiomeric arginine derivatives have different geometries. This structural difference does not translate into significantly different complex stabilities, however. # 2003 Elsevier Science Ltd. All rights reserved.
The guanidinium group in the side chain of the amino acid arginine is responsible for the strong and selective anion binding of many proteins. In the phosphate binding protein, for example, substrate recognition is mediated by an arginine residue strategically placed inside the active center.1 Arginine-rich regions in the backbone of RNA binding proteins interact with the phosphodiesters of the polynucleotide,2 and electrostatic contacts between phosphate moieties and basic residues of the peptide chain induce the folding of DNA around histone proteins.3 Finally, arginine/carboxylate interactions occur in the active centers of the enzymes thrombine and trypsin, both of which cleave peptides directly behind an arginine residue.4 Small molecules that interfere in these processes can potentially lead to new drug candidates. Various host molecules have therefore been synthesized in recent years that specifically recognize guanidinium ions or arginine in solution. An early example is a tartaric acid based crown ether that binds guanidinium chloride in water with high affinity (Ka=9000 M 1).5 Methylguanidinium is already bound significantly less efficiently, however (Ka=450 M 1). Potent receptors with a high affinity also toward alkylguanidinium ions or arginine
*Corresponding author. Fax: +49-211-81-5840; e-mail: kubik@ uni-duesseldorf.de
are based on, for example, fused azaheterocycles,6 cyclophanes,7 or bisphosphonates,8 all of which contain negatively charged phosphonate or carboxylate groups that interact with the cationic guest by a combination of hydrogen-bonding and electrostatic interactions. Additional binding mechanisms such as, for example, cation–p interactions are in some cases also possible and contribute favorably to complex formation. We are aware of only few receptors, however, that are capable of enantioselective arginine recognition.9 Recently, we described a cyclic hexapeptide 1 containing l-proline and 3-aminobenzoic acid derived subunits with three carboxylate groups arranged around the macrocyclic cavity that binds monosaccharides in 4% methanol/chloroform.10 We speculated that due to the three well preorganized anionic binding sites, this host should also be able to interact with guanidinium ions. Moreover, its chirality could induce an enantioselective substrate complexation. We therefore tested the ability of 1 to interact with different guanidinium derivatives, and report on the results of these binding studies in this communication. Receptor 1 was obtained by coupling of l-aspartic acid 3-benzyl 1-isopropyl ester to a cyclic hexapeptide containing alternating l-proline and 3-aminoisophthalic acid subunits followed by hydrogenative cleavage of the benzyl esters, and deprotonation of the resulting carboxylic acids
1472-7862/01/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. PII: S1472-7862(02)00073-4
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J. Bitta, S. Kubik / Journal of Supramolecular Chemistry 1 (2001) 293–297
Figure 1. Intramolecular stabilization of the aspartic acid substituents in 1.
guest molecules, the fact that 1 forms defined 1:1 complexes with monosaccharides shows that in order to allow for optimal interactions with the guests, the peptide is able to adopt conformations with converging binding sites.10
with tetra-n-butylammonium hydroxide.10 Investigations on the receptor properties of 1 were performed in deuterated DMSO, methanol, and water (10 mM aqueous [(n-Bu)4N]H2PO4/[(n-Bu)4N]2HPO4 buffer, pD 7.2) by using guanidinium hydrochloride 2+.Cl , methylguanidinium hydrochloride 3+.Cl , Na-benzoyll-arginine p-nitroanilide hydrochloride L-4+.Cl , and the corresponding d enantiomer of this arginine derivative D-4+.Cl as guests.11
Previous investigations have shown that 1 preferentially adopts conformations in solution with all aromatic subunits tilted into the same direction so that they line the wall of a hydrophobic cavity.12 Another characteristic structural feature of 1 in CDCl3 and DMSO-d6 are intramolecular hydrogen bonds between the terminal carboxylate groups and the amide protons of the peripheral substituents (Fig. 1).10 These interactions stabilize a cyclic arrangement of the aspartic acid side chains, and thus improve receptor preorganization. Although the orientation of the carboxylate groups with respect to the cyclopeptide cavity is not fixed in the absence of
The signals of the amide protons whose shift indicates the presence of hydrogen bonds in the aromatic substituents of 1 are not visible in polar protic solvents. Therefore, NMR spectroscopy does not allow an assignment of whether the cyclic arrangement of the aspartic acid residues is retained in these solvents or not. Since protic solvents compete strongly for hydrogen bonds it is reasonable to assume, however, that the flexibility of 1 is higher in CD3OD or D2O than in DMSO-d6. Electrospray ionization mass spectrometry (ESIMS) provided a first indication on interactions between 1 and arginine. The mass spectrum of a solution of 1 in CH3OH (0.1 mM) recorded in the negative ion mode contains mainly three signals that can be attributed to monoanions of the compositions [1-3(n-Bu)4N+ +2H+] (calcd 1250.4), [1-2(n-Bu)4N++H+] (calcd 1491.7), and [1-(n-Bu)4N+] (calcd 1733.0). In the spectrum of a solution also containing an equivalent of L-4+.Cl , two additional signals appear that correspond to the complexes [1+L-4+-3(n-Bu)4N++H+] (calcd 1648.6) and [1+L-4+-2(n-Bu)4N+] (calcd 1889.9) (Fig. 2). A similar spectrum results when D4+.Cl is present instead of L-4+.Cl . No signals of higher aggregates containing more than one guest are visible, which indicates than 1 preferentially forms 1:1 complexes with the arginine derivatives, a host/guest ratio that could be confirmed by NMR spectroscopy. Complex formation between 1 and the different guests in DMSO-d6 causes downfield shifts of the guanidinium NH signals in the NMR, that are due to hydrogen bonding interactions between the corresponding protons and the carboxylate groups of 1. In addition, the signal of the methyl protons of 3+ and, for example, those of the protons indicated Ha, Hb, and Hc in the structure of 4+ experience small but reproducible upfield shifts, similar to the ones observed when 1 interacts with monosaccharides.10 Such upfield shifts of guest signals in the NMR are quite common when suitable substrates interact with artificial receptors containing aromatic subunits, and they are usually explained by the close proximity of guest protons to the receptor p-systems in the complex.13 To examine whether similar reasons might also be responsible for the NMR shifts observed here, we analyzed the interactions between 1
J. Bitta, S. Kubik / Journal of Supramolecular Chemistry 1 (2001) 293–297
and the guanidinium ions by molecular modeling. These calculations were based on our structural assignment of the preferred solution structure of 1.10,12 They showed that 1 is indeed able to form complexes, in which all three receptor carboxylate groups are involved in hydrogen bonds with the guanidinium moiety of the guest. Since in these complexes, an aromatic residue of 4+ can reside inside the cavity of the cyclopeptide, complex formation does bring certain guest protons in close proximity to the p-systems of the carbonyl groups or the aromatic subunits of 1. An energy minimized geometry of such a complex is depicted in Figure 3. It shows additional attractive interactions between the NH groups of the guest and carbonyl groups in the cyclo-
Figure 2. ESI mass spectrum of an equimolar mixture of 1 and L-4+ (0.1 mM) in CH3OH.
295
peptide ring that should influence the overall stability of the aggregate. In solution, a complex between 1 and an arginine derivative is certainly quite flexible and most probably equilibrates between a number of energetically similar conformation, for example, ones with the benzoyl or the nitrophenyl group of 4+ included into the cavity of 1. The observed complexation induced signal shifts of the guest protons in the NMR therefore represent an averaged response to the altered environment of the guest in the complex. According to our calculations, they most probably do account for specific interactions between 1 and the guanidinium derivatives, however. Moreover, they enabled us to follow complex formation of 1 in solvents, in which the guanidinium NH protons exchange with deuterium of the solvent molecules. Thus, a convenient determination of complex stoichiometry by Job’s method of continuous variation, and of complex stability by NMR titrations was possible.14 No shifts of signals corresponding to protons of the tetra-nbutylammonium ions were observed in the NMR when 1 interacts with 2+, 3+, L-4+, or D-4+, which shows that the (n-Bu)4N+ cations do not interfere with guest binding. The Job plots describing the binding equilibria in CD3OD between 1 and the two enantiomers of 4+ exhibit a maximum at an equimolar host/guest ratio, a result which, in accordance with the one obtained by mass spectrometry, indicates a 1:1 stoichiometry for both complexes (Fig. 4). Complex stability could thus be calculated from the saturation curves obtained by NMR titrations by using the mathematical description of 1:1 binding equilibria. This treatment also describes the binding of 1 to the other guests and in other solvents very well so that the 1:1 stoichiometry can safely be assumed for all complexes investigated. The results of our NMR titrations are summarized in Table 1. As expected, guanidinium hydrochloride 2+ binds strongly to 1 in DMSO-d6. The smaller stability constant
Figure 3. Optimized structure of a complex between 1 and L-4+. The calculation was performed at the AM1 level without considering solvent molecules. All hydrogen atoms except the ones at the aromatic residues of the guest and at nitrogens as well as the three isopropyl ester groups in the aromatic side chains of 1 have been omitted for reasons of clarity. Intra- and intermolecular hydrogen bonds are depicted as dotted lines.
Figure 4. Job plots of the complexes between 1 and L-4+ (circles) and D-4+ (squares) (in both cases, the shift of the signal corresponding to Hc of the guests was used for the evaluation of complex stoichiometry).
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Table 1. Stability of the complexes of 1 with guests 2+, 3+, L-4+, and D-4+ in different solvents at T=298 K
DMSO-d6 CD3OD
D2O (pD 7.2)
2+ Ka/dmax
3+ Ka/dmax
L-4+ Ka/dmax
D-4+ Ka/dmax
52,000/+1.94 (NH)
19,300/ 0.08 (CH3)
31,000/ 0.07 (Hb) 37,000/ 0.05 (Hc)
29,300/ 0.09 (Hb) 37,000/ 0.04 (Hc)
3400/ 0.07 (CH3)
8200/ 0.14 (Ha) 8300/ 0.07 (Hb) 8300/ 0.04 (Hc)
8400/ 0.17 (Ha) 8800/ 0.08 (Hb) 8800/ 0.10 (Hc)
210/ 0.22 (Hb)
215/ 0.22 (Hb)
No binding
a
Ka stability constants in M 1; errors in Ka <20%; dmax maximum chemical shifts in ppm of the guest signal followed during the titration.
of the complex of 3+ accounts for the reduced number of hydrogen bonding interactions between 1 and the alkylated guanidinium ion. The fact that L-4+ and D-4+ are considerably better bound than 3+ indicates that besides carboxylate/guanidinium interactions, additional contacts between 1 and the arginine derivatives, possibly similar to those we have detected by molecular modeling, contribute favorably to complex stability. Not surprisingly, complex stability decreases in CD3OD and D2O. While no interactions between 1 and 3+ could be observed in water, interactions with the arginine derivates are clearly visible. This is another indication that in the latter case, not only the interactions between the carboxylate groups of 1 and the guanidinium moiety of the guest are responsible for complex formation but also other effects such as the inclusion of an aromatic protecting group of 4+ into the cyclopeptide cavity. Unfortunately, however, the affinity of 1 toward both enantiomers of 4+ is almost the same within the error limits, and arginine binding is thus essentially nonenantioselective. A close inspection of the saturation curves obtained by the NMR titrations reveals clear differences in the interactions of 1 with L-4+ and D-4+. Table 1 and Fig. 5 illustrate, for example, that the complexation induced signal shifts are larger in the NMR spectrum of D-4+ than the ones in the spectrum of L-4+. This difference is also evident in the Job plots (Fig. 4), and is most pronounced for Hc of L/D-4+ in CD3OD.
Complexation induced shifts of guest or host signals in the NMR reflect the altered environment of the corresponding protons in the complex formed. Therefore, the different signal shifts observed for L-4+ and D-4+ indicate that the two guests form structurally slightly different complexes with 1, which is consistent with their diastereomeric nature. Unfortunately, we were not able to experimentally assign solution structures to the complexes of L-4+ and D-4+ by, for example, NOESY or ROESY NMR spectroscopy. We can therefore currently only speculate that both complexes mainly differ in the orientation of the benzoyl and p-nitroanilide protecting groups and in secondary interactions, for example, between the amide groups of 1 and 4+. These differences are obviously not sufficient, however, to translate into a measurable difference in complex stability. In summary, we could show that 1 represents a new yet non-enantioselective platform for the complexation of guanidinium ions or arginine derivatives in polar solvents. Substrate affinity is of the same order of magnitude as that previously described for other receptors.5,8,15 Binding selectivity has still to be improved, however. This can most probably be achieved by introducing additional functional groups in the periphery of the macrocyclic cavity with which the guests can interact.16 We currently pursue this approach and we will report on results of this work in due course. Acknowledgements Thanks are due to S. Zhang for recording the ESI mass spectra. S.K. also thanks Prof. H. Ritter for his support, and the Deutsche Forschungsgemeinschaft for financial funding. References and notes
Figure 5. Complexation induced shifts during an NMR titration of the resonance of the Hc protons of L-4+ (circles) and D-4+ (squares) in CD3OD.
1. Luecke, H.; Quiocho, F. A. Nature 1990, 347, 402–406. 2. Varani, G. Acc. Chem. Res. 1997, 30, 189–195. 3. Arents, G.; Moudrianakis, E. N. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 10489–10493. 4. Keil, B. In The Enzymes, Hydrolysis: Peptide Bond; Boyer, P. D., Ed.; Academic: New York and London, 1971; pp 250– 277. Magnusson, S. In The Enzymes, Hydrolysis: Peptide Bond; Boyer, P. D., Ed.; Academic: New York and London, 1971; pp 278–322. 5. Lehn, J.-M.; Vierling, P.; Hayward, R. C. J. Chem. Soc., Chem. Commun. 1979, 296–298.
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6. (a) Bell, T. W.; Liu, J. Angew. Chem., Int. Ed. Engl. 1990, 29, 923–925. (b) Bell, T. W.; Khasanov, A. B.; Drew, M. G. B.; Filikov, A.; James, T. L. Angew. Chem., Int. Ed. Engl. 1999, 38, 2543–2547. 7. Ngola, S. M.; Kearney, P. C.; Mecozzi, S.; Russell, K.; Dougherty, D. A. J. Am. Chem. Soc. 1999, 121, 1192–1201. 8. Schrader, T. Chem. Eur. J. 1997, 3, 1537–1541. Schrader, T. H. Tetrahedron Lett. 1998, 39, 517–520. Rensing, S.; Arendt, M.; Springer, A.; Grawe, T.; Schrader, T. J. Org. Chem. 2001, 66, 5814–5821. 9. (a) Maruyama, K.; Sohmiya, H.; Tsukube, H. Tetrahedron 1992, 48, 805–818. (b) Famulok, M. J. Am. Chem. Soc. 1994, 116, 1698–1706. (c) Weiß, T.; Leipert, D.; Kaspar, M.; Jung, G.; Go¨pel, W. Adv. Mater. 1999, 11, 331–335. (d) Wehner, M.; Schrader, T.; Finocchiaro, P.; Failla, S.; Consiglio, G. Org. Lett. 2000, 2, 605–608. 10. Bitta, J.; Kubik, S. Org. Lett. 2001, 3, 2637–2640.
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11. The arginine derivative was chosen because both of its enantiomers are commercially available (BACHEM). In addition, its sterically demanding protecting groups could be advantageous for enantioselective recognition. 12. Kubik, S.; Goddard, R. J. Org. Chem. 1999, 64, 9475– 9486. 13. Ma, J. C.; Dougherty, D. A. Chem. Rev. 1997, 97, 1303– 1324, and references cited therein. 14. Connors, K. A. Binding Constants; Wiley: New York, 1987. 15. Some artificial arginine receptors also possess a significantly higher substrate affinity than 1.6b,7 It has to be noted, however, that many investigations are not directly comparable to ours because other arginine derivatives, protected as well as unprotected, were used as guests. 16. T. Schrader has recently shown that for enantioselective arginine complexation with a chiral bisphosphonate, at least a three-point binding is required.9d
Complexation of Arginine with a Cyclopeptide in Polar Solvents and Water Joachim Bitta and Stefan Kubik* Institut fu¨r Organische Chemie und Makromolekulare Chemie, Heinrich-Heine-Universita¨t, Universita¨tsstr. 1, D-40225 Du¨sseldorf, Germany
We dedicate this article to Professor J. L. Atwood on the occasion of his 60th birthday
Abstract—A cyclic hexapeptide containing free carboxylate groups in the periphery of a macrocyclic cavity binds guanidinium ions and arginine derivatives in polar solvents and even in water. Guest binding can be demonstrated by NMR spectroscopy and electrospray ionization mass spectrometry. The spectroscopic results indicate that in CD3OD, the complexes of enantiomeric arginine derivatives have different geometries. This structural difference does not translate into significantly different complex stabilities, however. # 2003 Elsevier Science Ltd. All rights reserved.
The guanidinium group in the side chain of the amino acid arginine is responsible for the strong and selective anion binding of many proteins. In the phosphate binding protein, for example, substrate recognition is mediated by an arginine residue strategically placed inside the active center.1 Arginine-rich regions in the backbone of RNA binding proteins interact with the phosphodiesters of the polynucleotide,2 and electrostatic contacts between phosphate moieties and basic residues of the peptide chain induce the folding of DNA around histone proteins.3 Finally, arginine/carboxylate interactions occur in the active centers of the enzymes thrombine and trypsin, both of which cleave peptides directly behind an arginine residue.4 Small molecules that interfere in these processes can potentially lead to new drug candidates. Various host molecules have therefore been synthesized in recent years that specifically recognize guanidinium ions or arginine in solution. An early example is a tartaric acid based crown ether that binds guanidinium chloride in water with high affinity (Ka=9000 M 1).5 Methylguanidinium is already bound significantly less efficiently, however (Ka=450 M 1). Potent receptors with a high affinity also toward alkylguanidinium ions or arginine
*Corresponding author. Fax: +49-211-81-5840; e-mail: kubik@ uni-duesseldorf.de
are based on, for example, fused azaheterocycles,6 cyclophanes,7 or bisphosphonates,8 all of which contain negatively charged phosphonate or carboxylate groups that interact with the cationic guest by a combination of hydrogen-bonding and electrostatic interactions. Additional binding mechanisms such as, for example, cation–p interactions are in some cases also possible and contribute favorably to complex formation. We are aware of only few receptors, however, that are capable of enantioselective arginine recognition.9 Recently, we described a cyclic hexapeptide 1 containing l-proline and 3-aminobenzoic acid derived subunits with three carboxylate groups arranged around the macrocyclic cavity that binds monosaccharides in 4% methanol/chloroform.10 We speculated that due to the three well preorganized anionic binding sites, this host should also be able to interact with guanidinium ions. Moreover, its chirality could induce an enantioselective substrate complexation. We therefore tested the ability of 1 to interact with different guanidinium derivatives, and report on the results of these binding studies in this communication. Receptor 1 was obtained by coupling of l-aspartic acid 3-benzyl 1-isopropyl ester to a cyclic hexapeptide containing alternating l-proline and 3-aminoisophthalic acid subunits followed by hydrogenative cleavage of the benzyl esters, and deprotonation of the resulting carboxylic acids
1472-7862/01/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved. PII: S1472-7862(02)00073-4
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Figure 1. Intramolecular stabilization of the aspartic acid substituents in 1.
guest molecules, the fact that 1 forms defined 1:1 complexes with monosaccharides shows that in order to allow for optimal interactions with the guests, the peptide is able to adopt conformations with converging binding sites.10
with tetra-n-butylammonium hydroxide.10 Investigations on the receptor properties of 1 were performed in deuterated DMSO, methanol, and water (10 mM aqueous [(n-Bu)4N]H2PO4/[(n-Bu)4N]2HPO4 buffer, pD 7.2) by using guanidinium hydrochloride 2+.Cl , methylguanidinium hydrochloride 3+.Cl , Na-benzoyll-arginine p-nitroanilide hydrochloride L-4+.Cl , and the corresponding d enantiomer of this arginine derivative D-4+.Cl as guests.11
Previous investigations have shown that 1 preferentially adopts conformations in solution with all aromatic subunits tilted into the same direction so that they line the wall of a hydrophobic cavity.12 Another characteristic structural feature of 1 in CDCl3 and DMSO-d6 are intramolecular hydrogen bonds between the terminal carboxylate groups and the amide protons of the peripheral substituents (Fig. 1).10 These interactions stabilize a cyclic arrangement of the aspartic acid side chains, and thus improve receptor preorganization. Although the orientation of the carboxylate groups with respect to the cyclopeptide cavity is not fixed in the absence of
The signals of the amide protons whose shift indicates the presence of hydrogen bonds in the aromatic substituents of 1 are not visible in polar protic solvents. Therefore, NMR spectroscopy does not allow an assignment of whether the cyclic arrangement of the aspartic acid residues is retained in these solvents or not. Since protic solvents compete strongly for hydrogen bonds it is reasonable to assume, however, that the flexibility of 1 is higher in CD3OD or D2O than in DMSO-d6. Electrospray ionization mass spectrometry (ESIMS) provided a first indication on interactions between 1 and arginine. The mass spectrum of a solution of 1 in CH3OH (0.1 mM) recorded in the negative ion mode contains mainly three signals that can be attributed to monoanions of the compositions [1-3(n-Bu)4N+ +2H+] (calcd 1250.4), [1-2(n-Bu)4N++H+] (calcd 1491.7), and [1-(n-Bu)4N+] (calcd 1733.0). In the spectrum of a solution also containing an equivalent of L-4+.Cl , two additional signals appear that correspond to the complexes [1+L-4+-3(n-Bu)4N++H+] (calcd 1648.6) and [1+L-4+-2(n-Bu)4N+] (calcd 1889.9) (Fig. 2). A similar spectrum results when D4+.Cl is present instead of L-4+.Cl . No signals of higher aggregates containing more than one guest are visible, which indicates than 1 preferentially forms 1:1 complexes with the arginine derivatives, a host/guest ratio that could be confirmed by NMR spectroscopy. Complex formation between 1 and the different guests in DMSO-d6 causes downfield shifts of the guanidinium NH signals in the NMR, that are due to hydrogen bonding interactions between the corresponding protons and the carboxylate groups of 1. In addition, the signal of the methyl protons of 3+ and, for example, those of the protons indicated Ha, Hb, and Hc in the structure of 4+ experience small but reproducible upfield shifts, similar to the ones observed when 1 interacts with monosaccharides.10 Such upfield shifts of guest signals in the NMR are quite common when suitable substrates interact with artificial receptors containing aromatic subunits, and they are usually explained by the close proximity of guest protons to the receptor p-systems in the complex.13 To examine whether similar reasons might also be responsible for the NMR shifts observed here, we analyzed the interactions between 1
J. Bitta, S. Kubik / Journal of Supramolecular Chemistry 1 (2001) 293–297
and the guanidinium ions by molecular modeling. These calculations were based on our structural assignment of the preferred solution structure of 1.10,12 They showed that 1 is indeed able to form complexes, in which all three receptor carboxylate groups are involved in hydrogen bonds with the guanidinium moiety of the guest. Since in these complexes, an aromatic residue of 4+ can reside inside the cavity of the cyclopeptide, complex formation does bring certain guest protons in close proximity to the p-systems of the carbonyl groups or the aromatic subunits of 1. An energy minimized geometry of such a complex is depicted in Figure 3. It shows additional attractive interactions between the NH groups of the guest and carbonyl groups in the cyclo-
Figure 2. ESI mass spectrum of an equimolar mixture of 1 and L-4+ (0.1 mM) in CH3OH.
295
peptide ring that should influence the overall stability of the aggregate. In solution, a complex between 1 and an arginine derivative is certainly quite flexible and most probably equilibrates between a number of energetically similar conformation, for example, ones with the benzoyl or the nitrophenyl group of 4+ included into the cavity of 1. The observed complexation induced signal shifts of the guest protons in the NMR therefore represent an averaged response to the altered environment of the guest in the complex. According to our calculations, they most probably do account for specific interactions between 1 and the guanidinium derivatives, however. Moreover, they enabled us to follow complex formation of 1 in solvents, in which the guanidinium NH protons exchange with deuterium of the solvent molecules. Thus, a convenient determination of complex stoichiometry by Job’s method of continuous variation, and of complex stability by NMR titrations was possible.14 No shifts of signals corresponding to protons of the tetra-nbutylammonium ions were observed in the NMR when 1 interacts with 2+, 3+, L-4+, or D-4+, which shows that the (n-Bu)4N+ cations do not interfere with guest binding. The Job plots describing the binding equilibria in CD3OD between 1 and the two enantiomers of 4+ exhibit a maximum at an equimolar host/guest ratio, a result which, in accordance with the one obtained by mass spectrometry, indicates a 1:1 stoichiometry for both complexes (Fig. 4). Complex stability could thus be calculated from the saturation curves obtained by NMR titrations by using the mathematical description of 1:1 binding equilibria. This treatment also describes the binding of 1 to the other guests and in other solvents very well so that the 1:1 stoichiometry can safely be assumed for all complexes investigated. The results of our NMR titrations are summarized in Table 1. As expected, guanidinium hydrochloride 2+ binds strongly to 1 in DMSO-d6. The smaller stability constant
Figure 3. Optimized structure of a complex between 1 and L-4+. The calculation was performed at the AM1 level without considering solvent molecules. All hydrogen atoms except the ones at the aromatic residues of the guest and at nitrogens as well as the three isopropyl ester groups in the aromatic side chains of 1 have been omitted for reasons of clarity. Intra- and intermolecular hydrogen bonds are depicted as dotted lines.
Figure 4. Job plots of the complexes between 1 and L-4+ (circles) and D-4+ (squares) (in both cases, the shift of the signal corresponding to Hc of the guests was used for the evaluation of complex stoichiometry).
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Table 1. Stability of the complexes of 1 with guests 2+, 3+, L-4+, and D-4+ in different solvents at T=298 K
DMSO-d6 CD3OD
D2O (pD 7.2)
2+ Ka/dmax
3+ Ka/dmax
L-4+ Ka/dmax
D-4+ Ka/dmax
52,000/+1.94 (NH)
19,300/ 0.08 (CH3)
31,000/ 0.07 (Hb) 37,000/ 0.05 (Hc)
29,300/ 0.09 (Hb) 37,000/ 0.04 (Hc)
3400/ 0.07 (CH3)
8200/ 0.14 (Ha) 8300/ 0.07 (Hb) 8300/ 0.04 (Hc)
8400/ 0.17 (Ha) 8800/ 0.08 (Hb) 8800/ 0.10 (Hc)
210/ 0.22 (Hb)
215/ 0.22 (Hb)
No binding
a
Ka stability constants in M 1; errors in Ka <20%; dmax maximum chemical shifts in ppm of the guest signal followed during the titration.
of the complex of 3+ accounts for the reduced number of hydrogen bonding interactions between 1 and the alkylated guanidinium ion. The fact that L-4+ and D-4+ are considerably better bound than 3+ indicates that besides carboxylate/guanidinium interactions, additional contacts between 1 and the arginine derivatives, possibly similar to those we have detected by molecular modeling, contribute favorably to complex stability. Not surprisingly, complex stability decreases in CD3OD and D2O. While no interactions between 1 and 3+ could be observed in water, interactions with the arginine derivates are clearly visible. This is another indication that in the latter case, not only the interactions between the carboxylate groups of 1 and the guanidinium moiety of the guest are responsible for complex formation but also other effects such as the inclusion of an aromatic protecting group of 4+ into the cyclopeptide cavity. Unfortunately, however, the affinity of 1 toward both enantiomers of 4+ is almost the same within the error limits, and arginine binding is thus essentially nonenantioselective. A close inspection of the saturation curves obtained by the NMR titrations reveals clear differences in the interactions of 1 with L-4+ and D-4+. Table 1 and Fig. 5 illustrate, for example, that the complexation induced signal shifts are larger in the NMR spectrum of D-4+ than the ones in the spectrum of L-4+. This difference is also evident in the Job plots (Fig. 4), and is most pronounced for Hc of L/D-4+ in CD3OD.
Complexation induced shifts of guest or host signals in the NMR reflect the altered environment of the corresponding protons in the complex formed. Therefore, the different signal shifts observed for L-4+ and D-4+ indicate that the two guests form structurally slightly different complexes with 1, which is consistent with their diastereomeric nature. Unfortunately, we were not able to experimentally assign solution structures to the complexes of L-4+ and D-4+ by, for example, NOESY or ROESY NMR spectroscopy. We can therefore currently only speculate that both complexes mainly differ in the orientation of the benzoyl and p-nitroanilide protecting groups and in secondary interactions, for example, between the amide groups of 1 and 4+. These differences are obviously not sufficient, however, to translate into a measurable difference in complex stability. In summary, we could show that 1 represents a new yet non-enantioselective platform for the complexation of guanidinium ions or arginine derivatives in polar solvents. Substrate affinity is of the same order of magnitude as that previously described for other receptors.5,8,15 Binding selectivity has still to be improved, however. This can most probably be achieved by introducing additional functional groups in the periphery of the macrocyclic cavity with which the guests can interact.16 We currently pursue this approach and we will report on results of this work in due course. Acknowledgements Thanks are due to S. Zhang for recording the ESI mass spectra. S.K. also thanks Prof. H. Ritter for his support, and the Deutsche Forschungsgemeinschaft for financial funding. References and notes
Figure 5. Complexation induced shifts during an NMR titration of the resonance of the Hc protons of L-4+ (circles) and D-4+ (squares) in CD3OD.
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11. The arginine derivative was chosen because both of its enantiomers are commercially available (BACHEM). In addition, its sterically demanding protecting groups could be advantageous for enantioselective recognition. 12. Kubik, S.; Goddard, R. J. Org. Chem. 1999, 64, 9475– 9486. 13. Ma, J. C.; Dougherty, D. A. Chem. Rev. 1997, 97, 1303– 1324, and references cited therein. 14. Connors, K. A. Binding Constants; Wiley: New York, 1987. 15. Some artificial arginine receptors also possess a significantly higher substrate affinity than 1.6b,7 It has to be noted, however, that many investigations are not directly comparable to ours because other arginine derivatives, protected as well as unprotected, were used as guests. 16. T. Schrader has recently shown that for enantioselective arginine complexation with a chiral bisphosphonate, at least a three-point binding is required.9d