DFT study of N H O hydrogen bond between model dehydropeptides and water molecule

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1 This article was downloaded by: [Uniwersytet Opolski] On: 14 January 2014, At: 03:44 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Molecular Physics: An International Journal at the Interface Between Chemistry and Physics Publication details, including instructions for authors and subscription information: DFT study of N H O hydrogen bond between model dehydropeptides and water molecule Aneta Buczek a & Małgorzata A. Broda a Department of Physical Chemistry and Molecular Modeling, Faculty of Chemistry, University of Opole, Opole, Poland Accepted author version posted online: 23 Sep 2013.Published online: 14 Oct To cite this article: Aneta Buczek & Małgorzata A. Broda, Molecular Physics (2013): DFT study of N H O hydrogen bond between model dehydropeptides and water molecule, Molecular Physics: An International Journal at the Interface Between Chemistry and Physics, DOI: / To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the Content ) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at

2 Molecular Physics, RESEARCH ARTICLE DFT study of N H O hydrogen bond between model dehydropeptides and water molecule Aneta Buczek and Małgorzata A. Broda Department of Physical Chemistry and Molecular Modeling, Faculty of Chemistry, University of Opole, Opole, Poland (Received 29 August 2013; accepted 18 September 2013) The strength of the hydrogen bond formed between a water molecule and two α,β-dehydroalanine derivatives including Ac- Ala-NMe 2 (1) and Ac- Ala-NHMe (2) in comparison with standard amino acid Ac-Ala-NMe 2 (3) is studied by density functional theory (with M06-2X and B3LYP functionals). Calculations were conducted for two different conformations of the peptides: extended (C 5 ) and bent (β) with polyproline II backbone dihedral angles. The obtained results show that both dehydro and standard peptides in bent conformation form stronger hydrogen bonds with water than in the extended ones. Moreover, due to higher polarity of the N H group of α,β-dehydroalanine residues, the H-bond in their complexes with water are stronger than for standard alanine. Keywords: dehydroamino acids; hydrogen bond; DFT; M06-2X; B3LYP 1. Introduction The hydrogen bonds play a crucial role in stabilising folded structure of proteins [1 4]. For a detailed understanding of these intermolecular and intramolecular interactions, the conformational preferences and proton donor/acceptor properties of small model peptides have been theoretically studied [5 9]. Correlated ab initio calculations have revealed that the strength of N H O bond in peptide water [10,11] or peptide formamide [12,13] complexes varies with the conformation of the polypeptide chain. The NH group is a much less potent proton donor in extended conformation (C 5 ) than in the bent ones because of the close proximity of the O atom of the neighbouring peptide group in C 5 conformer [11]. The introduction of conformationally constrained amino acid residues into bioactive peptides is a useful and frequently applied modification method for studying the resulting active conformations and to design new and potent analogues [14]. One group of such residues, found in naturally occurring peptides [15 23], are α,β-didehydroα-amino acids (in short: dehydroamino acids, Xaa). As a result of the presence of a double bond between C α and C β is their unique features such as a fixed value of the χ 1 torsion angle and an enlarged C C α N valence angle. The introduction of an α,β-dehydroamino acid into a peptide chain not only changes the conformational properties of the backbone and side chain but, in some cases, may also affect the strength of the H-bonds formed by these residues and alter the association tendency of the peptide [24,25]. As can be seen from our previous FTIR studies [26,27], model dehydropeptides Ac- Xaa-NHMe demonstrate lower tendency to form aggregates in non-polar solvents than standard peptides Ac-Xaa-NHMe. On the other hand, α,β-dehydroamino acid residues with tertiary C-terminal amide bond (Ac- Xaa-NMe 2 ) associate significantly easier than the standard analogue Ac-Xaa-NMe 2 [24,25]. For example, the dimerisation constant K D, determined on the basis of average molecular weights for Ac- Ala-NMe 2 (3), is one order of magnitude larger than that for Ac-L-Ala- NMe 2 (1) [24]. The lower positions of ν s (N H) dimmer bands in the FTIR spectra of Ac- Ala-NMe 2 and higher interaction energy obtained by B3LYP calculations show that an increased tendency of association for Ac- Ala- NMe 2 results from the higher strength of hydrogen bonds in the bimolecular complexes than in dimmers of standard alanine derivative. Theoretical calculations indicate that the low-energy cyclic dimmers of Ac- Ala-NMe 2 are primarily built of conformers β (ϕ, ψ = 43, 123 )[24]. In these structures the N H and C=O groups are the most exposed ones, pointing outside and are readily available for the intermolecular interactions. Hence, this conformation indicates a great potential for intermolecular hydrogen bonding. Moreover, these groups in dehydro residue show larger polarisability due to their participation in the resonance with the C α =C β double bond. Although previously mentioned experimental facts indicate that α,β-dehydroamino acid derivatives in certain conformations form stronger intermolecular H-bond than the standard amino acids, this problem so far has not been carefully examined by theoretical methods. In this study, using the density functional theory (DFT) methods, we investigated the energies and geometries of Corresponding author. Małgorzata.Broda@uni.opole.pl C 2013 Taylor & Francis

3 2 A. Buczek and M.A. Broda Figure 1. General formulas, atom numbering and selected torsion angles for the studied compounds. hydrogen bonds between the water molecule and model peptide or dehydropeptides in two different conformations: extended (C 5 ) and bent ones, with polyproline II (PPII) backbone dihedral angles. In DFT methods, the correlation energy is treated as a functional of threedimensional electron density. The combination of speed and sufficient accuracy makes them particularly effective for large molecules. In this work the calculations were carried out using hybrid density functional B3LYP and metahybrid M06-2X one to test their performance for modelling intermolecular hydrogen bonds of the peptides with water. 2. Computational details All calculations in this work were performed using the Gaussian 09 package [28]. The three model dipeptides examined in this study are alanine and dehydroalanine derivatives illustrated in Figure 1. The extended and bent conformations of the model compounds were selected. The conformation minima (initially taken from B3LYP/ G gas-phase optimisation [29 31]) were obtained from unrestricted optimisation in water using polarisable continuum model (PCM) developed by Tomasi and coworkers [32,33] to account for solvent effect at the same level of theory. Additionally, to better describe dispersion interaction apart from B3LYP/ G [34,35] the M06-2X/ G [36,37] method was used. Good performance of M06-2X functional for peptide conformation prediction was frequently reported [38 40]. In the next step a 1:1 complex of the studied alanine derivatives (with blocked ϕ, ψ angles) and one water molecule approaching the N 1 H 1 group were partly optimised. In order to ensure the linearity of the H-bond, the valence angle N H O w was frozen at 180. To avoid the formation of additional H- bond of water molecule with O 2 oxygen atom, the dihedral angles between the water H atoms and the carbonyl O 2 atom were held to be ±90. The basis set superposition error was corrected using the counter point (CP) method [41]. 3. Results and discussion The energy of water interaction with the studied peptides in different conformations was tested in this work using both implicit and explicit solvation models. The results of the former and latter approaches are gathered in Table 1 and 2, respectively. Table 1 presents relative energies and backbone angles ϕ, ψ of the C 5 and β conformers of studied diamides in water (modelled as continuous dielectric within PCM method) obtained by M06-2X and B3LYP methods. In the gas phase, according to the previous B3LYP calculations [29 31], the lowest energy conformer of Ac- Ala- NMe 2 (1) is the extended structure C 5 (ϕ, ψ = 179.7, ). Next in energy order is conformer β (ϕ, ψ = 39.5, ) with relative energy E = 3.67 kcal/mol. As can be seen from the DFT/PCM results shown in Table 1, aqueous environment has no effect on the energy order of (1) conformers, but distinctly diminish the energy gap between them to 0.4 or 1.1 kcal/mol by M06-2X or B3LYP methods. The M06-2X/PCM stabilisation energies due to interaction with water (E sol ) are 8.4 and 10.8 kcal/mol for C 5 and β conformer, respectively. The corresponding energies obtained by B3LYP/PCM are very similar: 8.5 and 11.2 kcal/mol, respectively. Thus, DFT methods combined with dielectric continuum model of solvent predict higher stabilisation of β conformer with increasing solvent polarity which is in good agreement with infrared (IR) [42] and X-ray [43] experimental data obtained for (1). The analysis of IR spectra of (1) solutions (in CCl 4,CH 2 Cl 2 and CH 3 CN) in the ν s (N H) region has revealed the presence of two conformers (intramolecularly H-bonded and the open one) in solutions and increasing share of the open conformer with rising solvent polarity [42]. In crystal state, Ac- Ala-NMe 2 (1) adopts β conformation (ϕ, ψ = 43, 127 ) stabilised by N 1 H O 2 intermolecular H-bonds [43]. Conformational properties of (2) molecule are rather different. However, according to the B3LYP calculations [29], (2) in the gas phase is also able to adopt C 5 and β conformations but the energy difference between them is very high, i.e kcal/mol. Water, just as in the case of

4 Molecular Physics 3 Table 1. Selected torsion angles ( ), relative energies ( E) and water stabilisation energies (E sol ) (kcal/mol) of local minima for the studied molecules in water fully optimised by M06-2X and B3LYP methods. M06-2X/ G B3LYP/ G Conformer ϕ ψ E E sol a ϕ ψ E E sol Ac- Ala-NMe 2 (1) C β Ac- Ala-NHMe (2) C β Ac-Ala-NMe 2 (3) C β a E sol stabilisation due to interaction with water, calculated as a difference of electronic energy in the gas phase and in solvent environment modelled with PCM method. previously discussed diamide (1), strongly stabilises β conformation and diminishes energy difference between the studied conformers to 2.14 or 2.90 kcal/mol according M06-2X or B3LYP methods (see Table 1). The M06-2X/PCM water interaction energies for C 5 and β conformers of (2) are equal to 8.9 and 12.3 kcal/mol, respectively. Thus, according to the DFT calculations, even in a polar environment, (2) has a strong tendency to adopt extended conformation. This is in agreement with the available experimental data. Both in solution and in the solid state, (2) adopts extended conformation [26,44]. The X-ray conformational study of ( Ala) 1 6 homopeptides [45] alsorevealed the strong tendency of Ala residue to adopt the fully extended conformation. For comparison, we also examined the conformational properties of N-methylated, at C-terminal amide group, alanine derivative Ac-Ala-NMe 2 (3). In the gas phase the B3LYP/ G calculation revealed two low-energy conformers and five high-energy ones [31]. The lowest in energy is extended conformer C 5 (ϕ, ψ = 154, 160 ), and the second in energy order, with relative energy E = 1.3 kcal/mol, is C 7eq conformer (ϕ, ψ = 101, 113 ). In the crystal state, (3) adopts the β conformation with the torsion angles ϕ, ψ = 82, 157 for Ac-L-Ala-NMe 2 and ϕ, ψ = 66, 158 for Ac-DL-Ala-NMe 2 [43]; however, this β conformation was not found on the gas-phase Ramachandran map of (3) calculated at B3LYP level [30,31]. Moreover, our B3LYP calculations with the implicit Table 2. Counterpoise-corrected H-bond energies and selected parameters of diamide water systems obtained by M06-2X and B3LYP methods. Complex Distances (Å) Atomic charges H-bond energy (kcal/mol) N 1 H 1 O w N 1 H 1 O2 N 1 H 1 O 2 M06-2X/ G 1(β) (β) (β) (C 5 ) (C 5 ) (C 5 ) B3LYP/ G 1(β) (β) (β) 1(C 5 ) (C 5 ) (C 5 )

5 4 A. Buczek and M.A. Broda Figure 2. M06-2X/ G geometries of the diamide water systems of 1 3 compounds. Dashed lines represent the intramolecular and intermolecular hydrogen N H O bonds. solvent model (PCM) did not lead to β conformation of (3) dipeptide. On the other hand, M06-2X/ G calculation of C 5 and β conformers of (3) in the gas phase revealed extended structure C 5 with torsion angles ϕ, ψ = 161, 165 and bent conformation slightly modified as compared to the B3LYP results, particularly with regard to the ϕ torsion angle, β conformation (ϕ, ψ = 85, 120 ). The energy difference between these two conformers equals

6 Molecular Physics kcal/mol. According to the M06-2X/PCM calculations, in the aqueous environment, both C 5 and β conformations are maintained with ϕ, ψ torsion angles and relative energies shown in Table 1. The backbone torsion angles of (3)in β conformation (ϕ, ψ = 68, 158 ) are almost identical to those found in the crystal state of Ac-DL-Ala-NMe 2 [43]. Stabilisation energy due to interaction with water is, again as for the previously discussed compounds, lower for the C 5 than for the β conformation, 9.0 and 9.6 kcal/mol, respectively. However, as can be seen, the energy difference in this case is not very high. Additionally, the water stabilisation energy of (3) in β conformation is clearly smaller than in the case of two previously discussed dehydro compounds while for the extended conformations of 1 3 compounds these values are similar. Figure 2 presents partially optimised geometries of complexes diamide water calculated by M06-2X method for extended (C 5 ) and bended (β) conformations of studied 1 3 molecules. In Table 2 are summarised the water peptide hydrogen bond energies and H O distances of intermolecular and intramolecular H-bonds obtained by M06-2X and B3LYP methods. For all studied compounds, N 1 H 1 O w hydrogen bond with water is shorter and stronger when model peptide is in β conformation than in the extended one. The highest difference in H-bond energy between those two conformations is observed for (2) and equals 2.3 kcal/mol (3.2 kcal/mol) according to the M06-2X (B3LYP) method. This lower N 1 H 1 proton donor disposition when peptide is in C 5 conformation is consistent with previous MP2 calculations for For-Gly-NH 2 [10] and is caused by close proximity of the negatively charged O 2 atom [11]. The results of our DFT calculations, presented in Table 2, for extended conformations of studied compounds are in agreement with this statement. Generally, the higher the O 2 negative charge, the shorter the N 1 H 1 O 2 intramolecular hydrogen bond, and at the same time, the longer and weaker the intermolecular N 1 H 1 O w H-bond. However, when we look at the details of the M06-2X results, there are some distortions of the overall relationship. In the complex of (3)inC 5 conformation with water, both the distances N 1 H 1 O 2 and N 1 H 1 O w are longer than those in the complex of 2(C 5 ). In our opinion, this is due to a greater polarisation of the N 1 H 1 group in dehydro compounds (see partial charges assigned to N 1 and H 1 atoms using Mulliken population analysis shown in Table 2) and thus stronger electrostatic attraction between water molecule and N 1 H 1 group of dehydro residue. The atomic charges reported in Table 2 indicate that charge on N 1 atom is more negative for 1 and 2 compounds than for standard amino acid derivative 3. On the other hand, the positive charge of the H 1 atom is almost independent of peptide conformation and the amino acid residue. Also for β conformations of Ala residue derivatives (1 and 2 compounds), we can observe the higher negative partial charge on N 1 atom which is probably the main cause of a stronger hydrogen bond with the water molecule formed by those molecules. It is worth noting that although 2(β) forms a slightly shorter H-bond with water, the calculated interaction energy is smaller. The reason for this is the additional C β H O w interaction which is somewhat shorter in the case of 1(β). The H O w distance is 3.47 and 3.61 Å for 1(β) and 2(β), respectively. Comparing the M06-2X and B3LYP results can be concluded that both methods are qualitatively consistent regarding the conformational properties of the studied model dehydropeptides in water and the strength of H-bond formed between water molecule and these dehydropeptides in two different conformations. Both methods predict that the hydrogen bond formed by dehydroalanine derivatives in β conformation is stronger and shorter in comparison to H-bond formed when peptide is in extended conformation. However, M06-2X calculated energies of N H OH 2 bonds for all studied complexes are higher than those obtained by B3LYP method (Table 2). The difference between the CPcorrected energies of the H-bonds calculated by these two methods varies from 1.4 to 2.6 kcal/mol. In the context of this work, the greatest failure of B3LYP method is that it cannot model the PPII conformation of Ac-Ala-NMe 2 neither in vacuum nor in a water environment. This is a more general problem of B3LYP functional, which sometimes has problems with finding conformations stabilised mainly by dispersion interaction and not by intramolecular N H O hydrogen bonds [39]. 4. Conclusion Introduction of the α,β-dehydroamino acid residue into the peptide not only changes the conformation of this peptide fragment but also modifies the distribution of electron density in both amide systems adjacent to the C α =C β double bond. This may affect the proton acceptor and proton donor properties of the C=O and N H groups in the proximity of dehydro residue and influence peptide intermolecular and intramolecular hydrogen bond pattern. DFT calculations performed in the present study allow us to formulate the following conclusions. α,β-dehydropeptides in β conformation (ϕ, ψ = 40, 130 ) form a stronger hydrogen bond with water than in the extended one. This relationship is analogous to that observed for the standard peptides. Moreover, the energies of hydrogen bonds in dehydroalanine complexes are higher than in complexes of alanine in similar conformations. This is due to the more negative charge on the nitrogen atom of α,β-dehydroalanine N H group. The results of our calculations indicate that meta-hybrid functional M06-2X in comparison with B3LYP allows us to obtain more reliable results regarding the peptide conformation, especially for N-methylated peptides for which dispersion interactions are more important in the stabilisation of bent structures.

7 6 A. Buczek and M.A. Broda Acknowledgements Calculations were carried out in Wrocław Center for Networking and Supercomputing, and in the Academic Computer Center CYFRONET, AGH, Kraków. Funding A. Buczek is recipient of a PhD fellowship from a project funded by the European Social Fund. References [1] L. Pauling, R.B. Corey, and H.R. Branson, Proc. Natl. Acad. Sci. USA 37 (4), (1951). [2] W. Kauzmann, Adv. Protein Chem. 14, 1 (1959). [3] T. Steiner and G. Koellner, J. Mol. Biol. 305, 535 (2001). [4] C. Toniolo, CRC Crit. Rev. Biochem. 9, 1 (1980). [5] T. Head-Gordon, M. Head-Gordon, M.J. Frisch, C.L. Brooks,, and J.A. Pople, J. Am. Chem. Soc. 113, 5989 (1991). [6] I.R. Gould, W.D. Cornell, and I.H. Hillier, J. Am. Chem. Soc. 116, 9250 (1994). [7] V. Cruz, J. Ramos, and J. Martínez-Salazar, J. Phys. Chem. B 115, 4880 (2011). [8] M. Nagaraju, and G.N. Sastry, Int. J. Quantum Chem. 110, 1994 (2010). [9] C.-L. Sun and C.-S. Wang, Int. J. Quantum Chem. 112, 2336 (2012). [10] S. Scheiner, J. Phys. Chem. B 111, (2007). [11] S. Scheiner and T. Kar, J. Mol. Struct. 166, 844 (2007). [12] S. Scheiner, J. Phys. Chem. B 110, (2006). [13] S. Scheiner, J. Phys. Chem. B 109, (2005). [14] C. Adessi and C. Soto, Curr. Med. Chem. 9, 963 (2002). [15] R. Rink, J. Wierenga, A. Kuipers, L.D. Kluskens, A.J.M. Driessen, and O.P. Kuipers, Appl. Environ. Microb. 73, 1792 (2007). [16] C. Chatterjee, M. Paul, L. Xie, and W.A. van der Donk, Chem. Rev. 105, 633 (2005). [17] N. Loiseau, F. Cavelier, J.-P. Noel, and J.-M. Gomis, J. Peptide Sci. 8, 335 (2002). [18] C. Malbrouck and P. Kestemont, Environ. Toxicol. Chem. 25, 72 (2006). [19] L. Herfindal and F. Selheim, Mini-Rev. Med. Chem. 6, 279 (2006). [20] M. Namikoshi, B.W. Choi, F. Sun, K.L. Rinehart, W.W. Carmichael, W.R. Evans, and V.R. Beasley, J. Org. Chem. 60, 3671 (1995). [21] G.A. Codd, L.F. Morrison, and J.S. Metcalf, Toxicol. Appl. Pharm. 203, 264 (2005). [22] J.R. Parkhurst, and D.S. Hodgins, Arch. Biochem. Biophys. 152, 597 (1972). [23] R.B. Wickner, Biol. Chem. 244, 6550 (1969). [24] M.A. Broda, M. Rospenk, D. Siodłak, and B. Rzeszotarska, J. Mol. Struct. 740, 17 (2005). [25] M.A. Broda, and B. Rzeszotarska, Acta Biochim. Pol. 53, 221 (2006). [26] M.A. Broda, B. Rzeszotarska, L. Smełka, and M. Rospenk, J. Peptide Res. 50, 342 (1997). [27] M.A. Broda, B. Rzeszotarska, L. Smełka, and G. Pietrzyński, J. Peptide Res. 52, 72 (1998). [28] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, G. Scalmani, V. Barone, B. Mennucci, G.A. Petersson, H. Nakatsuji, M. Caricato, X. Li, H.P. Hratchian, A.F. Izmaylov, J. Bloino, G. Zheng, J.L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao, H. Nakai, T. Vreven, J.A. Montgomery,, J.E. Peralta, F. Ogliaro, M. Bearpark, J.J. Heyd, E. Brothers, K.N. Kudin, V.N. Staroverov, R. Kobayashi, J. Normand, K. Raghavachari, A. Rendell, J.C. Burant, S.S. Iyengar, J. Tomasi, M. Cossi, N. Rega, J.M. Millam, M. Klene, J.E. Knox, J.B. Cross, V. Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A.J. Austin, R. Cammi, C. Pomelli, J.W. Ochterski, R.L. Martin, K. Morokuma, V.G. Zakrzewski, G.A. Voth, P. Salvador, J.J. Dannenberg, S. Dapprich, A.D. Daniels, Ö. Farkas, J.B. Foresman,J.V.Ortiz,J.Cioslowski,andD.J.Fox.GAUS- SIAN 09, Revision B.01 (Gaussian, Inc., Wallingford, CT, 2009). [29] D. Siodłak, M.A. Broda, and B. Rzeszotarska, J. Mol. Struct.: THEOCHEM 668, 75 (2004). [30] D. Siodłak, M.A. Broda, B. Rzeszotarska, A.E. Kozioł, and I. Dybała, Acta Biochim. Polon. 48, 1179 (2001). [31] D. Siodłak, B. Rzeszotarska, and M.A. Broda, Acta Biochim. Polon. 51, 137 (2004). [32] S. Miertus, and J. Tomasi, Chem. Phys. 65, 239 (1982). [33] J. Tomasi, B. Mennucci, and R. Cammi, Chem. Rev. 105, 2999 (2005). [34] A.D. Becke, J. Chem. Phys. 98, 5648 (1993). [35] C. Lee, W. Yang, and R.G. Parr, Phys. Rev. B 37, 785 (1988). [36] Y. Zhao and D.G. Truhlar, Theor. Chem. Acc. 120, 215 (2008). [37] Y. Zhao and D.G. Truhlar, Acc. Chem. Res. 41, 157 (2008). [38] T. Mourik, J. Chem. Theor. Comput. 4, 1610 (2008). [39] D. Siodłak, M. Bujak, and M. Staś, J. Mol. Struct. 1047, 229 (2013). [40] Y. E. Kang and B.J. Byun, J. Comput. Chem. 31, 2915 (2010). [41] S.F. Boys and F. Bernardi, Mol. Phys. 19, 553 (1970). [42] M.A. Broda, D. Siodłak, and B. Rzeszotarska, J. Peptide Sci. 11, 546 (2005). [43] B. Rzeszotarska, D. Siodłak, M.A. Broda, A.E. Kozioł, and I. Dybała, J. Peptide Res. 59, 89 (2002). [44] D.E. Palmer, C. Pattaroni, K. Nunami, R.K. Chadha, M. Goodman, T. Wakamiya, K. Fukase, S. Horimoto, M. Kitazawa, H. Fujita, A. Kubo, and T. Shiba, J. Am. Chem. Soc. 114, 5634 (1992). [45] M. Crisma, F. Formaggio, C. Toniolo, T. Yoshikawa, and T. Wakamiya, J. Am. Chem. Soc. 121, 3272 (1999).