Vol. 53 No. 1/2006, 65–72 Regular paper on-line at: www.actabp.pl Chelating ability of proctolin tetrazole analogue *

The aim of the investigation was to establish the chelating ability of a new proctolin analogue of the sequence Arg-Tyr-LeuPsi[CN(4)]Ala-Thr towards copper(II) ions. The insertion of the tetrazole moiety into the peptide sequence has considerably changed the coordination ability of the ligand. Potentiometric and spectroscopic (UV-Vis, CD, EPR) results indicate that the incorporation of 1,5-disubstituted tetrazole ring favours the formation of a stable complex form of CuH(-1)L. This 4N coordination type complex is the dominant species in the physiological pH range. The tetrazole moiety provides one of these nitrogens. The data indicate that Cu(II) ions are strongly trapped inside the peptide backbone. These findings suggest that Cu(II) can hold peptide chains in a bent conformation. This bent conformation may be essential for bioactivity of the tetrazole peptides.

Proctolin (Arg-Tyr-Leu-Pro-Thr) was the first neuropeptide isolated from insects (Brown & Starratt, 1975).This neuropeptide has been detected in various neuronal cell types of insects (Bishop et al., 1981;O'Shea & Adams, 1986, Orchard et al., 1989) and identified in neurones with a widespread distribution within the central nervous system of arthropods (Eckert et al., 1981).In order to determine structure-activity relationships for the myotropic activity of proctolin in insects, many modifications of the peptide have been made (Konopińska et al., 1988a;1988b;Kuczer et al., 1996;Konopińska & Rosiński, 1999;Woźnica et al., 2004).We have previously described analogues of insect kinins, another class of insect neuropeptides, that retain very significant diuretic activity, modified with a 1,5-disubstituted tetrazole ring (Ψ[CN 4 ]) (Fig. 1), which preferentially forms the type VI β-turn (Zabrocki et al., 1988;Nachman et al., 2002;2004).In this study, we have prepared a tetrazole analogue of proctolin.Proctolin was modified at position 3-4 by replacing the Leu-Pro dipeptide unit with the tetrazole dipeptide LeuΨ [CN 4 ]Ala.The interaction of Cu(II) with proctolin may have physiological relevance because copper content is especially high in synaptosomal fluids which are rich in neuropeptides (Linder & Goode, 1991).Studies on the Cu(II)-exorphin systems have shown that these exogenous opiate-like peptides are efficient chelating agents (Chruscinska et al., 1997;1998;Lodyga-Chruscinska et al., 1998;1999).Moreover, insertion of the tetrazole ring can effectively stabilize the metallopeptide structure (Lodyga-Chruscinska et al., 1999;2000;2004;Chruscinska et al., 2001).It has been found that tetrazole ring can be directly involved in the Cu(II) binding and then very stable complex species with 3N or 4N co-ordination mode are formed at physiological pH.The complex formation is the result of simultaneous formation of

2006
E. Łodyga-Chruścińska and others five-membered chelate rings by consecutive nitrogen donors.The unusual feature found for the Cu(II)tetrazole enkephalin analogue system has been the cooperative transition from a 2N to a 4N complex (Lodyga-Chruscinska et al., 1999).The position of the tetrazole ring system in the peptide backbone plays a critical role in the metallopeptide molecule stabilization.The insertion of the tetrazole between amide groups leads to enhanced complex stability and results in a very effective peptide chelating agent.These previous studies led us to investigate copper(II) complexes of proctolin (Scheme 1a) and its tetrazole analogue (Scheme 1b) in order to evaluate the factors governing their chelating ability.The copper(II) coordination to several proctolin analogues modified in the fifth position of the peptide chain have been studied (Kowalik-Jankowska et al., 2005).The presence of a proline residue in the fourth position of the proctolin analogues (RYLPP, RYLPI) led to the existence of the CuL and CuH -1 L complexes with 2N {NH 2 , N -, CO} and 3N {NH 2 , 2N -, CO} coordination modes, respectively, over a wide pH range.The amine group of the Dab residue of the RYLP-Dab proctolin analogue was coordinated to copper(II) ions, and a 3N {NH 2 , N -, CO, NH 2 Dab} complex was found.In this paper, we report the results of combined potentiometric and spectroscopic (UV-Vis, CD and EPR) studies on the copper(II) complexes of proctolin and its tetrazole analogue.The results suggest that the presence of the tetrazole ring enhances the metal binding ability of the peptide.

Reagents.
The tetrazole proctolin analogue (RYLΨ[CN 4 ]AT) was synthesized according to a previously reported procedure (Zabrocki et al., 1988).Their purity was verified by HPLC, mass spectrometry and potentiometry to be > 99%.Proctolin (RYLPT) was purchased from Bachem and used without purification, Cu(NO 3 ) 2 , KNO 3 , HNO 3 and NaOH were Merck products and were used without further purification.
Potentiometric studies.Protonation and coordination equilibria were investigated by potentiometric titration in aqueous solution, over the pH 3-11 range, at a constant ionic strength using 0.1 M KNO 3 and at constant temperature (298 K) under argon atmosphere with a total volume of 1.5-2 cm 3 of the sample.A 0.05 M solution of Cu(NO 3 ) 2 was used as the stock for the Cu(II) ion.An automatic titration set including autoburette meter (Molspin Ltd., Newcastle-upon-Tyne, UK), a semi-microcombined electrode (Russell CMAWL/S7) and an IBM-compatible PC were used to collect data.Alkali, about 0.1 M NaOH, free of CO 2 was added with a 0.250 cm 3 micrometer syringe, which was calibrated by both weight titration and titration of standard materials.The electrode was calibrated for hydrogen ion activity.The relationship between activity and concentration was calculated daily by titration with HNO 3 (Irving et al., 1967).Calculations were made with the aid of the SUPERQUAD computer program (Gans et al., 1985).This allows the refinement of total ligand concentrations.Therefore we were able to confirm the purity of the peptide, in particular the absence of acetate, a frequent impurity in peptide samples or of other coordinating ions.In all cases triplicate titrations (about 500 experimental points in one set of measurements) were carried out at the Cu/L ratio of 1:1.The ligand concentration was 1 × 10 -3 M. As usual, the stabilities of the metal complexes are reported as logarithms of the overall formation constants β pqr = [M p H q L r ]/ [M] p [H] q [L] r , where M stands for the metal ion, H is proton and L the deprotonated form of the ligand (Table 1).The standard deviations quoted were computed by SUPERQUAD and refer to random errors only.They are, however, a good indication of the importance of a particular species in the equilibrium.
Spectroscopic studies.UV-Vis spectra were recorded with a Perkin-Elmer Lambda 11 spectrophotometer.Circular dichroism spectra were obtained with a Jobin-Yvon CD-6 dichrograph over the range 200-750 nm, using 1 and 0.05 cm cuvettes.The spectra are expressed as Δε = ε l -ε r , where ε l and ε r are the molar absorption coefficients for left and right circularly polarized light, respectively.Electron paramagnetic resonance measurements were carried out with a Varian E-9 instrument at the X-band frequency (9.1 GHz) at 120 K; about 10% of ethanediol was added to the samples in order to obtain good glasses.Measurements were performed at the maximum concentration of each species found in titrations.The EPR parameters were read from the spectra (estimated uncertainties for A and g values are 1 × 10 -4 cm -1 and 0.002, respectively, in the spectra of a single species).

Protonation equilibria of free peptides
The protonation constants of the ligands are included in Table 1 together with some literature data for comparison.Two peptides studied (Scheme 1) contain an arginine at the first position of the peptide backbone.The arginine residue contains a very basic terminal nitrogen atom as a result of resonance stabilization of the protonated guanidine group (log K ≈ 12) (Clarke & Martell, 1970).Under the experimental conditions used, the pH is ≈11 and, at this value, the degree of deprotonation of the guanidine group is insignificant.Therefore the proton was considered as unionisable under the conditions used and its protonation constant ignored.Both pentapeptides investigated contain three functional groups: the l-tyrosine phenolic, the N-terminal l-arginine α-ammonium and the C-terminal threonine carboxyl groups, which deprotonate over the pH range 3-11.The first protonation constant, log β HL = 9.76 or 9.82 for RYLPT and RYLΨ[CN 4 ]AT, respectively, refers to the protonation of the tyrosine OH group.This value is comparable with those of proctolin analogues (Kowalik-Jankowska et al., 2005) and other literature values (Kozłowski et al., 1989;Lodyga-Chruscinska et al., 1999;2000).The protonation constant of N-terminal l-arginine α-ammonium group is similar to that found in RYLPP and RYLPI (Kowalik-Jankowska et al., 2005).For the C-terminal carboxyl group, the acidity decreases in the tetrazole derivative compared to the parent peptide and its analogues.This may be related to the specific conformational changes induced by the tetrazole ring, which makes the deprotonation of C-terminal threonine carboxyl more favorable.The same tendency was also observed in the [Leu 5 ]enkephalin tetazole analogue (see Table 1).

Complex formation
The peptide Arg-Tyr-Leu-Pro-Thr The stability constants and spectroscopic parameters of the copper(II) complexes of RYLPT and RYLΨ [CN 4 ]AT ligands are collected in Tables 2 and 3.
The best fit for the data obtained from the experimental titration curves for the Cu 2+ -RYLPT  Bal et al. (1993).
From the species distribution diagram one can see that the first species is formed above pH 5 (Fig. 2).It is a minor species with metal coordination to the N-terminal amino and nearby carbonyl groups {NH 2 , CO}.This species usually shows up in the earlier steps of Cu(II) complexation by peptides (Sovago, 1990).The CuHL complex cannot be supported by UV-Vis or CD spectroscopy data due to its very low concentration but it was distinguished by a set of EPR parameters: A || = 166 × 10 -4 cm -1 , g || = 2.307 suggesting the {NH 2 , CO} coordination mode (Table 3) (Lodyga- Chruscinska et al., 1999;Pettit et al., 1990).The CuHL species of the peptide with the tyrosyl oxygen protonated corresponds to the CuL of AAAAA-NH 2 .The log K * values for a 1N {NH 2 , CO} complex of proctolin ligand and the complexes of RYLPP, RYLPI and AAAAA-NH 2 are comparable to one another supporting the same set of donor atoms involved in copper(II) binding (Table 2).The next complex species with the stoichiometry CuL is dominant about pH 6.Its formation is a result of the CuHL deprotonation according to the equilibrium reaction: CuHL  CuL + H + .The deprotonation constant pK = 4.71 (Table 2) indicates the deprotonation and coordination of the first amide nitrogen atom to Cu(II) ion (Lodyga- Chruscinska et al., 1998;Kozlowski et al., 1999).
The CuL complex loses the next amide proton with increasing pH.The result of this process is the formation of CuH -1 L as a major complex species at pH about 8 (Fig. 2).It has three nitrogen donor atom centers arranged in the equatorial plane of the tetragonal geometry around Cu(II) ion (Fig. 5a).
The hypsochromic shift of the d-d absorption maximum to 571 nm in UV-Vis and 580 nm in CD spectra and the EPR parameters: A || = 178 × 10 -4 cm -1 , g || = 2.209 clearly indicate additional amide nitrogen atom binding and the {NH 2 , N -, N -, CO} coordination (Table 3) (Kozlowski & Micera, 1995).An increase in the pH above 9 results in the formation of a CuH -2 L species (Fig. 2).This complex has a similar binding mode to that of CuH -1 L as the CD, absorption and EPR parameters indicate (Table 3, Fig. 4).The protonation constant value of the CuH -2 L complex (log β(CuH -1 L)log β(CuH -2 L) = 9.70, see Table 2) is in good agreement with that corresponding to the Tyr side chain in the free ligand (pK = 9.76).This indicates that in CuH -2 L the tyrosine OH group is deprotonated.The phenolic O -of the Tyr residue does not coordinate to the metal ion as was the case in C-terminally modified proctolin analogues (Kowalik-Jankowska et al., 2005).This is supported by the absence of the characteristic charge transfer band at 400 nm in the UV-Vis or CD spectra (Figs. 3 and 4) (Livera et al., 1988).The next species CuH -3 L was detected by potentiometry above pH 11.The spectral parameters estimated at this pH range indicate that CuH −3 L still has the 3N coordination with an additional deprot-onated water molecule as OH − in the fourth coordination position of the copper(II) ion (Table 3).The protonation constant of this species is 11.09 and corresponds to the deprotonation of the coordinated water molecule (Onindo et al., 1995).The slight splitting observed in the CD spectra (Fig. 4) may reflect some constraints within the peptide backbone due to intraligand interactions following the deprotonation of the water molecule.
The peptide Arg-Tyr-LeuΨ[CN 4 ]Ala-Thr The stability constants and the species distribution diagram obtained for the tetrazole proctolin analogue is reported in Table 2 and Fig. 2. In the system involving Arg-Tyr-LeuΨ[CN 4 ]Ala-Thr, the fit of the titration curves indicates the same species as were observed for the proctolin: CuHL, CuL, CuH -1 L, CuH - 2 L and CuH -3 L (Fig. 2).The complex CuHL, with the usual 1N coordination mode, exists at a pH of about 5. It appears only as a minor species and therefore it has been not detected by UV-Vis, CD or EPR spectroscopy.However, with increasing pH, it evolves into the CuL complex which is the predominant one at a pH of about 6.The absorption band of the d-d transition at 685 nm and the CD parameters (Fig. 6, Table 3) clearly indicate the involvement of 2N coordination with the {NH 2 , N -, CO} bonding mode (Kozlowski & Micera, 1995).The EPR parameters show an increase of the hyperfine splitting constant (A || = 184 × 10 -4 cm -1 ) in comparison to those of RYLPT, RYLPP or RYLPI that may suggest a more stable tetragonal geometry around the copper(II) ion.Some stabilizing effect is also seen in the increase of log K * value in comparison to that of proctolin and its analogues (Table 2).At pH 8 the CuH -1 L complex was detected.It reveals a species with completely different coordination mode of copper(II) ion than that occurring in the parent peptide or its analogues (Fig. 5b).The deprotonation constant of the CuL complex (pK value for CuL  CuH -1 L + H + ) equals 6.69 and corresponds very well to the deprotonation and coordination of the third amide nitrogen to copper(II) ion (Kozlowski et al., 1999).The ligand with the phenolate group protonated coordinates the metal via an equatorial donor atom set of the {NH 2 , N -, N -, N tetr } type.The coordination mode is supported by spectroscopic parameters (Table 3) (Lodyga-Chruscinska et al., 1999).The value of A || = 200 × 10 -4 cm -1 is comparable to that characteristic for the 4N species formed in the other oligopeptide-Cu(II) ion systems (Kozlowski & Micera, 1995).The band of the d-d transition energy seen in UV-Vis or CD spectra of the CuH -1 L complex is slightly above that typical of the 4N complexes of simple peptides (about 530 nm) but this is due to the different nature of the fourth coordinated nitrogen atom, since it is expected that different parameters should be obtained for different nitrogen donor atoms.The log K * value for the CuH -1 L complex is 0.76, 1.07 and 1.24 orders of magnitude higher in comparison to those of RYLPT, RYLPP and RYLPI, respectively.Some gain in stability can arise from the replacement of the oxygen atom in the copper(II) coordination sphere by the nitrogen atom of tetrazole moiety (see Fig. 5) which exhibits a higher bonding affinity for Cu(II) ion than that of oxygen.This finding could be also attributed to the conformation of the peptide tetrazole analogue that is well suited for metal ion coordination by deprotonated amide nitrogens and the tetrazole nitrogen.The formation of the next complex CuH -2 L at a pH of about 10 is a result of the Tyr side chain deprotonation.The spectroscopic parameters are almost the same as for the CuH -1 L species supporting the {NH 2 , N -, N -, N tetr } coordination mode (Table 3).The last CuH -3 L complex species detected by potentiometric titrations is dominant above pH 11.The presence of the N -® Cu(II) charge transfer band at 304 nm in the CD spectrum (Fig. 6, Table 3) supports an involvement of the amide nitrogens in the metal coordination.However, the red shift of the d-d transition band and the decrease in the EPR parameters indicate the presence of {NH 2 , N -, N -, OH -} coordination (Table 3).In this complex the weakly basic nitrogen donor atom of the tetrazole moiety is substituted by the OH -group of a deprotonated water molecule (pK = 10.57)(Lodyga- Chruscinska et al., 1999).This coordination mode may lead to a distinct decrease in the geometric symmetry around the metal ion, which results in the splitting of the d-d band observed in the CD spectrum (Fig. 6).

CONCLUSIONS
The insertion of a tetrazole ring into the peptide sequence changes the coordination towards Cu(II) ion.Its insertion after the third amino-acid residue leads to the involvement of the tetrazole nitrogen in the metal binding as the fourth donor atom and a gain in the stabilization of the 4N complex species.The {NH 2 , N -, N -, N tetr } coordination mode makes the tetrazole derivative a more effective chelating ligand than proctolin and its C-terminally modified analogues.The enhancement of the bind-   The results indicate that Cu(II) could hold the peptide chains in a biologically active bent conformation.The extra stability may be attributed to a metal ion-induced conformational organization of the peptide molecule, involving a β-turn, depending on the nature of the amino-acid residues.This bent conformation achieved may be essential for binding of the tetrazole peptides at the receptor site.

a
Kowalik-Jankowska et al. (2005); b Lodyga-Chruscinska et al. (1999); c Bal et al. (1993); d log K * = log β(CuH j L) -log β(H n L) (where the index j corresponds to the number of the protons in the coordinated ligand to metal ion and n corresponds to the number of protons coordinated to ligand).

Figure 1 .
Figure 1.Structure of the tetrazole moiety (left), a mimic of a cis-peptide bond (right).Asterisks mark chiral centers.

Figure 3 .
Figure 3. Electronic absorption spectra of Cu(II)-RYLPT system as a function of pH at 1 mM Cu(II) concentration and metal to ligand ratio 1:1.

Figure 4 .
Figure 4. Circular dichroism spectra of Cu(II)-RYLPT system as a function of pH at 1 mM Cu(II) concentration and metal to ligand ratio 1:1.

Figure 5 .
Figure 5. Arrangement of donor atoms identified on the basis of spectroscopic data in the CuH -1 L complex of RYLPT (a) and RYLΨ[CN 4 ]AT (b).

Figure. 6 .
Figure. 6. Circular dichroism spectra of Cu(II)-RYLΨ[CN 4 ]AT system as a function of pH at 1 mM Cu(II) concentration and metal to ligand ratio 1:1.

Figure 7 .
Figure 7. Competition diagram for proctolin (solid line) and its tetrazole analogue (dashed line) in binding of Cu(II) ions as a function of pH for 1:1:1 (ligand/metal/ligand) molar ratio and metal concentration 1 mM.