DALTON FULL PAPER J. Chem. Soc., Dalton Trans., 1998, 3587–3600 3587 Oxovanadium(IV) complexes of the dipeptides glycyl-L-aspartic acid, L-aspartylglycine and related ligands; a spectroscopic and potentiometric study João Costa Pessoa,*a Tamás Gajda,b Robert D. Gillard,*c Tamás Kiss,*b Susana M. Luz,a José J. G. Moura,d Isabel Tomaz,a João P. Telo a and Ibolya Török e a Instituto Superior Técnico, Departamento de Engenharia Química, Av. Rovisco Pais, 1096 Lisboa, Portugal. E-mail: pcjpessoa@alfa.ist.utl.pt b Attila József University, Department of Inorganic and Analytical Chemistry, H-6701 Szeged, POB 440, Hungary c Department of Chemistry, University of Wales, POB 912, Cardiff, UK CF1 3TB d Centro de Química Fina, Universidade Nova de Lisboa, Quinta da Torre, 2825 Monte da Caparica, Portugal e Research Group for Biocoordination Chemistry of the Hungarian Academy of Sciences, Attila József University, Department of Inorganic and Analytical Chemistry, H-6701 Szeged, POB 440, Hungary Received 9th March 1998, Accepted 4th September 1998 The equilibria in the systems VO21 1 L (L = Gly-L-Asp, L-Asp-Gly, N-acetyl-L-aspartic acid or succinic acid) have been studied at 25 8C and 0.2 mol dm3 K(Cl) medium by a combination of potentiometric and spectroscopic methods (ESR, circular dichroism and visible absorption). Formation constants were calculated from pH-metric data with total oxovanadium(IV) concentrations of (0.6–4) × 1023 mol dm23 and ligand-to-metal (L : M) ratios of 2–8 (AspGly) or 4–15 : 1 (other systems).The position of the Asp residue in the peptide chain aVects the co-ordination mode of the ligands: while in the GlyAsp system bis complexes start to form at pH less than 2, for AspGly only 1 : 1 complexes form, with relatively high CD signal. The co-ordination behaviour of N-acetyl-L-aspartic and succinic acids is similar. The results of potentiometric and spectroscopic methods are self consistent.Isomeric structures are discussed for each stoichiometry proposed and the results compared with those for L-aspartic acid and dipeptides with non-coordinating side chains. Introduction To model potential binding sites for the oxovanadium(IV) cation, complexation by several a-amino acids and simple peptides has been investigated.1–21 For dipeptides containing Gly and/or Ala, at VO21 concentrations of ª1022 mol dm23, the hydroxide precipitates at pH ª 4–5, even when using high amino acid : metal (L :M) ratios, e.g. 150–180: 1, and at pH > 7.5–8 the hydroxide slowly dissolves to give brown solutions, indicating that oxovanadium(IV) is extensively hydrolysed. The present study of the systems VO21 1 L (L = Gly-L-Asp, L-Asp-Gly, N-acetyl-L-aspartic acid and succinic acid) combines the results of potentiometric and spectroscopic techniques (ESR, circular dichroism and visible absorption). These ligands, as compared with simple oligopeptides, are expected to be more eYcient VO21 binders, due to their extra carboxylate group. Part of this work has been presented in a preliminary form.22 Precipitation of VO(OH)2 is clearly not so important in these systems, as was also the case for L-aspartic acid,5 and much lower L :M ratios may be used.If lower oxovanadium(IV) concentrations are used e.g. ª (1–4) × 1023 mol dm23, results of pH-potentiometric titrations with L :M ratios of 1 to 15 : 1 up to pH ª 5.0–6.5 (depending on the ligand) may be used to calculate formation constants.Potentiometry must be ruled out for pH > ª8; pKa(NH3 1) ª 8.4 and 8.0 for Gly-L-Asp and L-Asp- Gly, respectively, limiting the use of samples with high L :M ratios in the pH range 8–9, and oxovanadium(IV) is extensively hydrolysed, particularly at pH > 10. Further, the very high absorbance values (especially for 450 < l < 650 nm) of the [(VO)n(OH)m] species present also preclude the use of visible spectra. Minor oxidation of VIV may also aVect spectral measurements. The ESR spectra may give important information about the groups co-ordinated to oxovanadium(IV).23–26 The CD and ESR spectra for solutions containing L-Ala-Gly, Gly-L-Ala and L-Ala-L-Ala with high L :M ratios gave clear evidence of peptidic Namide co-ordination,16 suggesting the formation of 1 (Y = H2O or OH2) as the important species contributing to these spectra in the pH range 7.5–8.5.The crystal-structure characterisation of [NEt4][VVO(O2)(Gly-Gly)]?1.58 H2O,27 of [VIVO(Gly-Tyr)(phen)] 28 [ Gly-Tyr = glycyl-L-tyrosinate(22), phen = 1,10-phenanthroline] and of [NHEt3][VIVO(mpg)- (phen)] 29 [H3mpg = N-(2-sulfanylpropionyl)glycine], where NH2, Namide and CO2 2 are equatorial, and the isolation of [VIVO(Gly-Gly)(phen)]?2CH3OH and [VIVO(Gly-Ala)(phen)]? CH3OH,29 and their characterisation by continuous wave ESR and 14N electron spin echo envelope modulation also indicate that the dipeptides Gly-Gly and Gly-Ala are bonded as in 1.Circular dichroism (CD) spectra are more informative than the corresponding isotropic absorption spectra 30–33 for systems containing optically active amino acids and peptides. Only the vanadium–peptide complexes contribute to DA values (DA = diVerential absorption) in CD spectra, and the sign patterns N V Y NH2 O Y O C O COO V W W W O R 1 23588 J. Chem. Soc., Dalton Trans., 1998, 3587–3600 of Cotton eVects can be compared for bands I, II (and III) of the diVerent oxovanadium(IV) peptide and/or amino acid systems 23 as pH is varied.Since three pKa values (terminal CO2H, b-CO2H of the aspartic side chain and terminal NH3 1) can be determined for these dipeptides (H3L1), and two for the carboxylic groups of N-acetyl-L-aspartic acid and succinic acid (H2L9), the formation constants correspond to the general reaction (1). pM21 1 qL22 1 rH1 MpLqHr (1) We abbreviate Gly-L-Asp as GlyAsp, L-Asp-Gly as AspGly and N-acetyl-L-aspartic acid as NacAsp.For the formulations (VO)p(ligand)qHr 2p 2 2q 1 r we normally use the abbreviation MpLqHr in the case of GlyAsp, AspGly and L-Asp, and MpL9qHr in the case of NacAsp, and other H2L9 ligands (e.g. alanine, succinic acid). For each MpLqHr or MpL9qHr proposed in our speciation models possible structures are discussed. Basicity corrected formation constants are used when needed to help in this discussion as in previous publications.4,5,7,9 Experimental All solutions were prepared and manipulated in an inert atmosphere (high purity dinitrogen or purified argon).Amino acids (from Sigma) were dried for several days (in a desiccator with silica gel in vacuo), and succinic acid was a Fluka product of puriss p.a. quality for pH-metric studies and Merck p.a. (GR) for spectroscopic measurements. The KOH and HCl solutions used were Reanal products of the highest purity. KOH was standardized with potassium hydrogen phthalate and HCl with KOH potentiometrically.The purity of peptides was checked as described below and the exact concentrations of solutions were determined by the Gran method.34 The experimental diYculties in the present systems were discussed in refs. 1 and 35. Stock solutions of VO21 were prepared and standardised as described in ref. 1. The studies have been performed in 0.2 mol dm23 K(Cl) ionic medium. For potentiometric measurements the temperature was 25.0 ± 0.1 8C, and for CD and VIS spectra 25.0 ± 0.3 8C.TLC Thin-layer chromatography was performed on Merck plates (Art. 5626, 10 × 20 cm). The compounds and solutions used for spectroscopic measurements were monitored throughout the whole pH range to check their purity and test for reactions (e.g. hydrolysis): neither contamination nor decomposition was detected. Usually 2 ml samples were applied to the plates and the eluent was butanol–ethanol–propionic acid–water (10:10:2:5). The chromatogram was developed with a ninhydrin–collidine (2,4,6-trimethylpyridine)–copper solution prepared according to MoVat and Lytle.36 In some cases, after such development, the plate was placed in an enclosed chamber for development with iodine vapour.Typically, samples were taken for TLC after dissolution of the ligand, and addition of VO21 at several pH values. pH Measurements Spectroscopic measurements. For preparation of the solutions and pH calibrations we used a special glass vessel with a double wall, with entries for the glass electrode (Orion Ross 81-01) and reference electrode (Orion Ross 80-05), thermometer, nitrogen and reagents (e.g.base). A computerised system developed locally (for an IBM-PCXT 286 computer) was used to control the titration conditions for pH calibrations. The emf measurements were made with a Crison 517 pH meter. Potentiometric titrations. Stability constants were determined by pH-metric titration of 10.0 cm3 samples. The ligand concentration was 0.004 and 0.008 mol dm23 and ligand-to metal ion molar ratios: 2 : 1, 4 : 1, 6 : 1 and 8 : 1 (AspGly), and 2 : 1, 4 : 1, 6 : 1, 10 : 1 and 15 : 1 (other ligands).Titrations were performed from pH 2.0 until precipitation, very extensive hydrolysis or slow equilibration: these problems occurred in the pH range 5.0–6.7, depending on ligand and L :M ratio (see Table 1). Titrations were with KOH solution of known concentration (ca. 0.2 mol dm23) under a purified argon atmosphere.In some cases pH equilibrium could not be reached within 10 min due either to precipitation or very slow complex formation. Those titration points were omitted. The reproducibility of the included points was within 0.005 pH unit over the whole pH range. The pH was measured with an Orion 710A precision digital pH meter equipped with an Orion Ross 8103BN type combined glass electrode, calibrated for hydrogen ion concentration as described earlier.37 The ionic product of water was pKw = 13.76.The concentration stability constants bpqr = [MpLqHr]/[M]p[L]q[H]r were calculated with the aid of the PSEQUAD computer program.38 The formation of the hydroxo complexes of VO21 was taken into account. The following species were assumed: [VO(OH)]1 (log b1–1 = –5.94), [{VO- (OH)}2]21 (log b2–2 = 26.95), with stability constants calculated from the data of Henry et al. 39 and corrected for the diVerent ionic strength using the Davis equation. Spectroscopic measurements The CD spectra were recorded with a JASCO 720 spectropolarimeter with a red-sensitive photomultiplier (EXWL-308), visible spectra with a Perkin-Elmer lambda 9 spectrophotometer. Unless otherwise stated, by visible (VIS) and circular dichroism (CD) spectra we mean a representation of em or Dem values vs.l [em = absorption/bCVO and Dem = diVerential absorption/bCVO where b = optical path and CVO = total oxovanadium( IV) concentration]. The spectral range covered was usually 400–900 (VIS) and 400–1000 nm (CD).The ESR spectra were usually recorded at 77 K with a Bruker ESR-ER 200D X-band spectrometer. The CD, VIS and ESR spectra for GlyAsp, NacAsp and succinic acid systems were recorded by varying the pH with approximately fixed total vanadium and ligand concentration, at L :M ratios of 15 and 30; for AspGly this was done for solutions with L :M = 9.8 : 1. Several CD and ESR spectra were also recorded at fixed pH at varying L :M ratios by addition of VO21 stock solution.the GlyAsp solutions at pH 6.1 (L :M = 35.0, 24.7, 14.5, 9.4 : 1) as well as at 7.5 (35.0, 15.0, 9.4 : 1), 7.3 (20.1 :1), and AspGly solutions at pH 4.9 (L:M = 10, 7.1, 6.3, 5.6, 4.6 : 1) as well as at 6.8 (L :M = 7.1:1) and 2.6 (L :M = 4.6 : 1) were used for these measurements. Results and discussion Protonation and formation constants calculated for the systems studied 38 are in Table 1. The protonation constants agree well with earlier results: 40–43 the presence of the N-acetyl group in NacAsp increases the acidity of the CO2H groups relative to succinic acid.These ligands all contain two carboxylate binding sites and VO21 has a strong aYnity for oxygen containing ligands,23 so their complexes may diVer from those of simple dipeptides such as GlyGly, AlaGly, GlyAla or AlaAla.16 Further, relatively low L:M ratios (e.g. 10 : 1) may be adequate to avoid precipitation of VO(OH)2 in the case of GlyAsp and AspGly.Complexation starts through monodentate carboxylate co-ordination (as in 2): MLH2 for GlyAsp and AspGly (one proton belongs to the terminal NH3 1, the other to the non-co-ordinated CO2H group) and ML9H for NacAsp and succinic acid (the proton belongs to the non-co-ordinated CO2H group). With high excesses of ligand, the bis complexes M(LH2)2 or M(L9H)2 may also be formed. However, their formation can hardly be detected by pH-metry due to the overlap between the pro-J.Chem. Soc., Dalton Trans., 1998, 3587–3600 3589 Table 1 Formation constants (log values a) of species formed in VO21–ligand systems at T = 298 K and I = 0.2 mol dm23 KCl MpLqHr HL2 H2L H3L1 pKa1 pKa2 pKa3 MLH2 21 MLH1b ML MLH21 2 ML2H3 1b ML2H2 ML2H2 pH range studied L:VO ratio GlyAsp 8.36(1) 12.58(2) 15.24(3) 2.66 4.22 8.36 15.1(5) 11.52(8) 1.7(1) 26.6(2) 22.5(3) 17.1(2) 2.0–5.5 4–15 AspGly 7.93(1) 11.50(2) 14.32(3) 2.82 3.57 7.93 13.4(2) 10.46(3) 6.42(2) 0.83(4) 2.0–5.5 2–8 MpL9qHr HL92 H2L9 pKa1 pKa2 ML9H1 ML9 b ML9H22 22 ML92H2b ML92 22 NacAsp 4.52(2) 7.61(5) 3.09 4.52 6.2(2) 2.7(2) 27.32(5) 9.56(6) 5.0(3) 2.0–6.2 4–15 Succinic acid 5.19(1) 9.17(3) 3.98 5.19 7.2(2) 3.20(4) 27.25(3) 10.76(4) 5.6(1) 2.0–6.7 4–15 a Formation constants correspond to bpqr = [MpLqHr]/[M]p[L]q[H]r where L is the ligand in its deprotonated form (L22).Oxovanadium(IV) hydrolysis products are VO(OH)1 and [(VO)2(OH)2]21 with log b10 2 1 = 25.94 and log b20 2 2 = 26.95.The numbers in parentheses apply to the last digit included; it defines the range of log b values in refinements with PSEQUAD for the several plausible models obtained. b Formation constants for stoichiometries MLH (or ML9) and ML2H3 (or ML92H) could not be refined simultaneously (see text). cesses of co-ordination and deprotonation of the carboxylate group and to the high buVering eVect of large excesses of ligand. Accordingly, most of the resultant pH change belongs to ligand deprotonations and only a relatively small part to VO21 complexation.It is also worth mentioning that b111 and b123 could not be refined simultaneously; if both were included in the same calculation; one was always rejected, even if only data obtained at high excesses of ligand were included in the calculation. Probably ML2H3 does not exist in significant concentration at low L:M and the same applies to MLH at high L :M. For the corresponding stoichiometries ML9 and ML29H for the NacAsp and succinic acid systems, it was similarly not possible to refine b110 and b121 simultaneously.This is because, in the pH range 3–4.5 in which bis complexes are mostly formed, the ligand has already lost a proton from its more acidic carboxylic function, hence the complex formation involves no H1, eqns. (2A) and (2B). Under the conditions used for pHVOLH1 1 H2L VOL2H3 1 (2A) VOL9 1 HL92 VOL92H2 (2B) potentiometry (except for AspGly) the formation of 2 : 1 species is assumed above pH ª 2–3; when using high L:M, no polymeric complexes are expected till pH ª5.Fig. 1 gives calculated 44 distributions of concentration. The X-band ESR spectra of frozen ’solutions’ may be simulated as axial spectra. The field region corresponding to A|| and MI = 5/2 and 7/2 gives more information about the type and number of species. Fig. 2 shows ESR spectra in this range. When the pH is increased from ª1 to ª3.2 (AspGly), to ª3.5 (GlyAsp), to ª5.0 (NacAsp) or to ª 6.5 (succinic acid, not seen), the peaks shift slightly to lower field, so more than one species contribute to each spectrum.For higher pH, distinct species are detected, but for pH > 6–7 (for NacAsp and succinic acid) or > 8 (for GlyAsp or AspGly) the signal weakens significantly, increasing again at pH > 12, due to the formation of VO(OH)3 2. For VO21 and succinic acid with L :M = 30 : 1 and CVO ª 0.008 mol dm23, a new component appears from pH > ª6.7; it may correspond to ML9H23 (or indeed another stoichiometry, e.g.ML92H22/23). At pH 7.3 the components ascribed to ML9H22 and ML9H23 have relative intensities approximately 3 : 2. At pH 7.9 most VO21 is precipitated and the ESR signal is weak. Table 2 summarizes the spin Hamiltonian parameters obtained by simulating the whole spectrum using program EPRPOW,45 ascribing the ESR-active components to specific stoichiometries. Superscripts ’exptl’ and ’est’ refer to ’experimental’ and estimated parameters [eqn.(3)] where A||,i A|| est = S 4 i = 1 A||,i/4 (3) are the contributions to A|| est of each of the four equatorial groups (most presented by Chasteen 24 with estimated accuracy ±3 × 1024 cm21). Fig. 3 includes visible spectra for (A) GlyAsp 1 VO21 with L:M ratio 30 : 1 and (B) AspGly 1 VO21 with L:M ratio 9.8 :1. For pH < 2.5, they resemble (except for em values) oxovanadium(IV) solutions. Visible spectra for NacAsp or succinic acid 1 VO21 are similar and show the same trend as VIVO–GlyAsp.For pH > 2.5, as pH increases, band II gradually separates from band I revealing a progressive increase of the ligand field around VO21. However, while spectra for solutions of GlyAsp, NacAsp or succinic acid 1 VO21 are similar and their changes are like those found for dipeptides with non-coordinating side chains, the spectra for AspGly 1 VO21 diVer: band II shifts ª30–40 nm to the UV and its em values are about the same as those for band I.This means that the type of complexes formed and their co-ordination geometries diVer in the system with AspGly. In the pH range 1–4.5 the VIS spectra for solutions of AspGly resemble those for L-Asp.5 The CD spectra for the GlyAsp, AspGly and NacAsp systems modify as the pH is increased. In the pH range 1.5–5, all diVer from those for L-Asp.5 Fig. 4 includes spectra for (A) GlyAsp 1 VO21 with L :M 15 : 1 and (B) AspGly 1 VO21 with L:M 9.8. In the pH range 1–4, CD spectra of NacAsp 1 VO21 with L :M 30 : 1 are similar and show the same trend as those of GlyAsp: maximum values of Dem for band I are found in the pH range 4.1–4.6.The decrease in intensity of this band for pH > 4.8 is apparently due to the formation of ML92 and is more significant when ML9H22 forms [Fig. (1C)]. For pH > 4.5 the CD spectra for GlyAsp, AspGly and NacAsp diVer (Fig. 5) and also modify as pH is increased. For pH > 10 optical activity is low and becomes almost zero for pH > 11. The pattern of the CD bands and approximate lmax values ascribed to each stoichiometry and comparison with species distribution is in Table 2.The CD (e.g. Fig. 5) and ESR (Fig. 2) spectra of VO21 1 GlyAsp in the pH range 8–9.5 have the same profile and very similar spin-Hamiltonian parameters as those for simple dipeptides such as GlyAla, AlaAla, etc.16 As with them, this GlyAsp complex probably involves Namide equatorial co-ordination.While for GlyAsp with L:M 30 : 1,3590 J. Chem. Soc., Dalton Trans., 1998, 3587–3600 Fig. 1 Concentration distribution of the complexes formed in the (A) GlyAsp–, (B) AspGly–, (C) NacAsp– and (D) succinic acid–VO21 systems in solutions with CVO = 0.008 mol dm23 and L :M = 10 (AspGly) or 30 : 1 (other systems), calculated44 using bpqr of the equilibrium models of Table 1. The dashed lines indicate that the curves only represent an approximate estimation of the relative concentrations. The possible relative proportion of [(VO)m(OH)n] hydrolysis products is estimated assuming the formation of M2H25 with b2–5 = 10221.8.1,23 For MLH22 (GlyAsp) and ML9H23 (succinic acid) no b values are available, and concentrations were roughly estimated from the ESR spectra.maximum |Dem| values are for pH ª9.5, for AlaAla and GlyAla these are at pH ª7.5. The CD and ESR spectra have also been recorded at fixed pH for varying but high L :M ratios. The profile for the grey solution containing GlyAsp 1 VO21 at pH 6.1 with L :M = 35:1 and CVO = 6.0 × 1023 mol dm23 is very similar to spectrum 2 in Fig. 5(A), but the |Dem| are ª1.5 times larger. The corresponding ESR spectrum is like that in Fig. 2(A) (pH 5.97). On adding VO21 stock solution till L :M = 9.4 : 1 the solution becomes greenish (L :M = 24.7 : 1) and dark green (L :M = 14.5 or 9.4 : 1); the CD and ESR spectral profiles remain the same but the ESR signal increases and values of |Dem| decrease: at L :M = 9.4 :1 to ª20% of those for L:M = 35 : 1. These significant changes in Dem as CVO is varied can be explained only by equilibria between species with diVerent degrees of polymerisation.At pH 6.1 these could be as in Scheme 1. As long as the L :M ratios are high (e.g. > 12 : 1) the relative concentrations of monomeric complexes vary little with CVO but the % of vanadium in the form of monomeric vs. oligomeric species varies significantly. Since the pattern of the CD spectra changes little with CVO (and ill defined isodichroic points are observed at l ª 560 and 790 nm with Dem ª 0), the oligomeric species present at pH 6.1 must be optically inactive.Other Scheme 1 Equilibria and main species involved at pH ª 6.1 in the GlyAsp system. amino acid systems behave similarly.1–6 In similar experiments at pH 7.5, when extra VO21 was added to a sample of L :M = 35:1 (light yellowish brown) to change L :M to ª14.5 : 1 (pH 7.50, dark green) the CD profile changed, in particular, the Cotton eVect associated with the band at 700 nm became negative and the band at ca. 800 nm shifted to the red. Overall |Dem| decrease to ª30% of those for L :M = 35 : 1. On addition of more VO21 till L :M = 9.4 : 1 (pH 7.50, very dark green), the CD spectrum becomes very noisy but now the profile apparently changes little. The ESR profile for these experiments at pH 7.5 is always the same: the dominant components are those designated by MLH21 and/or MLH22. Therefore at this pH the changes in CD profile and Dem with CVO can be explained only by assuming equilibria between monomeric complexes (MLH21 and MLH22), optically and ESR active, and oligomeric species (e.g.M2L2H23, M2L2H24) which are optically active but ESR-inactive. Optically inactive oligomeric species, e.g. [(VO)n- (OH)m], probably also have significant concentrations at pH 7.5. Scheme 2 summarizes the processes expected to occur as pH is increased. For GlyAsp 1 VO21 the distribution of Fig. 1(A) describes the ESR spectra of Fig. 2(A) reasonably well till at least pH ª6. Scheme 2 Overall description of processes as pH is increased in the range 6.5–13 for the GlyAsp system. The optically active oligomeric species is expected to form in the pH range 6.5–10.J. Chem. Soc., Dalton Trans., 1998, 3587–3600 3591 Fig. 2 High field range (3800–4400 G) of the first derivative ESR spectra at 77 K of frozen “solutions” containing (A) GlyAsp and VO21 with L:M = 30.0 : 1 and CVO ª 0.008–0.011 mol dm23, (B) AspGly and VO21 with L :M = 9.8 : 1 and CVO ª 0.006–0.010 mol dm23 and (C) NacAsp and VO21 with L :M = 30.0 : 1 and CVO ª 0.008–0.014 mol dm23. pH Values, colours of the corresponding solutions and stoichiometries corresponding to the ESR components are indicated.For pH > 6–6.5 at least two diVerent ESR-inactive oligomers form, as well as an ESR and CD active complex: possibly MLH22. Some results for it are in Table 2.No value of b is available and its concentration in Fig. 1(A) was roughly estimated from the ESR results. For AspGly 1 VO21, till at least pH 6 the distribution of Fig. 1(B) also describes reasonably well the relative intensities of the ESR-active species in Fig. 2(B). The CD signal is weak and noisy under conditions corresponding to these ESR spectra for pH < 2.7. At pH 1.2 VO(OH2)5 21 predominates and for pH < 1.7 the dominant optically active species is MLH2, with negative De, as for amino acid complexes containing a mono-co-ordinated carboxylate function.1–8,16 Since the CO2 2 group is now from an achiral glycine residue the CD signal is very weak.The structure of this complex probably corresponds to 3 (Table 3) so the vicinal eVect is transmitted mainly through the axial (NH)C]] Oamide, not as eYcient as an equatorial CO2 2 group.16 For pH > ª2.0 Dem becomes positive due to the formation of MLH. The CD profile in the pH range 2.7–4.1 is approximately the same so the relevant spatial factors that determine the CD signal are similar for MLH and ML.Although the VIS spectra of solutions containing AspGly and VO21 for pH > 3 suggest increased ligand field strength, no such increase occurs for GlyAsp or NacAsp. This suggests that in ML the NH2 group is also co-ordinated (e.g. 12 and 13 in Table 3). For AspGly and VO21 with L:M = 9.8 :1, at pH 6 [Fig. 5(B)], the overall CD signals are intense with the band pattern: 1,1,2 for bands II, IB, IA and: Dem band II ª 2Dem band IB @ Dem band IA.As pH is increased in the range 6–8 the pattern changes to 1,1,1, bands II and IA now being about equal. These modifications indicate that besides MLH21 a new optically active species forms. In the same pH range the ESR signal decreases: in the range 8–10.5 oligomeric ESR inactive species {e.g. [(VO)n(OH)m]} form, causing further decrease. The spin-Hamiltonian parameters for the ESR-active species correspond to MLH21 (Table 2).For AspGly 1 CuII, bis complexes formed, presumably involving amino acid-like co-ordination.41 Our pH-metric results (till L :M = 8 : 1) suggest no formation of bis complexes till at least pH 5.2. The CD decreases for pH > 5 which indicates no significant formation of bis complexes at higher pH. Therefore, the ESR and CD active complex that forms for pH > 5–6 probably corresponds to a stoichiometry MLH22. For NacAsp 1 VO21 it is not straightforward to compare the distribution of Fig. 1(C) with the ESR [Fig. 2(C)] because ML9H, ML92H and ML92 cannot be detected separately. However, the agreement is reasonable: e.g. at pH 5.78 the distribution predicts ª55% of ML92 and ª37% of ML9H22, and this is in good accord with the intensities of the ESR components. The CD and VIS profiles for the solutions containing NacAsp corresponding to the ESR spectra of Fig. 2(C) at pH < 3.5 are very similar to those for GlyAsp under similar conditions.The main contributors to CD are ML9H and ML92H. These correspond to stoichiometries MLH2 and ML2H3 in the GlyAsp system. So, dominant factors that contribute to the optical activity must be similar for the corresponding complexes. For pH > 4 the CD spectra for GlyAsp and NacAsp diVer. For NacAsp 1 VO21 [Fig. 5(C)] the pattern of the CD is the same in the pH3592 J. Chem. Soc., Dalton Trans., 1998, 3587–3600 Table 2 Hyperfine coupling constants, g values, lmax of CD spectra (and corresponding signal) or of VIS spectra (and corresponding e value for succinic acid) for several stoichiometries of each of the systems studied.Most A and g values were obtained by simulation with the program EPRPOW45 of EPR spectra of frozen “solutions” at 77 K lmax (CD signals or e) a Stoichiometry A|| exptl × 104 g|| exptl A^ exptl × 104 g^ exptl band II band I b GlyAsp MLH2 21c ML2H3 1 ML2H2 c ML2H2c MLH21 2 MLH22 22c (?) ª177 174.0 ª172 ª171 160.7 162.5 ª1.936 1.938 ª1.940 ª1.942 1.953 1.951 ª65.0 63.0 ª61 ª60 53.5 53.7 ª1.978 1.977 ª1.975 ª1.977 1.981 1.985 590 (2) 600 (2) 550 (2) ?? (2) ª510 (2) 510 (2) 510 (2) 760 (2) 820 (2) 820 (2) ??? (1), ??? (2) d 730 (1) ? 700 (1),e 850 (2) e ? 730 (1) AspGly M21f MLH2 21c MLH1c ML MLH21 2 MLH22 22c (?) 181.2 ª177.0 173.5 169.8 165.0 163.5 1.934 ª1.937 1.939 1.944 1.950 1.949 68.2 ª65–66 62.8 60.8 57.5 54.0 1.978 ª1.977 1.977 1.977 1.979 1.980 ??? (2) 575 (1) 565 (1) 550 (1) 525 (1) ??? (2) d 750 (1) 730 (1) 700 (1), 860 (2) 735 (+). ª880 (1) NacAsp M21g ML9H1c ML92H2 ML92 22c ML9H22 22c (?) MH23 2h 181.5 ª177 173.5 ª172 ª168 162 1.934 ª1.937 1.938 ª1.940 ª1.945 1.955 68.8 ª65.2 62.4 ª61.5 ª60.0 49.5 1.978 ª1.977 1.977 ª1.978 ª1.977 1.977 610 (2) 600 (2) 575 (1) 570 (1) 770 (2) 810 (2) 813 (2) 810 (2) Succinic acid i M 1 ML9H1 (pH 2.2) M 1 ML9H1 1 ML29H2 pH 3.0 ML29H2 ML92 22 ML9H22 22 ML9H23 32 (?) 179 178 174 172 172 171 ª165 1.934 1.938 1.940 1.942 1.942 1.942 ª1.944 69 67 63 61 61 61 ª62 1.978 1.980 1.981 pH 3.80 1.980 pH 5.75 ª1.980 pH 6.32 j ª1.980 pH 6.95 j ª1.982 ?? d ?? d ª625 (ª12) 605 (18.5) 565 (21) 565 (113 k) 768 (19.0) 773 (22.6) 780 (26.1) 800 (32.5) 830 (23.0) 830 (64 k) a Based on qualitative analysis of experimental CD spectra and distribution diagrams.For succinic acid the lmax/nm and em/dm3 mol21 cm21 presented are those for VIS spectra at pH values where each stoichiometry is expected to be largely predominating.b When splitting of band I is clearly observed, two lmax values are given: bands IB and IA. c The ESR spectrum was diYcult to simulate due to noise or presence of a significant amount of more than one species. d The lmax cannot be estimated satisfactorily. e This species could have this pattern or the same as for MLH22 (see text). f From ESR spectra of solutions containing AspGly and VO21 (L:M = 9.8 and CVO ª 0.010 mol dm23) at pH 1.0 and 1.2.g From ESR spectra of solutions containing NacAsp 1 VO21 (L:M = 30 and CVO ª 0.010 mol dm23) at pH 0.6 and 1.2. h From the ESR spectra of a solution containing NacAsp and VO21 at pH 12.9 (L :M = 30 and CVO ª 0.008 mol dm23). i Each spectrum seems to correspond to only one component, but all correspond to at least two species. Only parameters for ML92H, ML92, ML9H22 can be determined with reasonable accuracy. j The lmax and e for ML9H22 are only rough approximations as at pH 6.32 the contribution of oligomeric species e.g.[(VO)n(OH)m] is significant. k The em of solutions containing succinic acid and VO21 (L:M = 30 and CVO ª 0.008 mol dm23) at pH ª 7 decreases continuously from 350 (e ª 606) to 900 nm (em ª 61 dm3 mol21 cm21). Here em values are given at 565 and 830 nm, the lmax observed at pH 6.32. range 4.2–6.3, with lmax 550–580 (band II) and 805–815 nm (band I), see Table 2. The value of |Dem| increases till pH ª4.7 (band I) or 5.8 (band II).At pH > 3.5, for NacAsp Dem > 0 in the region of band II, but negative till pH ª6.7 for GlyAsp. Formation constants could be calculated from titrations up to pH 6.2 (Table 1) but the ESR of Fig. 2(C) and the CD of Fig. 5(C) reflect no new ESR or CD active complexes till pH ª 9.3. However, intensities decrease markedly for pH > 7 indicating the formation of [(VO)n(OH)m] inactive in ESR and CD. So Fig. 1(C) includes [{(VO)2(OH)5 2}m] with m = 1, in accord with previous practice,1–9 but in reality m is probably more than 1.The ESR and VIS spectra for VO21–succinic acid at high L:M as pH is varied resemble those for NacAsp but at pH > ª3.5 bands I and II are more distinctly separate. This system was previously studied by spectroscopic 18,46 and pHpotentiometric 43,46 methods at L :M ratios of 1 and 2 : 1, and by ESR47 using L :M 100 : 1. The formation of a single complex ML9 (with stability constant 103.66) was assumed at 30 8C and I = 0.1 mol dm23 KNO3.43 At low excess of ligand, in the pH range 4.5–6, spectroscopy (VIS and ESR) gave evidence only for the monodentate carboxylato complex 2, besides the aqua ion and VO21 hydroxo complexes.It was characterised by absorption maxima at 590–600 (band II) and 780–810 nm (band I).18,46 Our VIS spectra at high excess of ligand show features apparently not then observed 18,46 at low L:M ratios. Two bands are distinct in the range 450–900 nm for pH > 3.5, with lmax(band II) ª565 nm and lmax(band I) ª830 nm: this suggests complexes with bidentate succinate (or the monodentate co-ordination of three or more succinate ligands).The conclusions of Ferrari et al. 18 based on ESR and VIS spectra are similar, except that they assumed 2 to form at lower pH. In their ESR study with CVO = 1023 mol dm3 and L:M 100 : 1, McPhail and Goodman47 detected four components in the pH range 3–6, finding A to decrease and g to increase as pH is increased.Our ESR results agree. With succinic acid at high L :M the distribution of Fig. 1(D) describes the spectroscopic results up to pH 6.7. The new ESR-active species at pH ª7 could correspond to a ML9H23 stoichiometry: its estimated spin-Hamiltonian parameters are in Table 2. As at least two distinct components are detected in the ESR spectra; the parameters were obtained by simulation of spectra but using equations 48 based on Chasteen9s24 iterative method.Lacking a b value, the relative concentration was estimated from ESR.J. Chem. Soc., Dalton Trans., 1998, 3587–3600 3593 Since the vanadyl ion is lop-sided many of its chelates may give rise to more isomers than do those of apparently analogous monoatomic metal centres such as CuII refs. 1 and 23 briefly discuss this. For monodentate ligands, such as an amino acid bonded via its carboxylate, this extra isomerism would be absent. However, if there are two such ligands geometric isomers may be present.We now discuss, on the basis of Figs. 2–5 and Tables 1 and 2, the dominant structure in solution for each stoichiometry. For a given stoichiometry and similar co-ordination geometry, CD spectra for GlyAsp, AspGly and NacAsp may diVer. Table 3 gives plausible structures and, for each, the main mechanisms for inducing optical activity. These systems are labile. Optical activity consequently arises from the chiral arrangement of the chelate rings, i.e.the conformational effect,30 and, since the ligands contain asymmetric carbon atoms, from the transmission through space and by way of the chemical bonds linking the asymmetric centre to the chromophore, i.e. the vicinal effect.30 The order of magnitude of the conformational eVect depends on the chelate-ring puckering and that of the vicinal eVect on the number of atoms between the asymmetric centre and the metal, and on the eYciency of the donor groups in transmitting dissymmetry, which for peptides has been suggested to be: 16,31 Namide > (CO)Namide > CO2 2 > C]] Oamide > (NH)C]] Oamide > NH2.We also assume here and elsewhere 1–8,16 that optical activity transmits better from the chiral centres of the ligand into optical transitions through Fig. 3 Visible absorption spectra of solutions containing (A) GlyAsp and VO21 with L :M = 30.0 : 1 and CVO ª 0.008–0.011 mol dm23 and, (B) AspGly and VO21 with L :M = 9.8 : 1 and CVO ª 0.006–0.010 mol dm23.The pH corresponding to each spectrum is indicated. Spectrum 1 in B approximately coincides with that of VO(OH2)5 21 (]] ] M21). The ESR spectra corresponding to some of these solutions are in Fig. 2. equatorial rather than axial donor ligands. Asymmetric deviations of ligating atoms from regular polyhedra (asymmetric distortions) may also make a significant contribution to the CD spectrum,32,33 particularly in VO21 complexes. In these systems several optically active species may contribute at each pH, so extracting structural information from CD is not straightforward.In particular, the fact that in the pH range 3–8 |Dem|AspGly are much greater than |Dem|GlyAsp cannot be easily understood. One possibility is that the dominant complexes in the AspGly system (ML and MLH21) are more rigid, because of extra ‘chelation’ 49 (e.g. via hydrogenbonding of a solvent water between two donor groups or the dipeptide behaving as tetradentate with one donor group axial): the configurational eVect may then contribute to optical activity.Another explanation is a significant contribution to optical activity arising from inherent dissymmetry and asymmetric distortions for complexes ML and MLH21 in the AspGly system. These eVects may 32 be much stronger than the conformational or the vicinal eVects. Vanadium in VO21 may become an optically active centre, so most complexes here contain two dissymmetric centres. The structures presented correspond to one of two possible diastereomers: absolute configurations for oxovanadium, either C or A,50 and for the a-carbon, L (e.g.discussion later for structure 17a). Their stability may diVer as found51 by 51V NMR for e.g. [VVO(sal-L-aa)(dl2)] (sal-L-aa = N-salicylidene-L-amino acidate of Gly, Ala, Val or Phe; dl2 = monoanion of glycerol, ethane-1,2-diol or propane-1,3-diol), the trends observed being consistent with steric control. If this is the case for complexes ML and MLH21 in the AspGly system, the Fig. 4 Circular dichroism spectra of solutions containing (A) GlyAsp and VO21 with L :M = 15.0 : 1 and CVO ª 0.018–0.020 mol dm23 and, (B) AspGly and VO21 with L :M = 9.8 : 1 and CVO ª 0.008–0.010 mol dm23. The pH corresponding to each spectrum is indicated. The ESR and VIS spectra corresponding to some of the AspGly solutions are in Figs. 2 and 3.3594 J. Chem. Soc., Dalton Trans., 1998, 3587–3600 contributions of inherent dissymmetry, asymmetric distortions and finally that of the configurational eVect may become important.MLH2 and ML9H The stoichiometry MLH2 (GlyAsp, AspGly) or ML9H (NacAsp, succinate) corresponds to structures with equatorially co-ordinated carboxylate (the terminal one in the case of GlyAsp and AspGly). The pattern of the VIS and CD spectra of this stoichiometry resembles spectra 2 of Fig. 3(A) and 4(A), and those for the corresponding VO21 complexes of various amino acids 1–6 and simple dipeptides.9,16 For GlyAsp and NacAsp, co-ordination involves the CO2 2 group near the Fig. 5 Circular dichroism spectra of solutions containing: (A) GlyAsp and VO21 with L :M = 15.0 : 1 and CVO ª 0.016–0.018 mol dm23, (B) AspGly and VO21 with L :M = 9.8 : 1 and CVO ª 0.006–0.008 mol dm23 and, (C) NacAsp and VO21 with L :M = 30.0 : 1 and CVO ª 0.008–0.012 mol dm23. The pH corresponding to each spectrum is indicated. The ESR spectra corresponding to some of these solutions are in Fig. 2. asymmetric centre, the CD showing its characteristic pattern (Fig. 3 and Table 2): for AspGly it involves the Gly residue, the CD being therefore extremely weak at pH 1–2. Chasteen’s method 24 (eqn. 3) gives g|| est ª 1.953 and A|| est ª 180 × 1024 cm21 for this geometry. Consequently, the values in Table 2 agree with those obtained for solutions containing mostly MLH2 (and ML2H4) in the case of GlyAsp and AspGly, or ML9H (and ML29H2) in the case of NacAsp and succinic acid.VO21 1 H2L KCO2 2 VO(H2L)21 (4A) VO21 1 HL92 K9CO2 2 VO(HL9)1 (4B) MLH or ML9 and ML2H3 or ML92H In both MLH and ML9 the basic binding mode is probably chelation via both carboxylate groups. Derived equilibrium constants b1 1 and b1* for MLH (GlyAsp, AspGly and Asp) or ML9, and also for corresponding complexes of some reference ligands, are in the second row of Table 4. The data show that 7- membered chelate formation is less favoured, log b1* = 23.8, than the 6-membered dicarboxylate chelate in the malonic acid system (log b1* = 21.99).The appropriate derived constants log b1* for NacAsp and succinic acid are ª25 and 26, respectively, revealing important stabilisation for GlyAsp and AspGly complexes. This can very likely be explained by the co-ordination of Oamide of the peptides. For several plausible structures no strain is apparent and the amide group remains planar (e.g. 4–9). This is the case of 4 for GlyAsp and 5–7 for AspGly.For AspGly, structures 6 and 7 and others containing two “free” equatorial positions are discarded; in fact, if they corresponded to MLH there would be no obvious reason for the absence of ML2H3 (or dimeric complexes where the equatorial positions occupied by W and/or Y could be shared with donor groups from the ligand of a second complex). Therefore MLH probably corresponds to 5 although the tridentate equatorial co-ordination is not apparent in the values of b1 1 and b1*.For ML9 (NacAsp or succinic) the structures can correspond to 8 and/or 9. Structures for ML2H3 (GlyAsp) or ML29H (NacAsp or succinic) can be regarded as comprising those for MLH or ML9 plus a monodentate carboxylate group from a second ligand. Therefore, in the schematic structures here (e.g. 4, 8 or 9), X represents either H2O or a RCO2 2 ligand. The spin- Hamiltonian parameters for ML2H3 or ML29H [ g|| exptl ª 1.938 and A|| exptl ª (173–174) × 1024 cm21] indicate equatorial coordination of three carboxylate groups, or two carboxylate groups and one Oamide; therefore structure 4, 8 or 9 (and other isomers) is consistent with the ESR data.The pattern of CD expected for MLH (GlyAsp) and ML9 (NacAsp), and ML2H3 (GlyAsp) and ML29H, is the same; therefore, the dominant dissymmetric factors persist in corresponding stoichiometries in these systems, i.e. if, say, structure 4 is correct for GlyAsp, then for NacAsp the structure would correspond to 9. However it is not yet possible to decide which geometry (4, 8, 9 or some other) actually predominates.ML (AspGly) The pK values for the formation of ML stoichiometries are included in the fourth row of Table 4. While for NacAsp, succinic and malonic acids the pK values correspond to the deprotonation/co-ordination of a carboxylate group, for AspGly and L-Asp this corresponds to a similar process for the NH3 1 group. The equatorial co-ordination of the NH2 group of AspGly is indicated by the ESR spectra of Fig. 2(B), and by the spin-Hamiltonian parameters obtained for ML (Table 2).TheJ. Chem. Soc., Dalton Trans., 1998, 3587–3600 3595 Table 3 Structures, corresponding A|| est, g|| est [eqn. (3)],a stoichiometries and comment about expected origin for the optical activity for complexes that may form in solutions containing oxovanadium(IV) and GlyAsp, AspGly, NacAsp or succinic acid (see text) Schematic representation b A|| est × 104/cm21 ( g|| est) Stoichiometry (and comments) c a X = H2O 180 (1.935) b X = Ocarboxylate 177 (1.937) MLH2 (GlyAsp or AspGly) (optical activity induced by the vicinal eVect through the CO2 2 group) ML2H4 (GlyAsp or AspGly) a X = H2O 177 (1.937) b X = Ocarboxylate 174 (1.939) MLH (GlyAsp) (optical activity induced by the vicinal eVect through the CO2 2 groups, and by the conformational eVect) ML2H3 (GlyAsp) a Y = H2O 175 (1.939) d b Y = OH2 168 (1.946) d MLH (AspGly) (optical activity induced by the vicinal eVect throught the b-CO2 2 and C]] Oamide, and by the conformational eVect) MLH21 (AspGly) a Y = H2O 174 (1.939) d b Y = OH2 167 (1.946) d MLH (AspGly) [Optical activity induced by the vicinal eVect through the b-CO2 2 (low) and C]] Oamide (very low) and by the conformational eVect] MLH21 (AspGly) a Y = H2O 174 (1.939) b Y = OH2 167 (1.946) MLH (AspGly) [optical activity induced by the vicinal eVect through the b-CO2 2 (low) and C]] Oamide (very low), and by the conformational eVect] MLH21 (AspGly) a X = H2O 180 (1.935) b X = Ocarboxylate 174 (1.939) MLH (AspGly) or ML9 (NacAsp or succinic e) [optical activity induced by the vicinal eVect through the a-CO2 2 and b-CO2 2 (low) and by the conformational eVect] ML2H3 (AspGly) or ML29H (NacAsp or succinic e) a X = H2O 177 (1.937) b X = Ocarboxylate 170 (1.944) MLH (AspGly) or ML9 (NacAsp or succinic e) [optical activity induced by the vicinal eVect through the a-CO2 2 and b-CO2 2 (low) and by the conformational eVect] ML2H3 (GlyAsp) or ML29H (NacAsp or succinic e) a Y = H2O 172 (1.942) b Y = OH2 165 (1.950) ML2H (GlyAsp) [optical activity induced by the vicinal eVect through the NH2 (low), C]] Oamide (low) and axial CO2 2 (low), and by the conformational eVect] ML2 (GlyAsp) a Y = H2O 172 (1.942) b Y = OH2 165 (1.950) ML2H (GlyAsp) [optical activity induced by the vicinal eVect through the NH2 (low), C]] Oamide (low) and a-CO2 2 and by the conformational eVect] ML2 (GlyAsp)3596 J.Chem. Soc., Dalton Trans., 1998, 3587–3600 Table 3 (Contd.) Schematic representation b A|| est × 104/cm21 ( g|| est) Stoichiometry (and comments) c a Y = H2O 171 (1.942) b Y = OH2 164 (1.950) ML (AspGly) [optical activity induced by the vicinal eVect through the NH2 and b-CO2 2 (low) and by the conformational eVect] MLH21 (AspGly) a Y = H2O 171 (1.942) b Y = OH2 164 (1.950) ML (AspGly) [optical activity induced by the vicinal eVect through b-CO2 2, NH2 and C]] Oamide (low), and by the conformational, asymmetric distortion f and configurational eVects] MLH21 (AspGly) a Y = H2O 175 (1.940) b Y = OH2 168 (1.947) MLH21 (AspGly) (optical activity induced by the vicinal eVect through C]] Oamide and NH2, and by the conformational eVect) a Y = H2O ª163 g (ª1.950) g b Y = OH2 ª158 g (ª1.960) g MLH21 (AspGly) [optical activity induced by the vicinal eVect through (CO)Namide and NH2, by the conformational (?), asymmetric distortion f and configurational eVects] MLH22 (AspGly) a Y = H2O ª165 g (ª1.950) g b Y = OH2 ª158 g (ª1.957) g MLH21 (AspGly) [optical activity induced by the vicinal eVect through (CO)Namide and b-CO2 2, conformational, configurational and asymmetric distortion f eVects] MLH22 (AspGly) a Y = H2O ª162 g (ª1.953) g b Y = OH2 ª158 g (ª1.960) g MLH21 (GlyAsp) [optical activity induced by the vicinal eVect through Namide and a-CO2 2 and by the conformational (?), configurational and asymmetric distortion f eVects] MLH22 (GlyAsp) a Y = H2O ª162 g (ª1.953) g b Y = OH2 ª158 g (ª1.960) g MLH21 (GlyAsp) [optical activity induced by the vicinal eVect through Namide and b-CO2 2 (low), by the conformational eVect and asymmetric distortions f] MLH22 (AspGly) a The A|| est presented were calculated using eqn. (3) and the g|| est using an equivalent equation.In these estimates we assume that ligands co-ordinated in axial position have no influence on the spin-Hamiltonian parameters.The A|| and g|| donor group contributions presented by Chasteen 24 assume axial co-ordination of water. bThe glycine residue is normally indicated with a G; the CO2 2 group of the side chain of the aspartic residue with a b. c The optical activity induced by the vicinal eVect through NH2 or CO2 2 of glycine residues, or from groups co-ordinated in axial position, is expected to be low. d Assuming the contribution of Oamide in eqn. (3) is 174.7 × 1024 cm21 as presented by Pecoraro and co-workers.26 e No optical activity for the succinato complexes.f The eVect of asymmetric distortions (see text) may be important whenever there are distortions in the otherwise symmetric regular structures of complexes. As a whole these distortions must be dissymmetric. 1–6,32,33 g Assuming that the contributions of Namide to A|| and g|| are 136 × 1024 cm21 and 1.983, respectively.16,28,29 CD and VIS spectra for ML approximately correspond to spectra 7 of Figs. 4(B) and 3(B), respectively. Structure 12a or 13a could correspond to ML. For 12a the axial co-ordination of a water molecule is apparently precluded by the a-proton of the Asp residue. There is no significant strain in tetradentate co-ordination of AspGly and we propose 13a as the structure of the AspGly complex corresponding to ML stoichiometry. ML2H2 and ML29 The pK values for the formation of ML29 from ML29H or ML2H2 from ML2H3 are included in the fifth row of Table 4, being in the range 3.2–5.2. Comparing these values with the corresponding pKa2 of the ‘free’ ligands, no significant decrease in the pK of the carboxylic groups is observed in ML92H or ML2H3 complexes.The changes observed in the ESR, VIS andJ. Chem. Soc., Dalton Trans., 1998, 3587–3600 3597 Table 4 Derived equilibrium constants for VO21 complex formation, partial processes of the ligands studied and some related compounds Monodentate CO2 2 co-ordination a Bidentate 2O2C? ? ?CO2 2 co-ordination b Bidentate (2O2C? ? ?CO2 2)2 co-ordination c MLH ML 1 H1 or ML9H ML9 1H1 ML2H3 ML2H2 1 H1 or ML92H ML92 1 H1 ML2H2 ML2H 1 H1 or ML92 ML92H21 1 H1 ML MLH21 1 H1 or ML9 ML9H21 1 H1 log KCO2 2 log b1 1 log b1* log b2 1 log b2* pK pK pK pK GlyAsp 2.5 3.16 (7-m.r.) 23.72 5.8 (7-m.r.) 27.98 — 4.1 5.4 — AspGly 1.9 2.53 (7-m.r.) 23.86 — 4.0 — — 5.6 NacAsp 1.7 2.7 (7-m.r.) 24.9 5.0 (7-m.r.) 210.2 3.5 4.6 — — Succinic acid 2.0 3.20 (7-m.r.) 25.97 5.6 (7-m.r.) 212.7 4.0 5.2 — — GlyGly9 1.8 — — — — — pKamide ª7 Ala1 1.19 — 25.62 212.6 4.3 5.2 8.0 ª5.3 Gly10 1.17 — 25.60 212.4 4.3 4.8 7.7 ª5.2 Malonic acid35 1.18 5.6 (6-m.r.) 21.99 9.5 (6-m.r.) — ª0.6 — — 5.1 Aspartic acid5 ª1.9 2.8 (7-m.r.) 23.22 ª5.0 (7-m.r.) 27.0 ª3.5 ª3.2 ª3.5 6–7 AspGly (Cu)41 — 2.1 — — 3.5 — — pKamide 4.9 GlyAsp (Cu)40 — 2.1 25.04 — 3.8 — — pKamide 4.8 a Ligand is protonated except at the carboxylate which co-ordinates; KCO2 2 is defined for reactions corresponding to eqns.(4A) (GlyAsp, AspGly, Asp) and (4B) for the rest of the ligands. b Ligand is protonated at amino group: b1 is defined for reactions VO 1 LH VOLH (GlyAsp, AspGly and Asp) or VO 1 L9 VOL9 (rest of the ligands). The basicity corrected b1* is defined for VO 1 H3L VOHL 1 2H1 (GlyAsp, AspGly and Asp) or VO 1 H2L9 VOL9 1 2H9 (rest of the ligands); 7-m.r., 6-m.r. = 7- and 6-membered ring respectively. c The same as for footnote b but for bis complexes: b2 1 is defined for reactions VO 1 2HL VO(LH)2 (GlyAsp, AspGly and Asp) or VO 1 2L9 VOL92 (rest of the ligands).The basicity corrected b2* is defined for VO 1 2H3L VO(HL)2 1 4H1 (GlyAsp, AspGly and Asp) or VO 1 2H2L9 VOL92 1 4H1 (rest of the ligands).3598 J. Chem. Soc., Dalton Trans., 1998, 3587–3600 CD spectra as pH is increased for GlyAsp, NacAsp and succinic acid do however suggest that the co-ordination geometries of the complexes alter. This may be explained assuming that the O atoms of the second CO2 2 group in ML92 or ML2H2 also coordinate to VO21.The co-ordination geometries either involve all carboxylate O atoms equatorial or three equatorial and one axial (in the case of GlyAsp with possibly Oamide equatorial). The ESR parameters for ML92 or ML2H2 cannot be determined accurately but the approximate values presented in Table 2 are consistent with these geometries if the contributions of Ocarboxylate and Oamide are similar. Formation constants b2 1 (corrected for the protonation of the NH3 1 group: b2 1 = b122/b011 2) for complexes involving two 7-membered chelate rings co-ordinated by carboxylate groups (assuming these are the donor groups) are ª105 (third row of Table 4), while for malonate, which involves two 6-membered chelate rings, b120 ª 109.5.Basicity corrected formation constants b*, defined assuming ligands totally protonated (for H3L ligands: b2* = b122/b013 2 and for H2L9 ligands b2* = b120/b012 2) are significantly higher for GlyAsp and Asp than for NacAsp and succinic acid.As for MLH and ML2H3 there is extra stabilisation for GlyAsp complexes possibly due to the co-ordination of Oamide of one of the ligands. The VIS spectra for ML2H2 (GlyAsp) and ML29 (NacAsp and succinic acid) approximately correspond to spectrum 7 of Fig. 3(A). The CD spectrum is dominated by a relatively intense and negative band I (GlyAsp and NacAsp: lmax ª 820–830 nm), a negative band II at lmax ª 550 nm (GlyAsp) or a relatively weak and positive band II at lmax ª 575 nm (NacAsp); all these diVer from the corresponding bands in the L-Asp system: this is also in accordance with its diVerent binding mode.ML2H (GlyAsp) The pK for the process ML2H2 æÆ ML2H 1 H1 (sixth row of Table 4) is 5.4 (ª3.5 for L-Asp). This is not the deprotonation of equatorial water as ML2H2 has no such group. While this pK gives little change in ESR, the pattern of CD shows drastic changes from pH 4.5 to 6.5 due to the formation of ML2H and MLH21.This may involve changes in the co-ordination of the equatorial and axial donor functions. The spin-Hamiltonian parameters for ML2H (Table 2) practically coincide with those expected for four equatorially co-ordinated Ocarboxylate and/or Oamide atoms,25,26 so the geometry may be either (CO2 2)4, (CO2 2)3(Oamide) or (CO2 2)2(Oamide)2. Analysing the CD spectra for L :M 15 and 30 : 1 in the pH range 4.5–6.5 (Fig. 5) we expect for ML2H a pattern 2,1,2 with lmax ª530 ± 20 nm (band II), ª630 ± 20 (band IB) and ª840 ± 30 nm (band IA).Therefore the symmetry for this complex is low and the structure such that bands IA and IB are separate. Axial co-ordination of OH2 is not expected to promote change in the CD. Alternative coordination geometries for ML2H2 are 10a and 11a (Table 3: Y = H2O). These correspond to A|| est ª 172 × 1024 cm21 and could help explain the low Dem values for the GlyAsp system relative to AspGly, as the equatorial NH2 and C]] Oamide are far from the asymmetric carbon and a low vicinal eVect is expected.It might be surprising that formation of bis complexes is disfavoured with AspGly, although N-terminal Asp oVers a b-Ala chelation site. However, this is not favoured for VO21 which binds N-donors rather weakly. Unlike GlyAsp, chelating O-donor sites, including carboxylate groups (strong binding functions for VO21), are not similarly available in AspGly. MLH21, ML9H22 and MLH22 The pK values for the formation of MLH21 (seventh row in Table 4) for the dipeptides can either correspond to the deprotonation/co-ordination of Npeptide, or to the deprotonation of an equatorially coordinated water molecule.These two processes correspond to the formation of complexes such as 5b, 6b, 7b, 12b, 13b, or 14–18 (Table 3). The stoichiometry MLH22 (GlyAsp) probably corresponds to a co-ordination geometry such as 17b (possibly OH2 axial instead of CO2 2).For L :M = 30 :1, maximum |Dem| are found at pH ª 9.5, conditions where MLH22 would dominate CD spectra for GlyAsp; the ESR and CD spectra similarly resemble those for the corresponding complexes with AlaAla and GlyAla [ESR: A|| exptl = (161 ± 1) × 1024 cm21, g|| = 1.954 ± 0.004. CD spectra: band II, lmax ª 500 nm, De < 0; band I, lmax ª 720–750 nm, De > 0).16 This is why structure 17b is assigned to MLH22 and not 18. This is also consistent with 5-membered rings being more stable than 6-membered ones.Over the pH range 6.5–8 where MLH21 (GlyAsp) is predominant, as pH is increased the ESR spectra change little apart from decreasing intensity, but the CD shows changes due to the processes of Scheme 2. Some of these equilibria take time to establish making the exact pattern of CD bands for MLH21 elusive. The band pattern 2,1,2 at L:M = 15 : 1 may be due to the optically active oligomeric species: increasing L :M, the CD spectra in the pH range 7–8.5 change towards a 2,1 pattern.So MLH21 and MLH22 may well have identical CD band patterns, i.e. both like the corresponding complexes in the AlaAla and GlyAla systems; 16 the co-ordination geometry for MLH21 therefore probably corresponds to 17a, although it is not possible to rule out geometries such as (CO2 2, NH2, CO2 2, OH2)equatorial. The few results available for the oligomeric optically active species that form make it impossible to predict their binding modes.Assuming 17a corresponds to the geometry of MLH21 in the GlyAsp 1 VO21 system, and taking into account that now the vanadium atom is an asymmetric centre, two diastereomers may be considered. The geometry for both complexes corresponds to a distorted polyhedron and the contributions of inherent dissymmetry and asymmetric distortions to the optical activity will roughly cancel if both diastereomers are present in equal concentrations; however, if in 17a the b-CO2 2 is co-ordinated axial, this makes 17a slightly more stable than 17a*, these contributions to optical activity and its magnitude then depending on the relative concentration of the diastereomers.Similar comments apply to most structures included in Table 3. For the MLH21 (and MLH22) stoichiometries in the AspGly system, besides 5b, 6b, 7b, the following basic binding modes may be envisaged all compatible with the ESR: (CO2 2, N2 amide, NH2, Y)equatorial, e.g. 15, (CO2 2, NH2, CO2 2, Y)equatorial, e.g. 12, 13 or (CO2 2, N2 amide, CO2 2, Y)equatorial, e.g. 16. The present results and earlier data do not suYce to define the geometry for MLH21 and MLH22 stoichiometries, 15 and 16 corresponding to what is normally assumed for Cu21 complexes.30,31 Structure 16 is more in agreement with the high |Dem| values obtained for the AspGly–VO21 system [Fig. 5(B)]. In the GlyAsp system MLH21 starts to form at pH higher than for AspGly; this may be due to its higher pKa2, pKa3, and to some steric factor making tetradentate co-ordination (e.g. 17a) unfavorable. In solutions containing NacAsp and VO21 [L:M = 15 or 30:1 e.g. Fig. 5(C)] the CD spectra for pH > 5 diVer from those of GlyAsp in similar conditions, and the |Dem| values decrease for pH > 5–5.5. If N2 amide co-ordinates, e.g.: (CO2 2, N2 amide, CO2 2, Y)equatorial, we would expect an increase in |Dem| with pH and lower values of A|| exptl for ML9H22 (Table 2). These observations indicate that, for the NacAsp system, N2 amide does not C O O V* NH2 N W O HC C CH2 O CH2 – OOCb * W *V N H2N O O C O O H2C C CH Cb CH2 O * ? O 17a (V chirality50: C.a-C:L) 17a* (V chirality50: A. a-C:L)J. Chem. Soc., Dalton Trans., 1998, 3587–3600 3599 co-ordinate, and that NH2, although not a good anchor for VO21, is involved in co-ordination for the corresponding stoichiometries in the GlyAsp system (in agreement with our conclusions there). Therefore, the stoichiometry ML9H22 (NacAsp and succinic) possibly involves complexes where co-ordination is (OH2, OH2, CO2 2, H2O)equatorial and (CO2 2)axial, which corresponds to A|| est ª 168 × 1024 cm21.This and the extensive hydrolysis of oxovanadium(IV) are also compatible with the very low CD and ESR signal at pH > 7 for NacAsp 1 VO21. The only evidence for the stoichiometry ML9H23 (succinic acid) is that a new ESR active species appears at pH ª 7 (see above) with A|| exptl ª (165 ±2) × 1024 cm21; this possibly corresponds to a geometry involving (2CO2 2, 2OH2)equatorial (OH2)axial.This would have A|| est ª163 × 1024 cm21 while geometries of the type (CO2 2, 3OH2)equatorial (CO2 2)axial would give A|| est ª159 × 1024 cm21. Summarizing the behaviour of the studied dipeptides, in the weakly acidic range only the carboxylate, the peptide CO and amino groups (this latter being somewhat less favoured) participate in the metal ion binding. The position of the tridentate Asp residue in the peptide chain aVects the co-ordination mode of the ligands: when Asp is C-terminal (GlyAsp) it rather behaves as a succinic acid favouring equatorial carboxylate chelation of two neighbouring groups with some involvement of the peptide CO in metal binding. In the N-terminal Asp dipeptide, AspGly, the involvement of either the peptide carbonyl or the terminal amino groups seems more essential, since the two carboxylates are much further apart.For pH > 6–7 the dominant stoichiometries for the ESR and CD active complexes are MLH21 and MLH22.Further, for GlyAsp the results indicate the basic binding mode: (CO2 2, N2 amide, NH2, Y)equatorial with Y = H2O or OH2. The axial coordination of the b-CO2 2 group is possible but unproven. The binding mode for AspGly diVers, particularly the fact that, at least till L :M = 8 : 1, only 1 : 1 complexes form. The distinction in the mode of 1 : 1 attachment to the VO21 ion of these isomeric dipeptides is a remarkable new property.Whereas with the ‘spherical’ copper(II) ion, the chelating stabilities of N-terminal GlyAsp40 and C-terminal AspGly41 are the same, with the lop-sided vanadyl ion they diVer markedly. Acknowledgements We thank Fundo Europeu para o Desenvolvimento Regional (FEDER), project PRAXIS 2/2.1/QUI/151/94), the National Research Fund (project OTKA T2273/97) and the Hungarian Ministry of Culture and Education (project FKFP OOB/97) for financial support, and the Hungarian–Portuguese Intergovernmental S & T Co-operation Programme for 1998–1999 for travelling funds.We thank B. Herold, L. Alcácer and R. T. Henriques for the use of their ESR facilities and Fundação Calouste Gulbenkian for travel grants. References 1 J. Costa Pessoa, L. F. Vilas Boas, R. D. Gillard and R. J. Lancashire, Polyhedron, 1988, 7, 1245. 2 J. Costa Pessoa, L. F. Vilas Boas and R. D. Gillard, Polyhedron, 1989, 8, 1173. 3 J. Costa Pessoa, L. F. Vilas Boas and R.D. Gillard, Polyhedron, 1989, 8, 1745. 4 J. Costa Pessoa, L. F. Vilas Boas and R. D. Gillard, Polyhedron, 1990, 9, 2101. 5 J. Costa Pessoa, R. L. Marques, L. F. Vilas Boas and R. D. Gillard, Polyhedron, 1990, 9, 81. 6 J. Costa Pessoa, J. L. Antunes, L. F. Vilas Boas and R. D. Gillard, Polyhedron, 1992, 11, 1449. 7 J. Costa Pessoa, S. M. Luz, I. Cavaco and R. D. Gillard, Polyhedron, 1994, 13, 3177. 8 J. Costa Pessoa, S. M. Luz and R. D. Gillard, Polyhedron, 1995, 14, 1495. 9 J. Costa Pessoa, S. M. Luz and R. D. Gillard, Polyhedron, 1993, 12, 2857. 10 I. Nagypál and I. Fábián, Inorg. Chim. Acta, 1982, 62, 193. 11 H. Sakurai, Y. Hamada, S. Shimomura, S. Yamashita and K. Ishizn, Inorg. Chim. Acta, 1980, 46, L119; H. Sakurai, S. Shimomura and K. Ishizn, Inorg. Chim. Acta, 1981, 55, L67; H. Sakurai, Z. Taira and N. Sakai, Inorg. Chim. Acta, 1988, 151, 85. 12 M. D. Sifri and T. L. Riechel, Inorg. Chim. Acta, 1988, 142, 229. 13 A. Dess, G. Micera and D Sanna, J.Inorg. Biochem., 1993, 52, 275. 14 T. Kiss, P. Buglyó, G. Micera, A. Dessi and D. Sanna, J. Chem. Soc., Dalton Trans., 1993, 1849. 15 K. Knüttel, A. Müller, D. Rehder, H. Vilter and V. Wittneben, FEBS, 1992, 302, 11. 16 J. Costa Pessoa, S. M. Luz and R. D. Gillard, J. Chem. Soc., Dalton Trans., 1997, 569. 17 M. Delfini, E. Gaggelli, A. Lepri and G. Valensin, Inorg. Chim. Acta, 1985, 107, 87. 18 R. P. Ferrari, E. Laurenti, S. Poli and L. Casella, J. Inorg. Biochem., 1992, 45, 99. 19 E. G. Ferrer, P. A. Williams and E. J. Baran, Biol. Trace Elem. Res., 1991, 30, 175; J. Inorg. Biochem., 1993, 50, 253; P. A. Williams and E. J. Baran, J. Inorg. Biochem., 1994, 54, 75. 20 E. G. Ferrer, P. A. Williams and E. J. Baran, Biol. Trace Elem. Res., 1996, 55, 153; Transition Met. Chem., 1997, 22, 589. 21 L. Xiaoping and X. Yuanzhi, Kexue Tongbao, 1985, 30, 340; L. Xiaoping and Z. Kangjing, J. Crystallogr. Spectrosc. Res., 1986, 16, 681. 22 J. Costa Pessoa, T.Gajda, S. M. Luz, T. Kiss, J. J. G. Moura and I. Torok, J. Inorg. Biochem., 1997, 67, 388. 23 L. F. Vilas Boas and J. Costa Pessoa, in Comprehensive Coordination Chemistry, eds. G. Wilkinson, R. D. Gillard and J. A. McCleverty, Pergamon, Oxford, 1987, vol. 3, pp. 453–583 and refs. therein. 24 N. D. Chasteen, in Biological Magnetic Resonance, eds. J. Lawrence, L. J. Berliner and J. Reuben, Plenum, New York, 1981, vol. 3, p. 53. 25 C. R. Cornman, E. P. Zovinka, Y. D. Boyajian, K. M. Geiser-Bush, P. D. Boyle and P. Singh, Inorg. Chem., 1995, 34, 4213. 26 B. J. Hamstra, A. L. P. Houseman, G. J. Colpas, J. W. Kampf, R. LoBrutto, W. D. Frasch and V. L. Pecoraro, Inorg. Chem., 1997, 36, 4866. 27 F. W. B. Einstein, R. J. Batchelor, S. J. Angus-Dunne and A. S. Tracey, Inorg. Chem., 1996, 3, 1680. 28 A. J. Tasiopoulos, Y. G. Deligiannakis, J. D. Woolins, A. M. Z. Slawin and T. A. Kabanos, Chem. Commun., 1998, 569. 29 A. J. Tasiopoulos, A. T. Vlahos, A. D. Keramidas, T. A. Kabanos, Y. G. Deligiannakis, C. P. Raptopoulou and A. Terzis, Angew. Chem., Int. Ed. Engl., 1996, 35, 2531. 30 R. Kuroda and Y. Saito, in Circular Dichroism. Principles and Applications, eds. K. Nakanishi, N. Berova and R. W. Woody, VCH, New York, 1994, ch. 9, pp. 223–225. 31 H. Sigel and R. B. Martin, Chem. Rev., 1982, 82, 385 and refs. therein. 32 N. C. Payne, Inorg. Chem., 1973, 12, 1151; K. Z. Suzuki, Y. Sasaki, S. Ooi and K. Saito, Bull. Chem. Soc. Jpn., 1980, 53, 1288; S. Okazaki and K. Saito, Bull. Chem. Soc. Jpn., 1982, 55, 785. 33 I. Cavaco, J. Costa Pessoa, M. T. Duarte, R. T. Henriques, P. M. Matias and R. D. Gillard, J. Chem. Soc., Dalton Trans., 1996, 1989. 34 G. Gran, Acta Chem. Scand., 1950, 4, 559. 35 I. Nagypál and I. Fábián, Inorg. Chim. Acta, 1982, 61, 109. 36 E. D. MoVat and R. I. Lytle, Anal. Chem., 1959, 31, 926. 37 T. Gajda, B. Henry and J. J. Delpeuch, J. Chem. Soc., Dalton Trans., 1992, 2301. 38 L. Zekany and I. Nagypal, in Computational Methods for the Determination of Stability Constants, ed. D. Leggett, Plenum, New York, 1985. 39 R. P. Henry, P. C. H. Mitchell and J. E. Prue, J. Chem. Soc., Dalton Trans., 1973, 1156. 40 A. Gergeley and E. Farkas, J. Chem. Soc., Dalton Trans., 1982, 381. 41 I. Sóvágó, E. Farkas, T. Jankowska and H. Kozlowski, J. Inorg. Biochem., 1993, 51, 715. 42 A. E. Martell and R. M. Smith, Critical Stability Constants, Plenum, New York, 1974, vol. 1; 1977, vol. 3; 1982, vol. 5; 1989, vol. 6. 43 S. P. Singh and J. P. Tandon, Acta Chim. Acad. Sci. Hung., 1974, 80, 425. 44 A. E. Martell and R. J. Motekaitis, Determination and Use of Stability Constants, VCH, Weinheim, 1988, p. 197. 45 EPRPOW, L. K. White and R. L. Belford, University of Illinois (modified by L. K. White, N. F. Albanese and N. D. Chasteen, University of New Hampshire to include both Lorentzian and Gaussian line shape functions, an I = 7/2 nucleus, a 4th hyperfine interaction and multiple sites having diVerent linewidths), 1978. 46 G. Micera and A. Dessi, J. Inorg. Biochem., 1988, 33, 99.3600 J. Chem. Soc., Dalton Trans., 1998, 3587–3600 47 D. B. McPhail and B. A. Goodman, J. Chem. Soc., Faraday Trans., 1987, 3513. 48 L. Casella, M. Gullotti and A. Pintar, Inorg. Chim. Acta, 1988, 144, 89. 49 R. D. Gillard, J. Inorg. Nucl. Chem., 1964, 26, 657; J. P. Mathieu, Contributions to the Study of Molecular Structure, (Victor Henri Commem. Vol), Désoer, Liège, 1947, p. 111. 50 G. J. Leigh (Editor), Nomenclature of Inorganic Chemistry, Blackwell Scientific Publications, Oxford, 1990, p. 186. 51 S. Mondal, S. P. Rath, K. K. Rajak and A. Chakravorty, Inorg. Chem., 1998, 37, 1713. Paper 8/01888J