Copper amino-acid complexes – towards encapsulated metal centres

Polyhedron 23 (2004) 1709–1717 www.elsevier.com/locate/poly

Copper amino-acid complexes – towards encapsulated metal centres Diane L. Stone, David K. Smith *, Adrian C. Whitwood Department of Chemistry, University of York, Heslington, York, YO10 5DD, UK Received 22 January 2004; accepted 2 April 2004 Available online 27 April 2004

Abstract This article reports the synthesis of two novel amino-acid derivatives in which the side chain of glutamic acid (Glu) is coupled with different amines – tert-butylamine (ligand A) or a first generation ‘Newkome’ type dendron (ligand B). Consequently, ligand B possesses a bulkier branch than ligand A. Ligands A and B bind Cu2þ ions in solution, generating copper complexes A–Cu (Mr ¼ 508) and B–Cu (Mr ¼ 1077). These complexes were fully characterised and, in particular, have been investigated by electrochemical and electron spin resonance techniques to determine the extent of encapsulation of the copper ions. There were significant differences between the two complexes, indicating that Cu2þ may be more deeply encapsulated by ligand B and hence less able to interact with its surroundings. This indicates that metal ions can be sensitive reporters of even relatively small amounts of organic functionality, and emphasises the important role which protein superstructures can play in modulating the function of active metal ions. Ó 2004 Elsevier Ltd. All rights reserved. Keywords: Amino acid; Copper; Dendrimers; Electrochemistry; Electron spin resonance; Ligand

1. Introduction Metal ions play a dominant role in controlling the behaviour of many biologically important macromolecules [1]. By changing the metal ion, biological systems are able to achieve a wide range of different functions. However, it is also clear that a metal ion alone is not sufficient to achieve the required range of functions. It is only by carefully tuning the ligands employed, and the surrounding organic functional groups within the protein superstructure that biology can achieve selective function. Many active sites are deeply encapsulated within protein and enzyme superstructures, and this has profound effects on factors such as the microenvironmental dielectric constant experienced by the metal and its accessibility to different substrates [2]. There is currently great interest in mimicking biological systems [3], and recently, encapsulation of an active unit has been probed using dendritic molecules, in *

Corresponding author. Tel.: +44-0-1904-434181; fax: +44-0-1904432516. E-mail address: [email protected] (D.K. Smith). 0277-5387/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.poly.2004.04.001

which a branched superstructure surrounds a central metal ion, with the effect of this on function being investigated [4]. There have, for example, been many studies of the effect of a dendritic microenvironment on the optical [5], redox [6–8] or catalytic [9] properties of an encapsulated active site. On encapsulation of a redox active unit, it is well-known that the electron transfer kinetics are significantly slower – a feature also observed in redox-active proteins. Furthermore, cytochromes and their dendritic mimics indicate that the encapsulated micro-environment is significantly different to that of the surrounding bulk solvent/electrolyte, leading to modification of the redox potential of the metal ion [7]. Copper complexes have considerable biological importance, but encapsulated copper centres are less well studied than encapsulated iron. Aida and co-workers reported a dendrimer capable of ligating a copper ion at its focal point, and showed that this dendrimer could react with oxygen to form a bridged bis-copper complex [10]. Multiple copper ions bound within a spherical dendrimer have also been used for O2 binding [11]. Chow and co-workers [12] used a dendritic bis(oxazoline) ligand to bind to copper triflate and then used the

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complex in Diels–Alder catalysis, while recently, functionalised bipyridine and phenanthroline ligands have been used to achieve effective encapsulation of copper ions and generate a wide range of interesting molecular architectures [13]. The coordination of amino acids to metal ions is wellknown [14], with strong binding to transition metals occurring via a chelate binding mode involving both carboxylate and amine groups (Fig. 1). We therefore reasoned that the side chain of an amino acid offered a suitable point to attach functionality capable of encapsulating the bound metal ion. For this reason, we became interested in the development of synthetic amino-acid derivatives with bulky side chains. Whilst this work was in progress, Krishna and Jayaraman [15] reported some different modified amino-acid ligands and described their ability to bind and encapsulate cobalt ions. In this paper, we report amino-acid derivatives functionalised with small, flexible branches, and illustrate their ability to bind Cu2þ ions. Using electrochemical and electron spin resonance (ESR) methods, we have investigated the metal complexes in order to probe the degree of isolation of the Cu2þ ion. We report that the copper ions were surprisingly sensitive reporter units, with their properties being modified even by the small amounts of branching used in this case. These preliminary results have significance for biological chemists interested in the role played by the organic

O

O

H2 N

R

O

O

superstructure in shielding the active sites of metalloproteins and enzymes.

2. Results and discussion 2.1. Synthesis and characterisation of ligands A and B Ligand A was prepared using standard protecting group and peptide coupling methodology (Scheme 1). The side chain of N -Boc, tert-butyl ester protected glutamic acid (1) was coupled with tert-butylamine (2) using 1,3-dicyclohexylcarbodiiimide (DCC), 1-hydroxybenzotriazole (HOBt) and triethylamine in CH2 Cl2 to yield compound 3 After purification of the product by flash chromatography on silica, the product was obtained in 50% yield. The amino-acid protecting groups were then removed simultaneously. Previous literature indicated that a mild organic acid such as CF3 COOH should be sufficient, but even after stirring for 18 h the reaction was incomplete. Harsher conditions (45% HBr in acetic acid) were therefore employed and after stirring for 2 min, deprotection was complete in near quantitative yield to provide ligand A. This ligand was used for the synthesis of copper(II) complexes without isolation (although NMR characterisation of the ligand was achieved by performing the deprotection on one occasion in D2 O). Initially, the synthesis of ligand B was attempted using the same approach (Scheme 1). The DCC/HOBt mediated coupling of first generation ‘Newkome’ type dendritic branch 4 to the protected glutamic acid 1 was very successful, providing compound 5 in 93% yield. Once again, however, CF3 COOH would not fully deprotect the amino-acid group, even on heating (the Boc group was rapidly removed but the t-butyl ester would not come off). Unfortunately, the alternative conditions

Cu R

N H2

Fig. 1. Amino acids bind to copper(II) in a chelate binding mode.

BocHN BocHN

H2N

H2N O

2

O O

O O

O b

O

O

HN

OH

HN

a

OH

A

3 1 c CO2Me

O H2N

BocHN

CO2Me

O

O O

O

CO2Me 4

O

HN 5

CO2Me

O O O

d

CO2Me CO2Me

B

Scheme 1. Synthesis of A and attempted synthesis of B using Boc/tert-butyl ester protecting group methodology. Reagents and conditions: (a) DCC, HOBt, NEt3 , CH2 Cl2 , 50%, (b) HBr/CH3 COOH, 2 min, quant., (c) DCC, HOBt, NEt3 , CH2 Cl2 , 93%, (d) HBr/CH3 COOH, 2 min, 0%.

D.L. Stone et al. / Polyhedron 23 (2004) 1709–1717

using HBr/CH3 COOH were too harsh, and NMR analysis of the product indicated that the methyl ester units on the branch had also been removed by acid catalysed hydrolysis (peak at d ¼ 3:65 ppm disappeared). An alternative strategy was therefore developed in which the amino-acid protecting groups were removed non-hydrolytically. Consequently, N -CBz, benzyl ester

protected glutamic acid (6) was used as the starting material (Scheme 2). DCC/HOBt coupling of this with first generation ‘Newkome’ type branch 4 was performed and product 7 was purified using gel permeation chromatography (Biobeads SX-1, CH2 Cl2 ). This compound was obtained in 60% yield and fully characterised. Simultaneous deprotection of the two amino-acid groups was then attempted using Pd/C under H2 in ethanol. The reaction had to be stopped after just 30 min, because over longer time-scales, mass spectral and NMR analysis indicated that (unusually) the benzyl ester had been transesterified to an ethyl ester. We do not have a good explanation for this side-reaction, which is unexpected (EtOH is indeed normally the solvent of choice for the deprotection of benzyl esters). Nonetheless, by employing a 30-min reaction time, ligand B could be obtained in 93% yield and was fully characterised.

CBzHN O O OBn

OH

6

CO2Me

O H2N

a

CO2Me

O O

CO2Me 4

CBzHN O O OBn

HN 7

2.2. Synthesis and characterisation of copper complexes A–Cu and B–Cu

CO2Me

O

CO2Me

O O

The usual literature technique for the preparation of copper complexes is to dissolve both the ligand and a copper salt in water, and to encourage complexation by the addition of base. This is normally followed by spontaneous precipitation or recrystallisation of the complex. For the synthesis of A–Cu, ligand A was dissolved in water which had been basified with 10% NaOH. Copper carbonate was added and the mixture stirred for 2 h. On cooling, a light blue precipitate of complex A–Cu was obtained in 45% yield (see Fig. 2). The complex was analysed using electrospray mass spectrometry (ESMS) and a peak corresponding to [M + Na]þ was observed at m=z 488, with an isotope peak at m=z 490. A peak was also observed at m=z 953, corresponding to [2M + Na]þ , with isotope peaks at m=z 955 and 957. These higher mass peaks were not,

CO2Me

b

H2N O O OH

CO2Me

O

HN B

CO2Me

O

O

1711

CO2Me

Scheme 2. Synthesis of B using CBz/benzyl ester protecting group methodology. Reagents and conditions: (a) DCC, HOBt, NEt3 , CH2 Cl2 , 60%, (b) H2 , 10% Pd/C, EtOH, 30 min, quant.

O

O

H2 N

O

N H

Cu

H N

N H2

O

O

O CO2Me CO2 Me

A-Cu O O

MeO2C

O

O O

MeO2C MeO2C

H2 N

O

N H2

O

O N H

Cu

H N

O

O

O

B-Cu

Fig. 2. Structures of complexes A–Cu and B–Cu.

O

CO2 Me

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however, observed by MALDI-TOF mass spectrometry. These peaks could indicate dimerisation of the complex in solution, or could alternatively be an artefact of the electrospray ionisation process – aggregation with Naþ ions in ESMS is not uncommon. Infra red spectroscopy of the complex showed the expected ligand peaks. Elemental analysis provided a good match for the composition of the complex. Initial attempts to synthesise complex B–Cu used the procedure described above. Although the reaction changed colour (from pale green to deep blue) indicating complexation may have occurred, no precipitate was formed on cooling. Attempts at recrystallisation from the liquor failed and the procedure was therefore modified. Copper carbonate and ligand 2 were dissolved in methanol and one equivalent of solid sodium hydroxide (relative to the ligand) was added to the mixture. The mixture was stirred for 6 days and a gradual colour change was observed. The product was purified by gel permeation chromatography (Sephadex LH-20, methanol). A blue band was collected from the column and evaporated to give a blue oil in 19% yield. Complex B–Cu was characterised by electrospray mass spectrometry, with the major peak in the mass spectrum being observed at m=z 1100 [M + Na]þ , and the correct intensity isotope peak being observed at m=z 1102. Peaks corresponding to [2M + Na]þ were not observed in this case. Infra red spectroscopy of the complex showed the expected ligand peaks. Elemental analysis provided a reasonable match for the composition of the complex. 2.3. Electrochemistry of copper complexes A–Cu and B– Cu As stated in the introduction, electrochemistry is a widely used method for probing the microenvironment experienced by encapsulated redox active units [6–8]. Copper(II) is a biologically important redox active metal, with the Cu(II)/Cu(I) redox couple being implicated in the function of a wide range of enzymes. Furthermore, Cu(II) can also be oxidised to Cu(III) under very oxidising conditions. The electrochemical properties of complexes A–Cu and B–Cu were therefore investigated using cyclic voltammetry in dimethylformamide (DMF) solution. Complex A–Cu was investigated at different scan rates (Fig. 3). Initially the solution was swept in the reducing direction and then subsequently swept to oxidising potentials. In each case the same components were observed in the cyclic voltammogram. The reduction peak which is tentatively assigned to the Cu(II) to Cu(I) transition was observed at a peak potential ca. )990 mV. Re-oxidation was then observed at ca. )140 mV. The large peak to peak separation and the broad reduction and oxidation peaks indicate a quasi-

Fig. 3. Cyclic voltammogram of complex A–Cu (2 mM) in DMF solution with a supporting electrolyte of 0.1 M [Bu4 N]þ [BF4 ]
. Scan rate ¼ 200 mV s
1 .

reversible process. On continued oxidation (scan rate: 200 mV s
1 ) a sharp peak was observed at +210 mV, probably a stripping peak. Stripping peaks are typically observed following electrodeposition of metals (e.g., Cu(0) from disproportionation of decomplexed Cu(I) ion). In fact, on decreasing the scan rate to 50 mV s
1 the relative peak current of this peak increased significantly, while on increasing the scan rate to 500 mV s
1 , this stripping peak disappeared, in agreement with the above analysis. On continuing the sweep in the oxidative direction an oxidation peak was observed at ca. 1310 mV. This could correspond to the oxidation of Cu(II) to Cu(III), however, given the peak height, it is possible that ligand oxidation is also occurring. This process was irreversible. It is well-known that very robust ligands would be required if Cu(III) were to be observed as a stable species [16]. Complex B–Cu was then investigated using cyclic voltammetry under the same conditions of solvent and scan rate (Fig. 4). Interestingly, the Cu(II)/Cu(I) couple was significantly more reversible than for A–Cu. At a scan rate of 50 mV s
1 , the peak to peak separation was only 150 mV (rather than ca. 850 mV as observed for A– Cu). Furthermore, the stripping peak (observed for A– Cu at +210 mV) was absent. These observations indicate that the steric bulk of ligand B enhances the stability of the reduced complex, hence preventing the release of Cu(I) ion and its subsequent disproportionation. Complex B–Cu has an additional oxidation peak at ca. 1.0 V, which can tentatively be assigned to oxidation of the ligand framework. The other oxidation peak was now observed at +1370 mV, which compares with +1310 mV for complex A–Cu. This might reflect differences between the oxidation of the ligand structures in these two complexes although it may also indicate that the oxidation of Cu(II) to Cu(III) is thermodynamically more

D.L. Stone et al. / Polyhedron 23 (2004) 1709–1717

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Fig. 5. ESR spectrum of complex A–Cu (1 mM) in frozen methanol solution.

Fig. 4. Cyclic voltammogram of complex B–Cu (2 mM) in DMF solution with a supporting electrolyte of 0.1 M [Bu4 N]þ [BF4 ]
. Scan rate ¼ 200 mV s
1 .

difficult for B–Cu than A–Cu. This would be in agreement with our previous observations of dendritic effects which have indicated that charge build up is disfavoured when the redox active unit is encapsulated within an organic shell, and is hence more distant from bulk solvent/electrolyte [8a,8b]. Interestingly, the comparison between A–Cu and B–Cu indicates that even relatively small amounts of organic functionalisation appear to have significant effects on the kinetics and thermodynamics of redox processes. 2.4. Electron spin resonance of copper complexes A–Cu and B–Cu As a d9 paramagnetic species, copper(II) is appropriate for characterisation by ESR experiments. This technique has frequently been applied for the analysis of copper complexes as well as copper ions in biological systems [17], and is a useful method of determining the geometry of a copper centre when X-ray quality crystals cannot be obtained. For a mononuclear copper(II) complex, the nuclear spin ðI ¼ 3=2Þ means that four lines would be expected in the hyperfine splitting pattern in solution. In the case of the actual axial symmetry, the spectrum is characterised by ak (G), a? (G), hai (G), g? , gk , hgi values, which can be obtained from the splitting pattern of the spectrum and compared with values for complexes of known geometry in order to define the composition of the equatorial coordination of the complex [18a]. ESR experiments were performed on 1 mM frozen solution of the complexes in methanol. The ESR spectrum of A–Cu shows the expected approximate four line hyperfine splitting (Fig. 5) The values derived from the analysis of this spectrum were gk ¼ 2:258, a ¼ 170 G and g? ¼ 2:063. These values are consistent with com-

plex A–Cu having Jahn-Teller distorted octahedral geometry. It would be expected that the amino-acid ligands will be bound in the equatorial positions in a chelate mode, whilst there will be two more distant axial ligands. The ESR spectrum of complex B–Cu was then measured (Fig. 6). The values derived from the analysis of this spectrum were gk ¼ 2:260, a ¼ 171 G and g? ¼ 2:057. These values are very similar to those obtained for A–Cu and indicate once again, an equivalent Jahn-Teller distorted octahedral geometry. This indicates that the additional steric hindrance of ligand B does not manifest itself through a change in coordination geometry. Most interesting, however, was the fact that the spectrum of B–Cu was much sharper than that of A–Cu. The broadening in the ESR spectrum of A–Cu could possibly be caused by interaction between Cu2þ ions. This ESR evidence indicates that interaction between copper(II) ions may be occurring for A–Cu. It is wellknown, for example, that carboxylate ligands can act as bridging ligands connecting two copper centres [18]. On the other hand, for complex B–Cu, where a more ideal ESR sepctrum with sharp well-defined peaks is observed, the copper centre will be more shielded from other copper nuclei by the ligand bulk, hence preventing copper–copper interactions. The mediation of copper–copper interactions is of key importance in biological chemistry [19], and these

Fig. 6. ESR spectrum of complex B–Cu (1 mM) in frozen methanol solution.

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results indicate that relatively small amounts of steric bulk (complex B–Cu only has approximately twice the molecular mass of complex A–Cu) can have a marked effect on the isolation of metal ions. The use of an organic shell to protect copper ions in this way is an important strategy in biological chemistry, and it appears that it is possible to mimic such an effect in a relatively simple manner. Work is now ongoing to extend the extent of functionalisation of these amino-acid ligands to achieve even more effective encapsulation of copper ions.

using standard (ca. 50 cm) columns packed with Sephadex LH-20 and eluted using MeOH. The electrochemical studies were carried out on an EG & G Princeton Applied Research potentiostat/galvanostat model 273 with a standard 3 electrode configuration, consisting of platinum working (0.5 mm diameter disk) and counter electrodes and an Ag/AgCl reference electrode. Each compound was studied at a concentration of 2 mM in a supporting electrolyte of 0.1 M [Bu4 N]þ [BF4 ]
using scan rates of 50–500 mV s
1 . ESR experiments were performed using a Bruker ESP300 spectrometer. Elemental analysis was performed by the microanalytical lab at The University of Manchester.

3. Conclusions 4.2. Synthesis and characterisation This article describes the synthesis of two novel amino-acid ligands and their characterisation. Furthermore, these ligands have been used in the assembly of two novel copper(II) complexes with different degrees of steric bulk at the periphery. These complexes have been fully characterised and subsequently investigated using electrochemical and ESR methods. The results indicate that even for these relatively small molecules, the differential encapsulation experienced by the metal ion in the two complexes is significant. This emphasises the important role which an organic superstructure can play (even in relatively small quantities) to shield and modify the properties of metal ions within metalloproteins and enzymes. Modelling the role played by the organic superstructure of metalloproteins remains a real challenge for bioinorganic chemists in the attempt to mimic the remarkable levels of function which can be obtained by highly evolved natural systems.

4. Experimental 4.1. Materials and methods All 1 H and 13 C NMR spectra were determined either on a Jeol-E270 (270 MHz) or a Bruker AMX 500 (500 MHz) instrument, according to requirements and referenced to residual solvent. Neat IR, carried out on oillike compounds, were measured on a Mattson Genesis Series FTIR spectrometer, whereas solid IR using KBr disks were performed using a Mattson Sirius Research FTIR spectrometer. Electrospray mass spectrometry was carried out on a Finnigan LCQ. MALDI-TOF mass spectrometry was performed at Central Science Laboratories, York. Silica column chromatography was carried out using silica gel provided by Fluorochem Ltd. (35–70 lm). Thin layer chromatography was performed on commercially available Merck aluminium backed silica plates. Preparative gel permeation chromatography was carried out using a 2 m glass column packed with Biobeads SX-1 (Biorad) eluted with CH2 Cl2 or

All protected amino acids were purchased commercially, and first generation ‘Newkome’ type branches were prepared according to literature methodology [20]. 4.2.1. N-(tert-Butoxycarbonyl)-L -glutamic acid 1-tertbutyl ester, 5-tert-butylamide, compound 3 N -(tert-Butoxycarbonyl)-L -glutamic acid 1-tert-butyl ester (1.0 g, 3.3 mmol), DCC (0.68 g, 3.3 mmol) and HOBt (0.44 g, 3.3 mmol) were dissolved in dry CH2 Cl2 (10 ml) and stirred at room temperature for 10 min. Then NEt3 (1 ml, 7.18 mmol) and tert-butylamine (0.64 g, 3.3 mmol) were added as a mixture in dry CH2 Cl2 (5 ml). The reaction was stirred under N2 at room temperature for 2 d. After this period, CH2 Cl2 (30 ml) was added to the mixture, which was then filtered into a separating funnel, and washed with sat. aq. NaHCO3 (2  50 ml) and H2 O (2  50 ml). The organic phase was dried over MgSO4 , filtered and evaporated to give the crude product. This was purified by silica gel chromatography (CH2 Cl2 :MeOH, 98:2) to yield the pure product (0.591 g, 1.65 mmol, 50%). White solid; Rf 0.45 (CH2 Cl2 –MeOH 98:2); mmax (KBr disk) 3338 m (NH), 2979 m and 2933 m (alkyl), 1721 m, and 1655 m (C@O), 1543 m, 1456 m, 1366 m, 1277 m and 1153 m, 1051 m, 848 s; dH (CDCl3 ) 1.35 (9H, s, NHC(CH3 )3 ), 1.44 and 1.46 (18H, 2s, OC(CH3 )3 ), 1.86 (2H, m, CH2 CH2 CO), 2.15 (2H, m, CH2 CH2 CO), 4.13 (1H, t, J ¼ 8.5, CH CH2 ), 5.16 and 5.20 (1H, d, J ¼ 10.0, NH CHCH2 ), 5.80 (1H, bs, NH C(CH3 )3 ); dC (CDCl3 ) 28.7, 28.3, 28.0 (CCH3 ), 29.3 (CH2 CH2 CONH), 33.8 (CH2 CONH), 51.2 (NHC(CH3 )3 ), 53.6 (NHCH), 79.8 and 82.1 (CO2 C(CH3 )3 ), 155.8 (CONH), 171.5 and 171.4 (CO2 C(CH3 )3 ); m=z (Electrospray) [M + Na]þ requires 381, found 381 (100%), 382 (19%); HRMS (CI+) (C18 H34 N2 O5 ): found 359.2552, calculated 359.2546. 4.2.2. L -Glutamic acid 5-tert-butylamide, ligand A N -(tert-Butoxycarbonyl)-L -glutamic acid 1-tert-butyl ester, 5-tert-butylamide (3) was dissolved in hydrobromic acid (45% in acetic acid) and stirred at r.t. for 2 min.

D.L. Stone et al. / Polyhedron 23 (2004) 1709–1717

The mixture was then added to ice water (20 ml), stirred for 10 min and evaporated to dryness. The resulting product was used without further purification for complex formation. Rf 0.0 (CH2 Cl2 –MeOH 90:10); dH (CD3 OD) 1.33 (9H, s, NHC(CH3 )3 ), 2.17 (2H, m, CHCH2 CH2 ), 2.47 (2H, t, J ¼ 7.0, CHCH2 CH2 ), 4.06 (1H, m, CH CH2 CH2 ); dC (CD3 OD) 28.8 (C(CH3 )3 ), 30.2 (CHCH2 ), 33.2 (CH2 CONH), 52.3 (C(CH3 )3 ), 53.6 (CHCH2 ), 170.6 (CONH), 173.5 (CO2 H); m=z (Electrospray) [M + H]þ requires 203, found 203 (100%). 4.2.3. Compound 5 N -(tert-Butoxycarbonyl)-L -glutamic acid 1-tert-butyl ester (1) (0.72 g, 2.37 mmol) was dissolved in dry CH2 Cl2 (5 ml) then DCC (0.49 g, 2.37 mmol) and HOBt (0.32 g, 2.37 mmol) were added. The reaction was stirred under N2 at r.t. for 5 min. Next, NEt3 (0.5 ml, 3.59 mmol) and the first generation ‘Newkome’ type branch 2 (0.9 g, 2.37 mmol) in dry CH2 Cl2 (5 ml) were added, and the mixture was stirred at r.t. under N2 for 2 d. After this period, CH2 Cl2 (30 ml) was added to the mixture, which was then filtered into a separating funnel and washed with sat. aq. NaHCO3 (2  50 ml) and H2 O (2  50 ml). The organic phase was dried over MgSO4 , filtered and evaporated to give product 5 (1.47 g, 2.21 mmol, 93%). Colourless oil; Rf 0.61 (CH2 Cl2 –MeOH 90:10); mmax (neat) 3370 w (NH), 2977 m and 2897 m (alkyl), 1740 s and 1675 s (C@O), 1518 s, 1438 s, 1367 s, 1254 s, 1175 s, 1112 s, 848 w; dH (CDCl3 ) 1.41 and 1.43 (18H, 2s, C(CH3 )3 ), 1.66 (2H, m, CHCH2 CH2 CO), 1.87 (2H, m, CHCH2 CH2 CO), 2.53 (6H, t, J ¼ 6.5, CH2 CO2 Me), 3.67 (21H, m, CH2 OCH2 and CO2 CH3 ), 4.10 (1H, m, CH CH2 ), 6.16 (1H, s, CONH ); dC (CDCl3 ) 27.9, 28.3 (OC(CH3 )3 ), 28.7 (CHCH2 ), 33.3 and 34.7 (CH2 CONH and CH2 CO2 ), 53.8 (CH), 57.7 (CO2 CH3 ), 59.7 (NHC(CH2 )3 ), 66.7, 69.1 (CH2 OCH2 ), 79.5, 81.8 (OC(CH3 )3 ), 155.7 (CONH), 171.6, 172.1, 172.3 (CO2 ); m/z (Electrospray) [M + Na]þ requires 687, found 687 (100%), 688 (32%); HRMS (FAB) (C30 H52 N2 O14 ): found 687.3313, calculated 687.3316. 4.2.4. Compound 7 N -(Benzyloxycarbonyl)-L -glutamic acid 1-benzyl ester (6) (1.00 g, 2.7 mmol) was dissolved in dry THF (10 ml), followed by the ‘Newkome’ type branch 4 (1.50 g, 4.0 mmol) in dry THF (10 ml). To it was added NEt3 (1 ml, 7.0 mmol) followed by DCC (0.84 g, 4.1 mmol) and HOBt (0.54 g, 4.1 mmol) in dry THF (5 ml). The reaction was stirred under N2 at r.t. for 72 h. The solvent was removed by evaporation and CH2 Cl2 (50 ml) was added. The mixture was filtered, then washed with saturated aqueous NaHCO3 (2  50 ml) and H2 O (2  50 ml). The organic phase was dried over MgSO4 for 1 h, filtered and evaporated to give the crude product (2.684 g). The crude mixture was separated on a Bi-

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obeads gel column, and the pure product 7 was isolated (1.195 g, 1.63 mmol, 60%). Colourless oil; Rf 0.7 (CH2 Cl2 –MeOH 90:10); mmax (neat) 3358 s (NH), 2952 s and 2879 s (alkyl), 1797 m, 1735 s and 1674 m (C@O), 1524 m, 1456 m, 1260 s, 1197 s, 1111 m, 744 m, 699 m; dH (CDCl3 ) 1.98 and 2.21 (4H, m, CHCH2 CH2 ), 2.50 (6H, t, J ¼ 6.5 Hz, CH2 CO2 Me), 3.63 (21H, m, CH2 OCH2 , CO2 (CH3 )3 ), 4.36 (1H, m, NHCH CH2 ), 5.07 and 5.15 (2H and 2H, 2s, PhCH2 O), 5.84 and 5.87 (1H, d, J ¼ 7.5 Hz, NH CHCH2 ), 6.15 (1H, s, CONH ), 7.31 (10H, s, C6 H5 CH2 ); dC (CDCl3 ) 27.8 (CHCH2 ), 32.9 (CH2 CONH), 34.6 (CH2 CO2 Me), 51.6 (CO2 CH3 ), 53.8 (CHCH2 ), 59.6 (NHC(CH2 )3 ), 66.6, 66.8, 67.1 and 69.0 (CH2 OCH2 and OCH2 Ph), 128.0, 128.0, 128.2, 128.3, 128.4 and 128.5 (Aryl CH), 135.3 and 136.2 (Aryl C), 156.2 (CH2 CONH), 171.9, 172.1 and 172.1 (CO2 Me and CO2 CH2 ); m=z (Electrospray) [M + Na]þ requires 755, found 755 (100), 756 (36); HRMS (FAB) (C36 H48 N2 O14 Na): found 755.3009, calculated 755.3003. 4.2.5. Ligand B Compound 7 (174 mg, 0.24 mmol) was dissolved in EtOH (10 ml) and 10% Pd-C (60 mg) was added. The reaction was stirred under H2 for 30 min, and the reaction followed by TLC (90:10 CH2 Cl2 –MeOH). The mixture was filtered through Celite, and evaporated to give the product in near quantitative yield (113 mg, 0.22 mmol, 93%). Colourless oil; Rf 0.0 CH2 Cl2 –MeOH 90:10; mmax (neat) 3362 w and 3261 w (NH and NH2 ), 3069 w, 2953 w and 2879 w (alkyl), 1737 s and 1654 m (C@O), 1543 w, 1438 w, 1364 w, 1266 w, 1198 m, 1178 m, 1109 m, 1075 m; dH (CD3 OD) 2.07 (2H, m, CHCH2 CH2 ), 2.44 (2H, m, CHCH2 CH2 ), 2.56 (6H, t, J ¼ 6.0 Hz, CH2 CO2 Me), 3.69 (22H, m, CO2 CH3 , CH2 OCH2 , CH CH2 ); dC (CD3 OD) 27.2 (CHCH2 CH2 ), 33.2 (CHCH2 CH2 ), 34.8 (CH2 CO2 Me), 51.4 (CH2 CO2 CH3 ), 54.9 CHCH2 CH2 ), 60.7 (NHC(CH2 )3 ), 67.2 and 68.8 (CH2 OCH2 ), 127.4 (CONH), 173.2 (CO2 Me), 174.5 (CO2 H); m=z (Electrospray) [M ) H]
requires 507, found 507 (100%), 508 (16%). 4.2.6. Complex A–Cu Ligand A (144 mg, 0.828 mmol) was dissolved in H2 O (20 ml) and CuCO3 (51 mg, 0.414 mmol) was added. The pH was measured and NaOH soln (10%) was added dropwise with stirring, until pH 11 was reached. The solution was stirred for 2 h, and a blue precipitate was formed. The solution was placed in an ice bath for 1 h and the precipitate was then collected by filtration to give complex A–Cu (72 mg, 0.15 mmol, 39%). Blue powder; mmax (solid KBr) 3434 s and 3296 s (NH), 2968 m and 2933 m (alkyl), 1648 s (C@O), 1456 m, 1365 s, 1226 s, 1091 s; m=z (Electrospray) [M + Na]þ requires 488, found 488 (90%), 490 (36%), 953 [2M + Na]þ (87%), 955 (100%), 957 (34%); HRMS (FAB) (C18 H35 N4 O6 Cu): found 466.1851, calculated 466.1853;

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D.L. Stone et al. / Polyhedron 23 (2004) 1709–1717

Elemental analysis C18 H34 N4 O6 Cu  2H2 O requires C, H, N: 43.06, 7.63, 11.16; found 43.62, 7.38, 11.10. 4.2.7. Complex B–Cu Ligand B (305 mg, 0.60 mmol) was dissolved in MeOH (10 ml) and CuCO3 (23 mg, 0.19 mmol) was added, followed by solid NaOH (22 mg, 0.60 mmol). The reaction was stirred at r.t. for 14 d. The resulting blue/green mixture was filtered twice and reduced in volume by rotary evaporation. The product was then purified over a Sephadex (LH-20) gel column with MeOH as eluent. The blue band was collected from the column and evaporated to dryness to give a blue oil (38 mg, 0.035 mmol, 19%). Blue viscous oil; mmax (neat) 3297 w (NH), 2954 w, 2924 w, and 2879 w (alkyl), 1739 m and 1652 m (C@O), 1559 w, 1439 w, 1370 w, 1261 w, 1176 m; m=z (Electrospray) [M + Na]þ requires 1100, found 1100 (100%), 1102 (55%), 1006 (50%); Elemental analysis C42 H70 N4 O24 Cu  2H2 O requires C, H, N: 45.26, 6.69, 5.03; found 44.77, 6.13, 4.92.

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