4.1 Metallocene (cobaltocenium) conjugate
Although much work has been devoted to ferrocene bioconjugates,35,43 the closely related cobaltocenium group has received considerably less attention although the cobaltcenium cation has a much higher redox potential and better chemical stability than ferrocene. Its unique electrochemical properties have, however, been exploited in enzyme biosensors and DNA detection,44 and in a more recent study on the cellular uptake and directed nuclear delivery of a cobaltocenium-NLS peptide bioconjugate.45 The lipophilic nature of the ferrocenyl moiety acts as a mimic for the bulky Trp residue, whereas the positively charged cobaltocenium moiety is isostructural to the neutral ferrocene thus allowing an assessment of additional positive charge, and thus acts as a bulky Arg-mimetic.46 Capping the N-terminus of Arg- and Trp-containing hexapeptide sequences results in a net loss of one unit of positive charge in the case of the ferrocenoyl bioconjugates, but the cobaltocenium analogues retain the overall charge of the peptide, which is favorable for their antibacterial activity.
Metzler-Nolte and co-workers reported the first nonradioactive organometallic–
peptide conjugate47 which specifically delivers the organometallic species into the nucleus of a cell. Solid-phase peptide synthesis was used to prepare the cobaltocenium conjugate of a nuclear localization signal peptide. The cobaltocenium–NLS conjugate significantly accumulates in the nucleus of HepG2 cells. The heptapeptide H-Pro-Lys-Lys-Lys-Arg-Lys-Val-OH48 was chosen as the antigen NLS which serves as an “address label” for proteins, and indicates their destination as being the cell nucleus. In addition, this heptapeptide enables the active transport of a variety of substrates through the nuclear pore complex.49 An additional protected lysine residue was introduced at the N-terminus of the NLS peptide using Wang resin as solid support. Fluorescein isothiocyanate (FITC) was used as a label to visualize the metal conjugate inside the cells. For this purpose, the N-terminal lysine residue was modified with a Mtt protecting group. After coupling of the cobaltocenium moiety to the peptide, the N-terminal Fmoc-protecting group was cleaved and cobaltocenium carboxylic acid was coupled by use of TBTU. Cleavage from the resin and deprotection of all amino acid side chains were
accomplished by concentrated trifluoroacetic acid yielding the cobaltocenium-NLS peptide bioconjugate 94 (Figure 5).
Co+ HN
N
HN NH
HN
O O
O
O
NH O
H2N
NH NH2 HN NH
O
O
HN HN
S
O
O OH
CO2H
H2N H2N
HN H2N
O
OH O
94
Figure 5. Cobaltocenium-NLS peptide bioconjugate 94.
4.2 Phenanthrenequinone diimine (phi) rhodium conjugate
Barton and co-workers have focused on the development of peptide conjugates of rhodium(III) complexes as models for sequence-selective DNA binding proteins.50 For this issue, a family of rhodium-peptide complexes (Figure 6) was synthesized by coupling short oligopeptides to the intercalating ([Rh(phi)2(phen´)]3+ (phi = phenanthrenequinone diimine; phen = phenanthroline) to explore whether the side-chain functionalities of small peptides may be used to augment metal complex recognition.51 To summarize this work, DNA site-specificity depends on the peptide side-chain functional groups. Moreover, the phi complexes of rhodium cleave DNA upon photoactivation.
Barton and co-workers used two complementary solid-phase peptide synthesis strategies for the covalent attachment of phi complexes of rhodium(III) complexes to a specific site on synthetic peptides.52 All natural amino acids except methionine were used in the synthesis, and peptides ranging from 5 to 30 amino acids were successfully coupled to the rhodium complex by standard solid-phase synthesis. The metal-peptide conjugates were either synthesized using the coordination method or by direct coupling.
In the coordination strategy the chelating ligand is first coupled onto the amino terminus of the peptide on the resin. Then, the resin-bound peptide containing the chelating ligand is reacted with [Rh(phi)2(DMF)2](OTf)3, in a manner similar to the synthesis of the parent rhodium complex. In the direct coupling strategy, the coordinatively saturated metal complex is assembled first. Then the functionalized metal complex and the terminal amine of the peptide bound to the resin are condensed in a way that is analogous to the addition of another residue to the growing peptide chain. Several conditions for the synthesis were examined where peptides are constructed using both Fmoc and t-Boc methodologies and manual as well as automated solid-phase techniques. Furthermore, a range of coupling agents was examined using both strategies. To summarize, in the case of the coordination method, several different coupling reagents were used with similar success. These reagents include DCC/HOBt, DSC, TBTU, and TSTU. With the direct coupling method, Barton et al. observed that the presence of the metal centre makes the coupling reaction less efficient. The metal-peptide complexes are more difficult to be cleaved off the resin than the metal-peptide alone.
Several linkages to the resins such as MBHA, PAM, and PEG-PAM were also examined, but variation in the linker does not affect the yield of the cleaved product.
The presence of the metal complex, does, however, significantly decrease the overall yield; furthermore it tends to inhibit the coupling reaction since coordination on the resin is of lower efficiency than the coordination of the metal complex alone in solution.
In conclusion, both strategies offer distinct advantages over solution phase methods, in that functionalization of side chains is precluded. Thus, selective attachment of the metal centre to a specific residue or to the N-terminus can be reliably accomplished.
O
4.3 Diphenylphosphineoserine (Pps) rhodium conjugate
Gilbertson et al. reported over the last decade important examples of resin bound peptide based phosphine transition metal complexes.53 Rhodium was used to prepare the first peptide-metal complexes. For the incorporation of a phosphine-containing amino acid building block it was necessary to prevent the undesirable formation of phosphine oxide. To overcome this problem, a temporary conversion of the phosphine to the phosphine sulphide54 gave rise to an amino acid that could be used in standard coupling procedures. The best route to the required amino acid involved the use of Evans´chiral oxazolidinone chemistry (Scheme 21). Addition of diphenylphosphine to acrylic acid proceeded smoothly using tetramethylammonium hydroxide as a base. Treatment with sodium thiosulfate converted the phosphine to the phosphine sulphide 97. Acid 97 is then converted to the amino acid by formation of the oxazolidinone 98. Cleavage of the chiral auxiliary and reduction of the azide 98 with tin(II) chloride gives amino acid 99 which is finally converted to the Fmoc protected amino acid 100 ready for peptide synthesis.
H OH
3) Fmoc-Cl, NaHCO3
Scheme 21. Synthesis of a phosphine-containing Fmoc protected amino acid building block for use in solid-phase peptide synthesis.
Once the desired peptide was assembled, the phosphine was regenerated by desulfurization with Raney nickel.55 The phosphine-containing amino acids were incorporated in i, i + 4 position to stabilize helix formation and thus be able to chelate one metal ion between them (Scheme 22). The peptide conjugate was synthesized by standard Fmoc solid-phase peptide synthesis on Wang resin and the diphenylphosphineoserine (Pps) was incorporated as a dipeptide with alanine
(Fmoc-Pps(sulphide)-Ala-OPfp).56 The resulting bis-phosphine ligand 101 was complexed with rhodium by stirring with RhCl(NBD)+ClO4- (NBD = norbonadiene).
N
Scheme 22. Complexation of the bis-phosphine ligand 101 with a rhodium-salt.
4.4 Mixed bidentate Pps,Cps-based rhodium conjugate
The diphenylphosphineoserine (Pps) group was later incorporated into a 12-residue peptide 103 along with a dicyclohexylphosphineoserine (Cps) (Scheme 23).57 The synthesis of the peptide conjugate, as well as the rhodium complexation, was done as described before, but on polystyrene resin.
N
-Scheme 23. Mixed bidentate Pps,Cps-based rhodium conjugates.