Stephan S. Isied

Long range electron transfer in helical polyproline II oligopeptides

Abstract A series of binuclear donor-acceptor complexes with helical polyproline bridges [(bpy) 2 Ru II L-(Pro) n -apy-Ru III (NH 3 ) 5 ] 5+ , n = 6, 7, 9, where L = 4-carboxy-4′-methyl-2,2′-bipyridine, bpy = 4,4′-bipyridine, and apy = 4-aminopyridine, were synthesized and characterized by absorption spectra, electrochemistry and HPLC. The CD spectra of the complexes confirm that they exist in the helical polyproline II structure. Intramolecular electron transfer within these complexes was studied by generating the [(bpy) 2 Ru I L · -(Pro) n -apy-Ru III (NH 3 ) 5 ] intermediate from the reaction of e aq (from pulse radiolysis) with the [(bpy) 2 Ru II L-(Pro) n -apy-Ru III (NH 3 ) 5 ] species in aqueous solution. The driving force for this reaction is estimated to be |Δ G 0 | ≈ 1.5 V. The rates ( k , 25°C) and activation parameters (Δ H ‡ (kcal/mol), Δ S ‡ (eu)) for the intra molecular electron transfer were found to be: 1.08×10 5 s −1 , 5.6, −17; 6.40 × 10 4 s −1 , 5.1, −19; 1.91 × 10 4 s −1 , 4.0, and −26 for n = 6, 7, 9 respectively. The rate ( k , 25°C) and activation parameters (Δ H ‡ (kcal/mol), Δ S ‡ (eu)) for the inter molecular reaction between [(bpy) 2 Ru I L · ] and [(NH 3 ) 5 Ru III -apy-Pro] were found to be 2.1 × 10 9 M −1 s −1 , 3.3 and −5. This series extends our studies of the distance dependence of rate versus the number of helical prolines bridging a donor and acceptor ruthenium site to a metal-to-metal distance ≈ 40 A. The weak dependence of rate versus the number of prolines observed for n = 6, 7, and 9 is very similar to that observed earlier for [(bpy) 2 Ru II L-(Pro) n -Co III (NH 3 ) 5 ], n = 4–6. The rapid rates observed at these long distances show that long range electron transfer can be observed between an appropriate donor and acceptor directly connected to the proline bridge via peptide bonds at distances similar to the diameter of a small protein.

Peptide formation in the presence of a metal ion protecting group. 4. Synthesis of penta- and hexapeptide sequences with the pentaamminecobalt(III) complex

Kinetically inert metal ion protecting groups provide radically different approaches to the protection of the terminals and side chains of amino acids and peptides. Pentaamminecobalt(III) is a useful C-terminal protecting group for sequential peptide synthesis. This is demonstrated by the synthesis of a number of amino acid complexes of the type NH 3x yc CH x O-Co(NH 3 )5 (cofAA),) where R = hydrophilic, hydrophobic, aliphatic, and aromatic amino acid side chains. The reaction of these complexes with Boc-amino acid active esters or Boc symmetric anhydrides results in the formation of (Co(AA) 1(AA)2 BoC). The Boc group is removed with 95% trifluoroacetic acid to form (Co(AA)1(AA)2), which is further used for sequential peptide synthesis. Alternatively, the ((NH3)5Com-) group is selectively removed by rapid reduction with NaBH4 or NaHS to form the N-protected peptide fragment, ((AA)1(AA)2BoC), under extremely mild conditions. Tests for racemization of the amino acid directly bound to cobalt showed no detectable loss of chirality of ((AA) 1). The detailed synthesis, monitoring, purification, and analysis of cobalt pentaammine derivatives of Leu-enkephalin (TyrGlyGlyPheL eu), Met-enkephalin (TyrGlyGlyPheMet), and the hexapeptide sequence HisGlyHisGlyHisGly are described and so is a general procedure for peptide synthesis with the cobalt(III) protecting group. The individual fragments of these peptide sequences are characterized by HPLC. The attractive features of the ((NH3)5Conl-) protecting group include its color, tripositive charge, ease of introduction, and removal. Such properties are invaluable in the purification of synthetic peptides.

New perspectives on long-range electron transfer in conformationally organized peptides and electron-transfer proteins: an experimental approach

Abstract Intramolecular electron-transfer studies across a series of peptides ranging from dipeptides to longer peptides with secondary structure (such as polyproline II and a 17-amino acid α helix) have been carried out. Metal ammine and bipyridine complexes have been used as donors and acceptors in these studies. These studies show that the rate of electron transfer is sensitive to the peptide structure and conformation, even for dipeptide bridges. For peptides with secondary structure, the connectivity of the donor and acceptor to the peptide is also important for the observation of long-range electron transfer. For example, for (bpy)2RuIIL(Pro)n-apyRuIII(NH3)5 (n = 9) (bpy2,2′ bipyridine, L4-carboxy-4′-methyl-2,2′-bipyridine, apy-4-aminopyridine), an electron transfer rate 2 × 104 s−1 was observed, while intramolecular electron transfer could not be observed for the α helix bridge in (bpy)2RuIIL[α-helical peptide]-(His)2RuIII(NH3)4 (α-helical peptide  Ala-Glu-(Ala)3Lys-Glu(Ala) 3Lys-His(Ala)3His-Ala). Comparative intramolecular electron-transfer experiments were also conducted with two cytochrome c derivatives: one modified at His 33 by [-Ru(NH3)4isn] and one modified at Met 65 by [-Fe(CN)5]. Although the His 33 and Met 65 sites are located at similar distances from the heme, and the two metal complexes possess similar reorganization energies and driving force, different rates of electron transfer varying by about 1000 were observed for the electron transfer from the heme to the metal complex. The experiments presented show that the shortest through-space distance is not always the most important determinent of the rate of electron transfer, and other factors such as the peptide structure and conformation and the connectivity of the donor and acceptor to the peptide bridge are very important.

Metal to Metal Intramolecular Electron Transfer Across Peptide and Protein Bridges

Important to the field of monoelectronics [1] and biocomputers is the study of donor-acceptor bridged complexes (Figure 1) where the donor and acceptor are held at fixed distances in well defined and characterized systems. The understanding and control of single electron transfer reactions in such molecular assemblies — be it in solution, thin-films, or on modified electrodes, should allow the development of molecular electronic components capable of use in modern computational devices. Our interest in this area has centered on the use of synthetic peptides and proteins as components of these molecular assemblies. In this lecture I will review the advances we have made in the understanding and control of long range electron transfer reactions in donor-acceptor complexes in three systems where synthetic peptides and proteins are modified with metal ion probes in solution.

Hydrogen bonding association of a ruthenium(II) bipyridine barbituric acid guest to complementary 2,6‐diaminopyridine amide hosts: guidelines for designing high binding hydrogen bonding cavities in both high‐and low‐polarity solvents

The binding between a ruthenium polypyridine guest RuG2, (where Ru = 4,4′-di-tert-butyl-bpy)2Ru (bpy = 2,2′-bipyridine) and G2 = 5-[4-(4′-methyl)-2,2′-bipyridyl]methyl-2,4,6-(1H,3H,5H)-pyrimidinetrione, and a series of host acyl derivatives of 3,5-bis[(6-aminopyrid-2-yl) amino]carbonylpyridine (R/H = n-Pr/H, phenyl/H, CF3/H, t-Bu/H, —(CH2)3-CO2−H) and 3,5-bis[(6-amino-4-isopropoxypyrid-2-yl)amino]carbonylpyridine diacetyl derivative (R/X = CH3/i-OPr) was studied by fluorescence and NMR titrations. The RuG2 (which exists in the enolate form in the presence of the hosts) forms a number of H-bonds involving the amide groups of the hosts and the carbonyl groups of the G2 for all the hosts studied. Specific 1:1 association between RuG2 and all the complementary hosts was observed with binding constants, Ka (l mol−1), for R/H in CH2Cl2 of 3 × 105 (t-Bu/H), 5 × 106 (Ph/H), 3 × 107 (n-Pr/H), 9 × 107 (CF3/H) and >108 [—(CH2)3CO2H) and for R/X of 4 × 108 (Me/i-OPr). Similar, but weaker, binding was also observed in solvents of higher donor number such as d6-acetone, d3-acetonitrile and d6-DMSO with R/X = Me/i-OPr host showing the highest binding constant in CH2Cl2, d6-acetone and d6-DMSO. Differences in the binding constants of the ruthenium guest RuG2 to these hosts are analyzed in terms of the steric, electronic and solvational changes in the structure of the host amide substituents and the polarity of the solvents used. Copyright

Peptide Formation in the Presence of Metal Ion Protecting Groups: III. The Separation of [(NH3)5Co(III)-] Amino Acids and Peptides by Reverse Phase High Pressure Liquid Chromatography

Abstract Pentaammine cobalt(III) amino acid and peptide complexes of the type can be separated rapidly under mild conditions by high pressure liquid chromatography (HPLC) using octadecyl silane derivatized columns (RP-18) with 0.2% sodium trifluoroacetate (NaTFA) in aqueous-methanol at pH 2.5. The retention time of these cobalt(III) complexes is a function of the hydrophobicity of the amino acid or peptide ligand and the total charge on the complex. In the trifluoroacetate media used for these separations, the hydrophobic ligand and the number of trifluoroacetate counter ions on the cobalt complex contribute to the selective elution of these complexes.

Preparation and charactereation of trans-bis(ethylenediamine)chloro(Boc-L-valine)-Co(III)fluoroborate: an Intermediate in Metal Mediated Peptide Synthesis

Abstract The synthesis, X-ray structure and electronic spectra of the title complex are presented,trans Bis(ethylenediamine)chloro(Boc-L-valine)Co(III)-fluoroborate (Boc=t-butyloxycarbonyl) was prepared from trans [Co(en)2OHCl]+ and an active ester form of Boc-L-valine. Blue crystals of CoCl (O4N5C14H34)·BF4 are orthorhombic, space group P212121 with a=13.170(2), b=14.545(2), c=12.6268(8)A, Z=4 and RF(RwF)=0.040 (0.042) for 2087 reflections. The structure consists of [Co(en)2(Boc-L-valine)Cl]+ cations and BF4- anions linked by an extensive hydrogen bonding network involving the BF4 F atoms and the N atoms of the cation. Co(III) is coordinated in a distorted octahedral fashion by four en N atoms, a Cl atom, and a carboxylate O atom from the Boc-L-valine ligand. The coordinated Cl and O atoms are trans and Boc-L-valine acts as a monodentate ligand. The CoN bond lengths (1.943(4)–1.955(4) A) are typical as are the axial CoCl (2.248(1) A) and CoO(CO2)(1.910(3)A) distances. The visible absorption spectrum shows two bands (λ=447, 578 nm) characteristic of trans Co(III)-(en)2L2 complexes.

Directional Electron Transfer in Ruthenium Modified Cytochrome c

Horseheart cytochrome c has been modified at His-33 with several different ruthenium redox reagents. The resulting modified proteins undergo intramolecular electron transfer where an electron is transferred between the ruthenium label and the heme c site. Depending on the redox potential of the ruthenium to the heme or vice versa. Using the two ruthenium complexes, [Ru(NH 3)5(OH 2)]2+ and [Ru(NH 3)4(N O —C—NH 2(OH 2)]2+, two proteins modified at the same site (His 33) were prepared. Based on the redox potentials of the two ruthenium reagents, intramolecular electron transfer is expected to occur with the same rate, but in opposite directions. In this poster our recent results on these modified proteins will be presented and its implication discussed.

Electron Exchange in Cytochrome c

Although the title of my talk is on cytochrome c, I would like to start out by introducing some of the results from my laboratory on intramolecular electron transfer reactions across polypeptides. Over the last several years we have synthesized and studied three series of binuclear transition metal complexes bridged by polypeptides (Table I) [1–3].