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J. Phys. Chem. 1996, 100, 14630-14636
Stability and Conformational Analysis of Tc-RC160 and Re-RC160: Experimental and Theoretical Analysis of the Influence of Metal Complexation on the Structural Requisites for Activity James M. Varnum and Matthew Thakur Department of Pharmacology and Structural Biology, Thomas Jefferson UniVersity, Philadelphia, PennsylVania 19107
Kevin H. Mayo Department of Biochemistry, Biomedical Engineering Center, UniVersity of Minnesota, Minneapolis, Minnesota 55455
Susan A. Jansen* Department of Chemistry, Temple UniVersity, Philadelphia, PennsylVania 19122 ReceiVed: January 3, 1996; In Final Form: March 26, 1996X
The biological significance of somatostatin and related synthetic peptide analogs is well documented. These peptide analogs have demonstrated inhibitory effects on the growth of certain tumor lines including prostate, breast, and pancreas. Metal-peptide analogs have been developed for imaging small tumors overexpressing somatostatin receptors, i.e. receptor-based scintigraphy. Receptor-based scintigraphy requires that the binding constant of the metal-peptide analog be competitive with that of the native somatostatin analog. For these applications an isotope of technetium, 99mTc is a particularly useful label. To address the effect of added label on the native peptide conformation and predicted impact on its efficacy as a therapeutic imaging agent, a combination of experimental and computational techniques were used. In this work, a complete analysis of an active Tc- or Re-labeled somatostatin analog is provided through comparison of optical, MS, and NMR data. Computational modeling has assisted in providing a profile of energetic preferences for the various coordination isomers. Coupled with optical spectral data this modeling also provides a description of the electronic states giving the characteristic bands in the optical spectra and helps to describe the redox activity and stability of the complex. On the basis of this information the structure of a major isomer is proposed. The analysis suggests that only certain coordination isomers will be accommodated by peptide without significant distortion of the “active” conformation. Furthermore, this work provides useful information for the rational design of labeled peptides with increased stability.
Introduction Somatostatin is a cyclic peptide that has a widespread inhibitory action. In the hypothalamus it inhibits the release of growth hormone (Patel 1993, Parmar 1993). In the brain, somatostatin is a important neurotransmitter and neuromodulator (Giannis 1993). In the pancreas and intestine, it functions as an autocrine regulator. Somatostatin has been demonstrated to have inhibitory affects on the growth of certain prostate, breast, and pancreatic tumors (Schally 1988, Sodah 1991, Oberg 1994). Several synthetic analogs of somatostatin have demonstrated clinically relevant inhibitory activity (Handmaker 1994). In this work, we will focus on the analog RC160, a cyclic octapeptide with the primary sequence as given below (Cai 1986): D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2
Many tumor types significantly overexpress somatostatin receptors compared to normal tissue (Reubi 1986, Lamberts 1993). This response has been exploited in the development of imaging procedures based on receptor-mediated labeling of the tumor cells. Imaging agents based on somatostatin analogs * Author to whom correspondence should be addressed. X Abstract published in AdVance ACS Abstracts, August 1, 1996.
S0022-3654(96)00024-X CCC: $12.00
show great promise in detection of small tumors and in the evaluation of metastatic progression (Oberg 1994). Effective receptor-mediated scintigraphic agents must include a “safe” and effective radiolabel such as 99mTc (Thrall 1995). In addition, the labeled peptide must bind with high affinity to the appropriate somatostatin receptor. This latter criterion has not been adequately addressed. This means that for this technique to be effective the radiolabel cannot significantly alter the important structural features of the peptide that determine receptor specificity or affinity, including steric, hydrophobic, and electrostatic interactions critical for binding. The structure of 99mTc-labeled RC160 has been inferred using NMR and molecular modeling techniques on a related rhenium-labeled peptide, Re-RC160 peptide (Varnum 1994). This study has shown that backbone conformation and side-chain topology of the analogous rhenium-labeled peptide structure closely resemble the unlabeled form of the cyclic peptide. The determined RC160 peptide conformation is shown in Figure 1. The portion of the structure in the immediate vicinity of the metal structure was not analyzed in depth. It is believed that the metal center is really an oxo-metal bound by the sulfides of the two cysteines and amine nitrogens from phenylalanine and the carboxy terminal amine. A solvent molecule completes the octahedral coordination sphere. However, a more complete characteriza© 1996 American Chemical Society
Stability and Conformational Analysis of RC160
J. Phys. Chem., Vol. 100, No. 35, 1996 14631
Figure 1. Average solution conformation of labeled RC160 as determined from restrained molecular dynamics (Varnum 1994).
tion of the metal center and analysis of the coordination isomers of the metal center have not been performed. In fact, few chemical studies have been devoted to the structural characterization of technetium peptide complexes. Some studies on manganese and rhenium homologues have offered insight into the technetium chemistry (Trop 1980). In general, the chemical similarities between rhenium and technetium correlate strongly. Greater differences are observed between manganese and technetium. For this pair, the stability of the oxidation states, atomic and ionic radii, and coordination stability vary significantly. For these reasons, many studies including this one will focus on technetium and rhenium complexes which demonstrate nearly parallel reactivities. A few reviews describe the preparation, electronic spectra, and electrochemical properties of related Tc and Re complexes (Tiatso 1994). Stability of five- and six-coordination species is well documented for both Tc and Re complexes. In general, the electronic features for these metal homologues are similar, though a bathochromic shift is observed in the electronic spectra for the technetium complexes relative to those of rhenium. In addition, technetium complexes are reduced more readily than their corresponding rhenium homologue. These reductions are typically reversible or pseudoreversible. Davison et al. have reported that the ease of reduction is somewhat modulated by the electron-withdrawing character of the ligand (Davidson 1980). Oxidation of rhenium and technetium complexes has not been reported to occur readily, and the observed oxidation appears almost wholly attributable to the ligand. Measurements of the magnetic moment have suggested a spin-paired 1A1 ground state for five-coordinate complexes of C4V symmetry. The sixth ligand allows for a Jahn-Teller distorted octahedral geometry in which no significant magnetism is observed. Such studies provide a foundation for the analysis of the rheniumor technetium-labeled somatostatin analog, metal-RC160. In this work, multiple experimental and computational objectives are applied to define important energetic terms in metal-peptide coordination. The mass spectral analysis helps to define the coordination number of the metal and ligand type. The electronic spectra of Tc and Re complexes are described by computational modeling tools. Orbital effects giving rise to stability and those implicated in the optical processes are the focus of this analysis. Electron paramagnetic resonance and electronic structure calculations help to further define frontier orbital effects. From this we can also describe the redox stability of the six-coordinate Tc and Re complexes. NMR-derived structural restraints and molecular modeling define the “steric” constraints of metal binding. Finally, this study also confirms the low-energy coordination isomer of the RC160 metal complex.
Experimental Section Preparation of the Rhenium-Labeled Somatostatin Analog, RC160. The cyclic somatostatin analog RC160, D-Phe1-Cys2-Tyr3-D-Trp4-Lys5-Val6-Cys7-Trp8-NH2
was synthesized using Merrifield solid phase methodology. The synthesis and purification procedures for the rhenium-labeled RC160 were reported elsewhere (Varnum 1994). The purity of the reaction product was analyzed by reverse phase HPLC. A single peak was observed. The molar ratio of peptide to rhenium as determined from an added technetium tracer was in the range 0.9-1.1, indicating a one-to-one molar ratio. Electronic Spectroscopy. Visible spectra of RC160, RC160 complexed with rhenium, and various other rhenium complexes were obtained in the ultraviolet/visible frequency range using a Perkin-Elmer Lambda-2 spectrophotometer. All samples were prepared in spectral grade dimethyl sulfoxide, DMSO (Fisher). The spectra were collected at room temperature using a 1 cm path length quartz cell. The results are reported in arbitrary absorption units or molar extinction coefficient (L/(mol cm)). Electron Paramagnetic Spectrometry (EPR). EPR experiments were carried out with a Bruker ER-200D spectrometer equipped with an ER035M NMR gaussmeter and VT4111 temperature control unit. The precision of the gaussmeter is 0.001 G/s, and the temperature was maintained within 0.1 °C. The experiments were performed at room temperature and 100 K in DMSO/H2O. Mass Spectrometry. Initial mass spectrometry analysis of RC160 and the metal complex was performed using either fast atom bombardment (FAB) or electrospray ionization techniques. The samples were dissolved in acetonitrile. Subsequent samples were analyzed on a LD1-7000 matrix-assisted laser desorption (MALDI) time-of-flight (TOF) mass spectrometer (Linear Scientific Inc.). The DMSO solvent was removed from the sample by repeated lyophilization, and the peptide was redissolved in acetonitrile. These solutions were then mixed with the appropriate matrix, sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid), and analyzed using standard protocols. The system was calibrated using a series of appropriately sized peptide standards. Computer Modeling. Initial modeling was performed using Discover and Insight II (Biosym Technologies Inc.), and the standard parameter set was supplied. This work was done on a Silicon Graphics 310 GTX workstation. Subsequent modeling was done using Sybyl (Tripos) and the standard Tripos force field. The latter modeling was performed using Sybyl for convenience and availability. Careful analysis of structures showed good agreement and reliability between the methods.
14632 J. Phys. Chem., Vol. 100, No. 35, 1996
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Figure 2. Representative starting model for the octahedral metal complex.
TABLE 1: Parameters Used for Extended Hu1 ckel Calculations slater exponent
element/symbol technetium/Tc
sulfur/S nitrogen/N carbon/C hydrogen/H
5s 5p 4d 3s 3p 2s 2p 2s 2p 1s
2.020 1.980 4.900 0.572 0.601 1.817 1.817 1.950 1.950 1.625 1.625 1.300
orbital energy (eV) -10.07 -5.40 -12.82
Figure 3. MALDI-TOF mass spectroscopy of the RE-RC160 complex.
-20.00 -13.30 -26.00 -13.40 -21.40 -11.40 -13.60
The starting structures were previously determined by restrained molecular modeling (Varnum 1994). These structures were modified by adding a suitable octahedrally coordinated metal. Several geometries were examined, including placing the thiolates in a cis or trans orientation. Sulfur-metal bonds were optimized to be in the range 2.255-2.314 Å. The nitrogenmetal bond lengths were restricted to the range 2.01-2.18 Å (Tiatso 1994). The M-RC160 geometry utilized in the quantum chemical modeling was optimized using the MM2/MM+ force field to normal convergence limits. A representative model structure is shown in Figure 2. The geometries of the various RC160 isomers were obtained in a similar manner. The electronic structure calculations have relied on applications of the extended Hu¨ckel method (EH). The application of the extended Hu¨ckel formalism has already demonstrated significant utility in describing hydrocarbon chemistry and has reliably produced experimental trends in organometallic and main group chemistry. The method developed by Hoffmann et al. is well suited for this study, as relative energy trends are valid and σ and π bonding terms are well described (Hoffmann 1963). Frontier orbital effects are well characterized through applications of the EH method also. The parameters used in this study are typical valence state ionization energies and Slater exponents. These are listed in Table 1. Results and Discussion Identification of the Re-RC160 Complex in Solution. A complete discussion of the M-RC160 complex requires a discussion of local and extended molecular features of the complex. Figure 3 shows the MALDI-TOF-MS obtained for the Re-RC160 complex. The highest mass peak in this spectrum is observed at m/z 1374. The structure associated with this peak is a six-coordinate Re-oxo complex in which four ligand sites are occupied by peptide-based ligands. This mass assignment is consistent with a solvent molecule, acetonitrile, coordinated to the complex as the sixth ligand. The sixth ligand appears to be acetonitrile, introduced during the preparation of the matrix for MALDI. Other solvents are known to exchange in the coordination sphere
Figure 4. Geometric isomers analyzed for an octahedrally coordinated rhenium or technetium complex.
of rhenium in solution; however, some reports suggest that fivecoordinate species are stable in solution (Tiatso 1994). Thus, the identification of the sixth ligand is critical for the analysis of the restrained molecular modeling and electronic spectra. The most intense peak occurs at m/z 1132 and corresponds to the free peptide. Characterization of Metal Complex Geometry. The solution structure of the RC160 complex has been discussed in detail elsewhere, and thus only the critical features of the structure will be described here (Varnum 1994). This structure was obtained by a combination of NMR and molecular modeling techniques, as described in the Experimental Section. The NMR experiment has defined the peptide coordination about the central metal atom in the Re-RC160. Two of the ligating agents are thiolate groups from the cysteines, and the other two are amino groups from the N-terminus and the added C-terminal amide group of the peptide. The molecular modeling refined structure is shown in Figure 4. Geometry and Stability of Coordination Isomers. Unfortunately the NMR studies did not provide a detailed analysis of the geometry or coordination isomerization about the central metal atom. However, the coordination isomerization can be assessed very effectively by the evaluation of electronic spectral data with quantum mechanical methods. In these complexes, it is understood and accepted that the rhenium is in the (+5) oxidation state. This means that the
Stability and Conformational Analysis of RC160
J. Phys. Chem., Vol. 100, No. 35, 1996 14633 TABLE 2: Summary of the Relative Energies for the Six-Coordination Isomers of Tc-RC160 from Extended Hu1 ckel Modeling isomer
total energy (eV)
stabilization energy (eV)
I-1 I-2 I-3 I-4 I-5 I-6
-1394.69 -1395.08 -1394.70 -1394.94 -1394.70 -1395.26
0.57 0.18*a 0.58 0.32 0.58 0.00*
a
* indicates the most stable isomer by the EH method.
TABLE 3: Summary of the Relative Energies for the Six-Coordination Isomers of Tc-RC160 from Molecular Mechanics Modeling
Figure 5. Jahn-Teller distortion of a d2 octahedral metal complex.
isomer
total energy (kcal/mol)
stabilization energy (kcal/mol)
I-1 I-2 I-3 I-4 I-5 I-6
0.762 -0.253 1.121 -0.481 1.127 1.910
1.243 0.228*a 1.602 0.000* 1.608 2.391
a
Figure 6. Three views of the average conformation of Re-RC160 as determined by molecular modeling. The octahedral metal complex and the complexing solvent molecule were explicitly included in these calculations.
formal metal d-orbital occupation is d2. It is well-known that partial occupation of the metal-based orbitals of six-coordination species leads to structural distortions. In fact, d1 and d2 complexes undergo significant Jahn-Teller distortions in which an asymmetry occurs in the M-L axial bonds. The Jahn-Teller distortion for a d2 complex is shown in Figure 5. The typical distortion gives a short axial M-L bond of 1.6-1.8 Å and a long axial bond ranging from 2.4 to 2.8 Å. This distortion generally reduces the antibonding nature of M-L π-states. The local symmetry about the metal is C4V, and the ground electronic state is expected to be 1A1. The model structures evaluated in this component of the analysis include significant distortion along the axial direction. This is the only structural constraint introduced in the model structure. Several probable coordination isomers were evaluated by the extended Hu¨ckel method. These isomers are shown in Figure 4. This method offers several parameters by which the stability of molecular species can be compared. Perhaps the most effective is the so-called cluster assembly energy (CAE). In extended Hu¨ckel analysis, the minimum energy is obtained when the maximum stability in covalent bonding is achieved; thus, one valid comparison of thermodynamic stability is a comparison of the total energy of the molecular system less that of its isolated atomic constituents. This effectively gives a numerical indicator of bond energy for the molecule or cluster. Since all of these species in this study have the same atomic constituents, the total energy is an valid indicator of stability. It is important to note that the extended Hu¨ckel analysis is derived from orbital overlap, and thus the differences in bonding and energy arise from covalent interactions. Those which will affect the energies of the various isomers most significantly are
* indicates the most stable isomers from MM modeling.
the metal-ligand interactions. Steric effects are not well described by EH, and therefore molecular modeling of the octahedral fragment was performed. The comparison of EH and MM data will give a more complete analysis, as the former does not predict geometry well. The data for the EH and MM are contrasted in Tables 2 and 3 for the coordination isomers of Tc-RC160. The data presented in Tables 2 and 3 suggest that the second and fourth structure should possess the greatest stability. The difference in the stabilization energies predicted by the EH method is fairly small for this application, and thus it is necessary to consider the MM data to further assess each isomer stability. Though the sixth isomer shows considerable stabilization of the electronic structure especially in the metalligand coordination sphere, it appears to be the most sterically comprised isomer and thus is not a likely choice for isomer stability. In general, greater stability is observed when the sulfides are cis to each other and the amines are cis to each other. The low-energy structures show these features and are differentiated by the relationship between the sulfide and amine coordination relative to the oxo and solvent ligands. This analysis of the isolated species clearly cannot satisfactorily define the isomer preference in the labeled peptide, especially since the peptide has a well-defined and biologically active solution structure. The conformational constraints of the peptide will define the coordination isomer preference for the active somatostatin label, I. Clearly, the peptide conformation must not be altered by metal binding if the maximum activity is to be preserved. The molecular mechanics analysis for the complete peptide shows that coordination types I-1, I-3, and I-4 are the only ones that can be accommodated in the peptide structure without significant distortion of the structure of the peptide. A calculation of the volume and surface area for these three coordination types shows striking similarity; however, the first structure shows a slightly smaller volume and surface area. The other coordination types, I-2, I-5, and I-6, show significantly larger volumes and corresponding larger surfaces areas. Structure types I-5 and I-6 are the least “sterically” stable. Table 4 shows a comparison of torsion angles determined by restrained molecular modeling for RC160 and Tc-RC160 for coordination type I-1. Five coordinate isomers were also considered in their “free” and complexed form; however, none appeared as stable as the sixcoordinate species.
14634 J. Phys. Chem., Vol. 100, No. 35, 1996
Varnum et al.
Figure 7. Isomer I-1 coordination of rhenium. The two constrained cyclic systems formed by metal complexation are labeled.
Figure 8. β turn region of Re-RC160 showing the average conformation of the biologically important side chains.
TABLE 4: Torsion Terms for RC160 and TcRC-160 from Molecular Modeling
ring (2) formed by Cys7-Phe8-NH is larger and is accordingly less strained. Only minor changes in Cys8 and Trp9 φ and ψ torsion angles were observed for the technetium complexed peptide. No significant deviations in the ω torsion angle were observed for this cyclic system. Similar effects were observed when rhenium was coordinated. After simulated annealing and extensive molecular minimization the difference between RC160 and Re-RC160 energies was less than 4 kcal/mol. None of the NOE-based distance constraints were violated in the final conformations of either the rhenium- or the technetium-labeled peptides. The proposed solution conformations of Re-RC160 and RC160 are very similar. The D-Trp-Lys side chain topology required for strong biological activity is maintained and is shown in Figure 8. Interestingly, the type II′ β turn and short antiparallel β sheet conformations of RC160 and metal-RC160 peptide structure I-1 are extraordinarily similar to one of the three conformations (Octreotide I, using the paper’s nomenclature) recently determined by X-ray crystallography for a closely related somatostatin analog octreotide (Pohl 1995). Curiously, the conformation of octreotide I appears to be influenced by intermolecular hydrogen bonding to the other two structurally distinct molecules (II and III) in the unit cell. Therefore, this may not be a favored conformation in aqueous solution. The proposed structure of RC160, labeled RC160, and octreotide conformation I may correspond to an energy minima accessible only in less polar environments, such as DMSO (strong hydrogen bond acceptor) or perhaps a somatostatin receptor. The receptor-binding site for RC160 can be viewed as a surface composed of both polar and hydrophobic regions, an environment probably better simulated by DMSO than aqueous solutions (Saulitis 1992, Seelig 1993). The proposed RC160 peptide backbone conformation may be a better estimation of the receptor-bound peptide backbone conformation. The structural information obtained in this study provides a rational basis for the design of metal-peptide complexes with increased stability. Decreasing the strain in ring 1 by increasing its size should increase stability. Alternatively, modifications of the metal-binding site may allow for the formation of different more stable coordination isomers. Analysis of the Metal Center Using EPR, UV-Visible Spectroscopy, and Molecular Modeling. A closer examination of the extended Hu¨ckel data for the metal center and the EPR spectra provides a description of the frontier orbital states of the Re-RC160 complex. The conclusions drawn from these studies explain effects identified in the electronic spectrum and further support the coordination isomerization suggested by the modeling studies. The electronic spectrum shows a fairly weak absorption, ≈ 158 L/(mol cm) at 530 nm. Another more intense absorption is observed at 380 nm. These data are for the Re complex and are shown in Figure 9. Similar features are expected for a Tc complex. The band at 380 nm has been observed in a variety of Re and Tc complexes and has been attributed to ligand to metal charge transfer.
residue
torsion angle
D-phenylalanine(1)
φ ψ χ1 χ2 φ ψ χ φ ψ χ1 χ2 f y c1 c2 φ ψ χ1 χ2 φ ψ χ φ ψ χ φ ψ χ1 χ2
cystine(2) tyrosine(3)
D-tryptophan(4)
lysine(5)
valine(6) cystine(7) tryptophan(8)
relative energy
RC-160
Tc-RC-160
-73 178 -143 146
-89 -99 -101 143 -153 143
-173 138 54 -87 51 -135 -78 97 -87 -25 -164 -176 -82 96 -58 -154 126
-158 112 61 -62 63 -135 -78 -100 -70 -21 -67 173 -108 130 -60 -170 113
-141 48 114
-141 85 67
36.1 kcal/mol
40 kcal/mol
The overall lowest energy conformation of the peptide occurs when the metal center is in coordination type I-1. The basic peptide structure was not altered by metal coordination. The type II′ β turn and short antiparallel β sheet arrangement were maintained. A comparison of the determined conformations for Tc-RC160 and the unlabeled peptide data is presented in Table 4. No appreciable differences are observed for the labeled peptide backbone for residues 3-6. The topology of the side chains of Trp4 and Lys5 are not affected by metal coordination. This is entirely consistent with the earlier NMR analysis (Varnum 1994). As a result of the complexation, two additional constrained cyclic systems 1 and 2 are formed. These are shown in Figure 7. A nine-membered ring (1) is formed by the Phe1-Cys2-SMetal. This cyclic system (1) is significantly sterically strained. The most stable conformation consistently obtained by molecular modeling contained a cis peptide bond between Phe1-Cys2 (Sukumaran 1991). The typical lower energy trans peptide bond was observed for the normal disulfide constrained peptide. The conformational space accessible to the aromatic side chain for Phe1 of Re-RC160 is similar to the unlabeled analog. The presence of the metal complex does not appear to interfere with any potential Phe1 side chain-receptor interactions. The other
Stability and Conformational Analysis of RC160
J. Phys. Chem., Vol. 100, No. 35, 1996 14635
Figure 9. Electronic spectra of RC160 and Re-RC160 dissolved in DMSO.
The intense absorption observed at 380 nm is due to ligand to metal charge transfer. The weak absorption at 530 nm is due to a Laporte allowed d-d transition for the rhenium center. This transition is characteristic of d-d transitions in sixcoordinate isomers only. Few related materials show the longer wavelength transition. The analysis of the electronic structure of the metal center suggests that this transition is a Laporte allowed d-d transition for the Re center. Analysis of the frontier states of the coordination isomers for varying oxidation states, I-1(0), I-1(+1), and I-1(+2), shows that the frontier orbitals are primarily metal based. The Jahn-Teller distortion produces a C4V metal center, and thus transitions between the frontier states become somewhat more intense as higher symmetry restrictions are removed. Though several authors suggest that five-coordinate species are stable in solution, the electronic structure calculations and MS and electronic spectral data do not suggest that such species are present in any significant quantity (Chi 1994). The long wavelength transitions appear to be characteristic of d-d transitions in six-coordinate isomers only. EPR Spectroscopy The EPR data further support this analysis. In general, EPR can be used to probe centers which have an unpaired electron. The resonance position and the g-value associated with the transition (g ) hν/βH) are determined by the chemical environment of the unpaired electron. Generally speaking, if the spinorbit contribution is high, the value of the g is shifted rather significantly from the free electron g of 2.002 319. For late transition metals, this is a positive shift; for early transition metals, a negative shift due to the sign of the spin-orbit coupling constant. The g is a tensor quantity and thus can give structural information as well. Electron spin-nuclear spin coupling is also diagnostic in EPR analysis. Frequently, this coupling gives rise to additional splitting, called hyperfine splitting, which can aid in the characterization of site geometry. The EPR spectrum observed from the Re system shows a partially oriented, well-resolved resonance characteristic of a sulfur radical. Such species have been observed in many biological systems containing disulfide connectivity or cysteine amino acids. This spectrum is shown in Figure 10. The spectral intensity suggests that a sizable fraction of the peptide is responsible for the signal. The shift in the g is from a SH• coupled to a metal center. These observations suggest that there must be a modest contribution of sulfur to the frontier states of the peptide and that a significant contribution to the total spin density must be strongly localized at the sulfur site. Only isomers 1 and 4 show a significant amount of spin density localized at the sulfur when the isomer has a modest positive
Figure 10. Electronic spin resonance spectrum of Re-RC160. The spectrum was run at 100 K. The spectrum shows a partially oriented, well-resolved resonance characteristic of a stable sulfur radical. The spectral intensity indicates that a sizable fraction of the metal-peptide complex is responsible for the signal. The shift of the g from that normally observed for a SH• is consistent with coupling to a metal center.
Figure 11. Contour plot of the spin density of the Re-RC160 I-1 isomer. The spin density was calculated using ZINDO methods. Only isomers I-1 and I-4 show a signifcant amount of spin density on the sulfur. The other isomers do not show significant sulfur contributions to the frontier orbital character or the spin density.
charge. Figure 11 shows the spin density calculated from both EH and ZINDO methods. The neutral isomer has all of its spin density localized at the metal center. The other isomers, 2, 3, 5, and 6, do not show any significant sulfur contributions to the frontier orbital character or the spin density. This suggests that a fraction of the metal-ligand species with the peptide is in a slightly oxidized state overall with coordination isomerization of either type I-1 or type I-4. Conclusions In this work we have established that the metal species in M-RC160 is six-coordinate, not five-coordinate as reported for similar species in the solid or solution state. It appears as though neutral solvent species can easily serve as the sixth ligand, and it appears that these ligating solvents are exchangeable. Multiple experimental and computational methodologies have shown that there is a strong coordination and geometric preference at the metal site. EPR analysis suggests that the metal complex may be positively charged in solution at very low temperature, ∼100 K. Treating the sulfur as bound sulfide (-1) and the oxygen as free oxide (-2) and keeping the amine and solvent ligands neutral, a complex with a net formal charge of (+1) is anticipated for ReV species. Citrate is present in the solution and remaining from the preparative procedure and thus can balance the charge. For the Re or Tc complex to exist as
14636 J. Phys. Chem., Vol. 100, No. 35, 1996 a neutral species, the amine would have to lose a proton to form an iminyl type ligand. There are no NMR data to support this; in fact, both amine protons are identifiable in the NMR spectrum of the bound Re species. The molecular mechanics simulations define conformational and coordination preferences for isolated and bound metalligand moieties. It has been noted that some of the computed coordination preferences for the metal-ligand complex will require significant distortion of the peptide if the normal metalbonding constraints are maintained. Though these structures may possess real stability in solution, they are not anticipated to be biologically active as the resulting peptide conformation is affected. A strong preference for isomers of structure type 1 and 4 was found. It is anticipated that type 1 will be more favorable, as the Jahn-Teller distortion will allow for facile solvent exchange of the ligand trans to the oxo substituent without significant interruption of the metal-peptide binding. The proposed most stable and active metal complexation geometry I-1 has little effect on peptide conformation. When either technetium or rhenium was complexed, the overall structure of the RC160 peptide was not significantly altered, and thus the biological activity should be maintained. The most stable electronic conformation is not the most stable overall, due to the steric factors. Peptide modifications that would favor the formation of the most stable electronic conformation could increase the stability of the complex. This approach provides a structural basis for the rational design of more stable metalRC160 analogs. References and Notes (1) Bystrov, V. F., Ivanov, V. T., Baisova, T. A., Ovchinnikov, Y. A. (1973) Tetrahedron 29, 873-877. (2) Cai, R. Z., Szoke, B., Lu, R., Fu, R., Redding, T. W., Schally, A. V. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 1869-1900. (3) Chi, D. Y., O’Neil, J. P., Anderson, C. J., Welch, M. J., Katzenellebogen, J. A. (1994) J. Med. Chem. 37, 928-937. (4) Coles, M., Sowemimo, V., Scanlon, D., Munro, S. A., Craik, D. J. (1993) J. Med. Chem. 36, 2658-2655. (5) Davidson, A., Orvig, C., Trop, H. S., Sohn, M., DePamphilis, B. V., Jones, A. G. (1980) Inorg. Chem. 19, 1988-1992. (6) Dewanjee, M. (1990) Semin. Nucl. Med. XX, 5-27. (7) du Preez, J. C. H., Gerber, T. I. A., Jacobs, R. (1994) J. Coord. Chem. 33, 147-160. (8) Elseviers, M., Jasper, J., Delaet, N., De Vadder, S., Pepermans, H., Tourwe, D., Van Binst, G. (1989) In Peptides, Chemistry, Structure and Biology, Proceedings of the 11th American Peptide Symposium; Rivier, J. E., Marshall, G. R., Eds.; pp 198-200. (9) Giannis, A., Kolter, T. (1993) Angew. Chem., Int. Ed. Engl. 32, 1244-1267. (10) Handmaker, H. (1994) Diagn. Imaging 94, 77-83. (11) Hoffmann, R. (1963) J. Chem. Phys. 39, 1397-1406.
Varnum et al. (12) Hoyer, D., Lubbert, H., Bruns, C. (1994) Arch. Pharmacol. 350, 441-453. (13) Huang, Z., Probstl, A., Spencer, J. R., Yamazaki, T., Murray, G. (1993) Int. J. Peptide Protein Res. 42, 352-365. (14) Huang, Z., He, Y.-B., Raynor, K.; Tallent, M., Reisine, T., Goodman, M. (1992) J. Am. Chem. Soc. 114, 9390-9401. (15) Kazmierski, W. M., Ferguson, R. D., Knapp, R. J., Lui, G. K., Yamamura, H. I., Hruby, V. J. (1992) Int. J. Pept. Protein Res. 39 (5), 401-414. (16) Kvols, L. K., Moertel, C. G., O’Connel, M. J., Schutt, A. J., Rubin, J., Hahn, R. G. (1986) New. Engl. J. Med. 315, 702-708. (17) Kvols, L. K., Brown, M. L., O’Connor, M. K., Lamberts, S. W. J. (1993) Radiology 187, 129-133. (18) Lamberts, S. W. J. (1988) Endocr. ReV. 9, 417-436. (19) Lamberts, S. W. J., Koper, J. W., Reubi, J. (1987) Eur. J. Clin. InVest. 17, 281-287. (20) Lamberts, S. W. J., Reubi, J. C., Krenning, E. P. (1993) Acta. Oncol. 32, 167-170. (21) Lee, M. T., Liebow, C., Kamer, A. R., Schally, A. V. (1991) Proc. Natl. Acad. Sci. 88, 1656-1660. (22) Oberg, K. (1994) Curr. Opin. Oncol. 6, 441-451. (23) Pan, M. G., Florio, T., Stork, P. J. S. (1992) Science 256, 12151217. (24) Patel, Y. C. (1983) New Engl. J. Med. 323, 1274-1276. (25) Parmar, H., Phillips, R. H., Lightman, S. L. (1993) Recent Results in Cancer Research: Peptides in Oncology II; Hoffken, K., Ed.; Springer-Verlag: New York, Vol. 129, pp 1-25. (26) Patel, Y. C. (1992) Somatostatin; Patel, Y. C., Yogesh, C., Eds.; Springer-Verlag: New York, pp 1-15. (27) Pohl, E., Heine, A., Sheldrick, G. M., Dauter, Z., Wilson, K. S., Kallen, J., Huber, W., Pfaffli, P. J. (1995) Acta Crystallogr. D51, 48-59. (28) Reubi, J. C., Hacki, W. H., Lamberts, S. W. J. (1987) J. Clin. Endocrinol. Metab. 65, 1127-1143. (29) Schally, A. V. (1988) Cancer Res. 48, 6977-6985. (30) Saulitis, J., Mierke, D. F., Byk, G., Gilon, C., Kessler, H. (1992) J. Am. Chem. Soc. 114, 4818-4827. (31) Seelig, J., Nebel, S., Ganz, P. Bruns, C. (1993) Biochemistry 32, 9714-9721. (32) Schubert, M. L. (1994) Curr. Opin. Gastroenterol. 10, 575-558. (33) Soudah, H. C., Hasler, C. (1991) New Engl. J. Med. 325, 15081514. (34) Srkalovic, G., Cai. R., Schally, A. V. (1990) J. Clin. Endocrinol. Metab. 70, 661-669. (35) Sukumaran, D. K., Porok, M., Lawrence, D. S. (1991) J. Am. Chem. Soc. 113, 706-707. (36) Thrall, J. H., Zeissman, H. A. (1995) Nuclear Medicine: The Requisites; Mosby Co.: St. Louis, MO, pp 555-565. (37) Tisato, F., Refosco, F., Bandoli, G., H. A. (1994) Coord. Chem. ReV. 135/136, 325-397. (38) Trop, H. S., Jones, A. G., Davison, A. (1980) J. Am. Chem. Soc. 100. (39) Van Binst, G., Tourwe, D. (1992) Pept. Res. 5 (1), 8-13. (40) Varnum, J. M., Thakur, M. L., Schally, A. V., Jansen, S. A., Mayo, K. H. (1994) J. Biol. Chem. 209, 12583-12588. (41) Veber, D. F. (1992) In Peptides: Chemistry and Biology, Proceedings of the 12th American Peptide Symposium; Rich, D. H., Gross, V. J., Eds.; ESCOM: Leiden, The Netherlands, pp 3-14.
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