NMR Studies Reveal Structural Differences between the Gallium and

Nuclear Medicine, Division of Radiological Chemistry, University Hospital Basel, CH-4031, Basel, Switzerland. Received May 19, 2004. The somatostatin ...
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NMR Studies Reveal Structural Differences between the Gallium and Yttrium Complexes of DOTA-D-Phe1-Tyr3-octreotide Mandar V. Deshmukh,† Georg Voll,† Angelika Ku¨hlewein,† Helmut Ma¨cke,‡ Jo¨rg Schmitt,‡ Horst Kessler,† and Gerd Gemmecker*,† Department Chemie, Technische Universita¨ t Mu¨ nchen, Lichtenbergstrasse 4, 85747, Garching, Germany, and Institute of Nuclear Medicine, Division of Radiological Chemistry, University Hospital Basel, CH-4031, Basel, Switzerland Received May 19, 2004

The somatostatin analogue DOTATOC, DOTA-[Tyr3]octreotide, is used for in vivo diagnosis and targeted therapy of somatostatin-receptor-positive tumors. DOTATOC consists of a disulfide-bridged octapeptide, D-Phe1-Cys2-Tyr3-D-Trp4-Lys5-Thr6-Cys7-Thr8-ol, connected to the metal chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). Two metal complexes, GaIII- and YIII-DOTATOC, were reported to differ significantly in somatostatin receptor affinity and tumor uptake. Our 1H and 13C solution NMR data and modeling studies of both compounds are in agreement with a fast conformational equilibrium of the peptide part, as previously reported for octreotide itself. However, the different coordination geometry of Ga3+ and Y3+ (6-fold and 8-fold, respectively, as known from model compounds) causes pronounced differences for the D-Phe1 residue. For YIII-DOTATOC this leads to two conformers exchanging slowly on the NMR time scale. From various NMR measurements, they could be identified as cis-trans isomers at the amide bond between DOTA chelator and first residue (D-Phe1HN) of the peptide. Introduction The radiolabeled analogues of regulatory peptides have been used recently for the contrast-enhanced diagnosis and radiotherapy of primary tumors and their metastases.1,2 The prototypes of these peptides are analogues of the naturally occurring family of somatotropin release inhibiting factors (SRIF), neuropeptides consisting of 14 and 28 amino acids. The natural peptides could not be used for clinical application because of their instability due to enzymatic degradation. This motivated the search for synthetic peptides such as octreotide, lanreotide, and vapreotide, which are successfully used in clinical applications.1,3 The basic feature of these SRIF-based peptides is the cyclization by a cysteine disulfide bridge, causing restricted conformational flexibility, and the introduction of the D-Trp4-Lys5 sequence (originally Trp8-Lys9 in the somatostatin sequence) in the i + 1 and i + 2 positions of the β-turn, respectively. Further modifications for radiolabeling of these peptides were used successfully for the in vivo localization of SRIF-receptor-positive tumors.4 The isotopes 111In, 99mTc, 186/188Re, 66/67/68Ga, 177Lu, and 90Y were among the radionuclides that were tested for this purpose. The primary structure of such peptides connected to a metal ion complex is shown in Figure 1 for the most widely used macrocyclic chelator, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). DOTA usually confers very high kinetic and thermodynamic stability to its metal complexes. 111InIIIDOTATOC (i.e., [111InIII-DOTA,Tyr3]octreotide) and 90YIII-DOTATOC have been shown to be excellent * Corresponding author: Phone: +49-89-28913308. Fax: +49-8928913210. E-mail: [email protected]. † Technische Universita ¨ t Mu¨nchen. ‡ University Hospital Basel.

Figure 1. Primary structure of DOTATOC. The peptidic part consists of D-Phe1-Tyr3-octreotide; the DOTA chelator is attached to the N terminus via an amide bond. In this study a metal ion (gallium or yttrium, for GaIII- and YIII-DOTATOC, respectively; not shown here) is complexed by the four DOTA nitrogens and additional carboxyl oxygens, depending on its ionic radius (cf. Figure 6).

targeting and therapeutic agents in animal models and in patients.2,5,6 The peptidic part of the MIII-DOTATOC compounds consists of [Tyr3]octreotide. The early NMR studies of van Binst et al. in water and in DMSO-d6 solution7-9 indicated that the octreotide adopts a predominant antiparallel β-sheet conformation characterized by a type II′ β-like turn across residues D-Trp4 and Lys5. Similar conformations were obtained in a watermethanol solvent system.10 In a recent study, Melacini et al.11 have used NOE restraints for molecular dynamics calculations. Violations of NOE distance and 3J(HNHR) dihedral angle restraints showed that the NMR data on octreotide could not be explained by a single conformation. Instead, Melacini et al. found an equilib-

10.1021/jm0496335 CCC: $30.25 © 2005 American Chemical Society Published on Web 02/10/2005

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rium between antiparallel β-sheet structures and conformations in which the C-terminal residues fold into a 310-helix-like array or a similar helical ensemble. In all these studies, the focus of structure determination was solely on the peptide sequence, without the metal chelator attached. In several studies the properties of DOTATOC labeled with 67Ga, 111In, and 90Y were investigated in vitro and in vivo. Specifically, the IC50 value of GaIII-DOTATOC, measured in a (subtype SSTR2) receptor binding assay with [125I]-[Leu8,D-Trp22,Trp25]somatostatin-28 as a radioligand, was about 5 times higher than the value of YIII-DOTATOC (0.46 nM vs 2.2 nM, respectively). 12 In the same study, biodistribution data in an AR4-2Jbearing nude mouse model also showed differences for the two radiopeptides, with a more than 2 times higher tumor uptake for 67GaIII-DOTATOC. Moreover, the kidney uptake of 67GaIII-DOTATOC was significantly lower than that for 90YIII-DOTATOC. The very good performance of 67GaIII-DOTATOC in vitro and in the animal model prompted different groups to study 68GaIII-DOTATOC as a PET tracer. 13,14 Gallium-68 is especially attractive, since it has a 68 min half-life time and is generator produced, with a very favorable 280 d half-life time of the parent isotope 68Ge. The human data look indeed very promising and parallel the preclinical results. The reasons for the significant differences between GaIII- and YIII-DOTATOC are far from being fully understood. The improved kidney clearance of the GaIII compound is most probably caused by its additional free carboxylate group in the DOTA part, as found in the crystal structures of the model compounds GaIII- and YIII-DOTA-D-Phe.12 However, so far there are no structural data available to explain the observed differences in receptor affinity and tumor uptake, since attempts to crystallize the complete DOTATOC complexes have failed. Here we present structural results based on 1H and 13C NMR data of the GaIII- and YIII-complexes of DOTATOC in aqueous solution. While the peptidic parts of GaIII- and YIII-DOTATOC exhibit similar solution conformations, i.e., a fast equilibrium of a 310-helical and a β-sheetlike structure, the specific metal coordination geometry in YIII-DOTA-D-Phe1 causes an additional slow cis-trans isomerization about the DOTA-D-Phe1 amide bond.

two signal sets is 67:33, as determined by integration of several carefully deconvoluted resonances. In the ROESY spectrum of YIII-DOTATOC, weak exchange cross-peaks can be observed between the two sets of signals, indicating the existence of slow exchange between them15 (Figure 3). Therefore, this double signal set clearly represents two different solution conformations for YIII-DOTATOC, slowly interconverting on the NMR time scale, which will henceforth be referred to as the major and minor conformations, denoting the more and less populated conformer, respectively. The existence of such separate signal sets has not been reported in earlier NMR studies on similar DOTA model compounds.16 The ratio of the two conformers was found to be temperature dependent (cf. Supporting Information). At lower temperature (275 K), clear and distinct resonances (average proton line width ∼ 4-6 Hz) are observed for both conformers, with a ratio of 55:45. Upon temperature increase, a broadening of the amide proton resonances is observed, with an average proton line width of ∼20 Hz at 310 K, due to an accelerated exchange of the HN protons with the solvent. However, no coalescence occurs between the various resonances of the major and minor conformers, even at temperatures well above 330 K, as can be judged from well-resolved 2D 1H-13C HSQC spectra (data not shown). Compared to the other amide signals, the D-Phe1-HN protons also show an unusual line broadening, in addition to their pronounced downfield shift. Both features can be attributed to a complex formation between the carbonyl oxygen of the DOTA-D-Phe1 peptide bond and the metal ion, as seen in the X-ray crystal structure of the model peptide YIII-DOTA-D-Phe-NH2.12 The association of the amide carbonyl with the metal ion causes the D-Phe1 amide proton to resonate further downfield, together with an increase of its acidity and hence solvent exchange rate, resulting in a larger line width compared to other amide resonances in the peptide. The 1H and 13C chemical shift differences between corresponding atoms of the two conformations are largest in the vicinity to DOTA (see Supporting Information for chemical shift tables). Specifically, the 1H chemical shift differences between the two conformers follow the order D-Phe1 ≈ Cys2 > Tyr3 > Cys7 > D-Trp4, whereas they are practically absent for Lys5 and Thr6. The 13C shifts behave in a similar way: the R and β carbons of D-Phe1, Cys2, Tyr3, and Cys7 show two wellseparated carbon chemical shifts, indicating two different environments, whereas the other carbon atoms of the peptide part remain essentially unaffected. Interestingly, L-Thr(ol),8 though being close to D-Phe1 in the primary structure of the peptide, nevertheless shows only one signal set, except for a small chemical shift difference for its amide proton. Chemical shifts could not be unambiguously assigned for the DOTA moiety due to its high symmetry and resulting spectral complexity; therefore, chemical shift differences between the two conformers in the DOTA ligand could not be determined. However, one of the DOTA protons in the major conformation resonates at a characteristic upfield shift of 1.54 ppm (corresponding carbon shift, 58.49 ppm). This shift can only be explained if the proton is spatially close to the D-Phe1

Results and Discussion 1H NMR and Spectral Assignments. The 1D proton NMR spectra of GaIII- and YIII-DOTATOC are shown in Figure 2. From a set of 2D homo- and heteronuclear NMR experiments (cf. Materials and Methods), all 1H and 13C resonances of GaIII- and YIIIDOTATOC could be assigned, except for the highly symmetric DOTA parts, where no unambiguous chemical shift assignment was possible. Characterization of GaIII- and YIII-DOTATOC. The 1D proton spectrum of GaIII-DOTATOC exhibits just a single set of NMR signals (Figure 2a). In contrast, the 1H spectrum of YIII-DOTATOC shows a second signal set consisting of weaker resonance lines, most clearly observable for the downfield signal of DPhe1-HN (Figure 2b). At 290 K the ratio between the

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Figure 2. 1D proton NMR spectra of GaIII-DOTATOC (a) and YIII-DOTATOC (b) at 290 K; only the amide region is shown. Amide proton shifts are quite similar for both compounds (cf. assignments shown here). However, for YIII-DOTATOC, the D-Phe1HN resonance appears at ca. 9.5 ppm, a much higher value than expected for an amide proton. In addition, the spectrum shows the presence of two signal sets, most clearly seen for the D-Phe1HN resonance (insert).

phenyl ring and located above the ring plane, thus being influenced by the anisotropic ring current. Indeed, an NOE cross-peak can be observed between this specific DOTA proton and the Hδ and H protons of D-Phe1, as well as NOEs between aromatic protons of D-Phe1 and other DOTA protons in the major conformation (data not shown). Identification of the Two Conformations of YIIIDOTATOC. There are three possible explanations for the two conformations in the YIII-DOTATOC, namely, two slowly exchanging conformations of the peptide part, the two well-known m/M diastereomeric conformations of the chelator often found in LnIII-DOTA complexes, or a cis-trans isomerization occurring at the amide bond in the linker (i.e., between the carboxylic carbon of the acetate side chain of YIII-DOTA and the amide nitrogen of D-Phe1).

A first clue can be derived from the observation that most of the carbon atoms of the peptide part of YIIIDOTATOC show only one single NMR signal. The chemical shifts of these atoms are practically identical with the corresponding carbons of the single signal set of GaIII-DOTATOC (Figure 4). For the rest of the peptidic YIII-DOTATOC carbons, the chemical shift differences in the two conformations are quite small. Together with the very similar NOE pattern of the GaIII and YIII complexes, this practically excludes a conformational change of the peptide part (see Supporting Information for NOE tables), and the second signal set must be caused by the DOTA or linker sections of the molecule. DOTA-lanthanide(III) complexes have already been extensively studied by various methods, and they are known to exhibit a square-antiprismatic geometry (8-

Ga and Y Complexes of DOTA-D-Phe1-Tyr3-octreotide

Figure 3. Contour plot of low field region of the 2D ROESY spectrum of YIII-DOTATOC (290 K). Positive exchange crosspeaks (black) and negative NOE cross-peaks (red) can be distinguished, demonstrating the existence of two slowly interchanging conformations for this peptide (cf. annotations).19

fold coordination with four nitrogens and four oxygens around the lanthanide ion). The arrangement of the ethylene bridges and the positioning of the acetate side chains give rise to four exchanging basic conformations, commonly denoted as m1, m2, M1, and M2. 17 Here, m1 and M1 (or m2 and M2) describe the different diastereomers, while m1 and m2 (or M1 and M2) constitute enantiomeric forms that normally cannot be distinguished by NMR. On the other hand, GaIII-DOTA-DPhe-NH2 shows a 6-fold octahedral coordination geometry, with four nitrogens and two oxygens of the carboxylate arm complexing the central ion.12 YIII-DOTA, as a pseudo-lanthanide complex, could be generally expected to show four conformations in solution (m1, m2, M1, and M2). However, it has been found thatsdue to its specific ionic radiussYIII-DOTA exhibits exclusively the M conformation in solution17 (while it adopts only the m conformation in the crystalline form12). Nevertheless, it is principally conceivable

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that addition of the bulky peptide part in YIII-DOTATOC might influence the conformational equilibrium to give rise to a second DOTA conformation. A comparison of the carbon chemical shifts of the DOTA region in the GaIII- and YIII-DOTATOC should give further insight into the probability of such a conformational equilibrium. In an overlay of the 1H,13C HSQC spectra of GaIII- and YIII-DOTATOC (Figure 4), the carbon chemical shifts of the DOTA part show a behavior similar to that observed for the peptide signals. For the DOTA part of GaIII-DOTATOC, 12 distinct carbon chemical shifts could be identified (corresponding to the 12 different proton-bearing carbon positions in the molecule), while for YIII-DOTATOC 16 carbon resonances could be identified. However, in case of the existence of two diastereomeric conformations (m and M), 24 distinct carbon chemical shifts should have been observed. If in our case this interconversion was slow enough to lead to split peptide signals, the effect on the DOTA signals should be even more pronounced, i.e., two clearly separated signals would be expected for all DOTA signals, not just for four out of 12. In addition, Aime et al. have studied the LuIII-DOTA complex by solution NMR.18 They have reported distinctive carbon chemical shifts for the m and M conformations in the DOTA ring: 57.6/56.9/67.4 ppm for NCCN/ NCCN/NCCO in the M form and 55.9/50.9/61.5 ppm for the m conformer, respectively. In YIII-DOTATOC, the carbon chemical shifts for the DOTA signals occur at 54.08-56.32, 60.76-63.63, and 65.24-66.70 ppm at 275 K (no more degenerate due to the attached peptide). This is in good agreement with the carbon chemical shifts found for the M conformer of LuIII-DOTA, but incompatible with the values for the m form.18 LuIII and YIII have very similar ionic radii (0.97 and 1.04 Å) and are both diamagnetic; hence, a direct comparison of the carbon chemical shifts should indeed be meaningful. Clearly, if YIII-DOTA would have adopted an m form as one of its conformations, the corresponding carbon chemical shifts should be pronouncedly shifted toward lower frequencies. For LuIII-DOTA it has also been reported that the coalescence of proton resonances occurs around 310 K, corresponding to an energy barrier of ∼60 kJ/mol for the m/M transition.18 If the same exchange between m

Figure 4. Comparison of the 1H,13C HSQC patterns of GaIII- (black) and YIII-DOTATOC (red). Panels a and b display the aliphatic region containing mainly CH3 and CH2 correlations. Panel c shows the CR-HR correlations and the crowded DOTA region (not assigned); panel d depicts the aromatic region (not assigned). Agreement between the chemical shifts of peptidic protons and carbons of GaIII- and YIII-DOTATOC (panels a, b, and d) suggests that the peptide conformation is very similar for both. Dispersed chemical shifts in the DOTA region (panel c) is indicative of the presence of different conformations near this region. The presence of only 16 carbons for the DOTA region of YIII-DOTATOC reveals that the conformational exchange affects only a certain region of DOTA, suggesting an amide cis-trans isomerization across the linker, i.e. the (DOTA)CH2CO-D-Phe1HN bond.

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and M was responsible for the second signal set in YIIIDOTATOC, then coalescence should occur in the same temperature range. However, in our measurements hardly any change was observed in the splitting of the 1H and 13C resonances of the two conformations over the whole temperature range up to 330 K (data not shown)sa clear indication that the energy barrier is significantly higher for the conformational exchange observed in YIII-DOTATOC than known for the m/M transition. Our NMR data suggest a coalescence temperature of g350 K for YIII-DOTATOC, corresponding to a lower limit for the energy barrier of 69 kJ/mol. Alternatively, from the integration of the Cys2 amide exchange cross-peaks, a rate constant of k ≈ 0.155 s-1 can be derived at 275 K, resulting in a value for ∆Gq of 71 kJ/mol. 19 These values are in good agreement with the approximately 80 kJ/mol expected for a peptide bond cis-trans isomerization. On the basis of all these facts, an amide cis-trans isomerization across the linker, i.e. the (DOTA)CH2COD-Phe1HN bond, seems the only possible explanation. This would also explain the observations that the NMR signals of D-Phe1 are most effected by the conformational exchange and that only four carbons in the DOTA part of YIII-DOTATOC resonate at two different frequencies. Cis-Trans Isomerism in YIII-DOTATOC. Upon closer inspection of the NMR data, a very weak NOE cross-peak between D-Phe1HN of the major conformation to the nearest CH2 group of DOTA could be observed, indicating that the major conformation is trans configured. To confirm these findings, an additional ROESY spectrum was recorded at 275 K for better resolution and higher intensity of the D-Phe1 amide signals. A set of 2D experiments (TOCSY; 1H,13C HSQC; 1H,13C HMBC) was run at this temperature to reassign all proton and carbon resonances. At 275 K, the above-mentioned NOE correlation appears as a relatively strong cross-peak in the spectrum (Figure 5). On the other hand, only a very weak cross-peak exists for the minor conformation. However, on the basis of purely geometric considerations, in the trans configuration D-Phe1HN should give rise to two NOE cross-peaks, one corresponding to an average distance of 2.5 Å (to the CH2 group of the covalently attached acetate arm) and the other with an average distance of 3.8 Å (to one of the CH2 groups of the cyclen backbone of DOTA). The latter NOE is absent in the ROESY spectrum, probably due to its weaker nature and the still large line width of the D-Phe1 amide. In a similar consideration, for the cis configuration two NOE cross-peaks should be observed corresponding to average distances of 3.6 and 4.5 Å (the first between D-Phe1HN and the CH2 group of the covalently attached acetate arm and the latter between D-Phe1HN and one of the CH2 groups of the cyclen backbone of DOTA). If the conformational exchange was occurring in the DOTA part (i.e., between the m and M form) and the amide bond was trans configured in both conformations, then two strong cross-peaks would be expected from the two D-Phe1HN resonances to the CH2 group of the covalently attached acetate arm, with a distance of ∼2.5 Å. Absence of this strong NOE in the minor signal set of YIII-DOTATOC again rules out the possibility of conformational exchange in the DOTA part.

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Figure 5. NOE cross-peaks between the acetate-CH2 of DOTA and the D-Phe1HN of YIII-DOTATOC (275 K). Left row (at 9.60 ppm): major conformation of D-Phe1HN. Right row (at 9.36 ppm): minor conformation of D-Phe1HN. The NOE pattern indicates that only the major conformation corresponds to a trans-configured amide bond, showing a strong cross-peak with a DOTA proton (and one of its own β protons). In contrast, the cis conformation (minor) displays only a much weaker cross-peak to a DOTA signal (and both its β protons). NOE intensities for distance calculation (see text) are corrected for the appropriate population ratio.

Interestingly, no NOE cross-peak could be detected between the two DOTA protons at 3.44 and 3.56 ppm (cf. Figure 5). This suggests that both belong to the same group in the two different conformations (although no exchange cross-peak could be observed). It seems plausible that the DOTA 1H resonances at 3.44 and 3.56 ppm belong to the CH2 group of the covalently attached acetate arm. The conversion of the NOE intensities measured for the D-Phe1HN-DOTA cross-peaks into distances (after correcting for the appropriate population ratios) resulted in some discrepancy from the distances expected from the geometric considerations. The NOEs correspond to distances of 3.5 Å (2.5 Å) for the major conformation and 4.5 Å (3.6 Å) for the minor conformation (expected values in parentheses). However, the D-Phe1HN signals are much broader than all other 1H resonances (line width major, 38 Hz; minor, 42 Hz; peptide amide protons, ∼12 Hz at 275 K). Obviously, the large line width and hence the existence of significant alternative relaxation mechanisms could readily explain the reduced absolute NOE signal intensities for the D-Phe1HN resonances. Nevertheless, the observed large intensity differences between the NOE cross-peaks of the two conformations agree very well with a cis-trans isomerization. The ROESY spectrum at 275 K also shows a correlation between D-Phe1HN and only one of the D-Phe1Ηβ protons in the major conformation, whereas in the minor conformation, D-Phe1HN correlates with both Hβ protons, pointing to different side chain conformations of D-Phe1. Structure Calculations of the Peptide Part. Structure calculations based on NOE-derived distance restraints were performed for the peptidic moieties of

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GaIII- and YIII-DOTATOC, to check for possible structural changes induced by the DOTA parts. For the calculations of GaIII-DOTATOC, 64 such restraints were used. Due to the excessive overlap between the two signal sets of YIII-DOTATOC, only 27 and 28 restraints could be unambiguously extracted and used for calculations for the major and minor signal set, respectively. Since the 1H signals of the DOTA chelator could not be assigned unambiguously, no restraints were included for this part for both GaIII- and YIIIDOTATOC (this also includes the very weak NOEs from D-Phe1HN). Initial conformational searches were performed with distance geometry (DG) calculations; further refinement was done by molecular dynamics (MD) simulations with the DISCOVER program package (version 2.9.8).20 To take the metal ions into account, all dynamics simulations were performed with the ESFF force field implemented in DISCOVER. To allow conformational transitions during the simulation runs, time-averaged distance restraint protocols21-24 were utilized in the form of an in-house written extension for the DISCOVER package.25,26 Due to the absence of HR(i)-HR(i+1) cross-peaks in the ROESY spectra, all peptide bonds (except for D-Phe1HN in the YIII-DOTATOC) were restricted to the trans configuration in all structure calculations. For the major and minor conformations of YIII-DOTATOC, calculations were performed separately, with the amide bond between D-Phe1HN and the DOTA moiety set to trans or cis, respectively. Since the peptide parts of GaIII-DOTATOC and both conformations of YIIIDOTATOC consist of more than a single conformation in solution, the initial DG calculations (100 structures for each dataset) led to somewhat distorted structures. Both the antiparallel β-sheet structures and the 310helical structures proposed by Melacini et al.11 were found in the DG ensembles of all three NMR datasets (GaIII-DOTATOC and YIII-DOTATOC in the minor and major conformation). Time-Averaged MD Simulations. Therefore, from each dataset those sheet and helical structures fulfilling the experimental data best were chosen as starting structures for further MD simulations. With each starting structure, a restrained dynamics simulation of 500 ps duration was performed with time-averaged distance restraint protocols. The resulting trajectories were then clustered with NMRCLUST. Since no experimental NOE data had been available for the DOTA sections, superposition and clustering were based on the peptide cycle, i.e., the backbone atoms of the fragment Cys2Tyr3-D-Trp4-Lys5-Thr6-Cys7 plus the disulfide bridge. The cluster analysis clearly revealed the highly flexible nature of the peptidic parts of GaIII- as well as YIIIDOTATOC. Both the sheet and helical conformations were represented in the trajectories of all three datasets, in addition to a variety of other conformations. A thorough variation of all critical parameters of the timeaveraged distance restraints (exponential decay time τ, the force constants of the restraints, and simulation time) did not change this finding. Because of overlap of the cysteine Hβ resonances, the experimental data do not give direct information on the dihedral angle CSSC of the disulfide bridge, which can be either +90° or -90°. Interestingly, both the helical

and the sheet conformations of GaIII- and YIII-DOTATOC are able to assume stable minima with both angles in the ESFF force field. To test the influence of this angle in the starting structures, a set of starting conformations was generated for both GaIII- and YIIIDOTATOC by minimization in ESFF, with all possible permutations of idealized helical and sheet geometry, as well as both disulfide bridge angles. With each of the starting structures a 500 ps dynamics simulation was performed and analyzed as described above. For YIII-DOTATOC, the disulfide bridge dihedral angle remained +90° in the most populated clusters of the minor conformation and -90° for the major conformation, regardless of its value in the starting structure. Similarly, the simulation of GaIII-DOTATOC resulted in an angle CSSC of -90° in the most populated cluster (sheet conformation), but also yielded an angle CSSC of +90° in the less populated clusters (helical conformation). Conclusions The results of these simulations indicate that the peptidic parts of both compounds can be characterized by a fast equilibrium of two predominant conformations, displaying a helical and a sheetlike structure, as had been shown for octreotide alone. Specifically, the peptidic moieties of both NMR signal sets of YIII-DOTATOC show essentially the same helical and sheetlike contributions as the GaIII complex; only a minor shift of the conformational equilibrium between the sheet and helical forms seems possible from the MD simulations. Therefore, the previously reported significant differences in bioavailability between GaIII- and YIII-DOTATOC12 must be due solely to the differences found in the D-Phe1 linker: in the YIII complex this residue is involved in the metal coordination sphere, giving rise to two separate NMR signal sets, while in GaIIIDOTATOC it assumes an essentially extended conformation. An investigation into the nature of the two observable signal sets of YIII-DOTATOC by variable temperature NMR and various 2D NMR experiments confirmed a cis-trans isomerization across the DOTApeptide linker, i.e., the (DOTA)CH2CO-D-Phe1HN amide bond (Figure 6). This phenomenon is a direct effect of the incorporation of the carbonyl oxygen of this amide bond into the coordination sphere of the YIII ion. The resulting conformational differences at the D-Phe1 residue represent the only plausible structural cause for the significant differences in the biological activities in vivo of GaIII- and YIII-DOTATOC. This is in agreement with the finding that not only the orientation of the side chains of Tyr3, D-Trp4, and Lys5 but also that of D-Phe1 play an important role in the binding of the peptide to the somatostatin receptor.11 In addition, our results agree with recent observations that large hydrophobic (specifically, Phe) side chains in positions 1 and 8 could be responsible for enhanced SSTR-2 receptor specificity (vs SSTR-5).27 Experimental Section Abbreviations: COSY, correlated spectroscopy; DG, distance geometry; DMSO, dimethyl sulfoxide; DOTA, 1,4,7,10tetraazacyclododecane-1,4,7,10-tetraacetic acid; DOTATOC, DOTA-Tyr3-octreotide; DQF-COSY, double quantum filtered COSY; E.COSY, exclusive correlation spectroscopy; ESI-MS,

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Figure 6. Stereo models showing the cis (a) and trans (b) forms of the amide bond between DOTA and D-Phe1 in YIII-DOTATOC. Due to the participation of the amide carbonyl oxygen in the metal coordination sphere, steric interactions are comparable for both isomers. electron spray ionization mass spectroscopy; ESFF, extensible systematic force field; Fmoc, 9-fluorenylmethyloxycarbonyl; HMBC, heteronuclear multiple-bond correlation; HSQC, heteronuclear single quantum correlation; HPLC, high performance liquid chromatography; IC50, inhibitory concentration 50%; MD, molecular dynamics; MHz, megahertz; NOE, nuclear Overhauser enhancement; NOESY, nuclear Overhauser and exchange spectroscopy; PET, positron emission tomography; ppm, parts per million; ROESY, rotating frame Overhauser effect spectroscopy; RP-HPLC, reverse phase HPLC; SRIF, somatotropin release inhibiting factors; SSTR, somatostatin seven transmembrane receptor; TSPA, 3-(trimethylsilyl)propionic acid; TOCSY, total correlation spectroscopy; WATERGATE, water suppression by gradient-tailored excitation. Sample Preparation. All chemicals were obtained from commercial sources and used without further purification. Trityl chloride-resin was obtained from Pep Chem (Tu¨bingen, Germany), and all Fmoc-protected amino acids were commercially available from Nova Biochem AG (La¨ufelfingen, Switzerland). The prochelator DOTA(tBu)3 was synthesized according to Heppeler et al. 12 Synthesis of Peptides. The peptide synthesis was performed on a semiautomatic peptide synthesizer available from Rink Combichem Technologies (Bubendorf, Switzerland) according to a general procedure described previously.28 Stan-

dard Fmoc chemistry on 2-chlorotrityl chloride resin was used, and the side chains of the amino acids were protected with the following groups: Cys, acetamidomethyl; Lys and D-Trp, tert-butoxycarbonyl; Thr and Tyr, tert-butyl. DOTA was coupled as its tris(tBu) ester. The DOTA conjugate was purified using preparative HPLC. The GaIII and YIII complexes were synthesized by heating DOTATOC with a two molar excess of Ga(NO3)3 and YCl3, respectively, in sodium acetate buffer (pH 5.0) at 100 °C for 25 min. The mixtures were loaded onto a Sep-Pak C18 cartridge (Millipore, Switzerland) preactivated with 5 mL of methanol followed by 10 mL of water. First, free metal ions were washed from the cartridge with water; the MIII-DOTA-peptide was then eluted with MeOH, followed by evaporation of the solvent. The metallopeptides were lyophilized and characterized by ESI-MS and RP-HPLC. The purity was >98%. NMR Experiments. MetalIII-DOTATOC samples were prepared in 90:10 H2O:D2O solvent (pH 6.0), with concentrations of 9.0 and 9.7 mM for GaIII- and YIII-DOTATOC, respectively. All NMR experiments were performed on a BRUKER 600 MHz NMR spectrometer, equipped with a triple resonance probe with gradient pulse facility and temperature control unit. Generally, NMR experiments were recorded at 290 K, yielding optimum resolution in the 1D spectrum; additional measurements at 275 K were performed for YIII-

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DOTATOC because of the narrower line width of the D-Phe1 amide signals. Standard pulse programs were used for data acquisition, but occasional modifications were incorporated in order to suppress artifacts. A WATERGATE29,30 sequence was used in all NMR experiments for effective water signal suppression. The D2O signal was used throughout all experiments for achieving a field frequency lock condition. All spectra were calibrated with 3-(trimethylsilyl)propionic acid sodium salt (TSPA) as an external standard at 0 ppm in the proton dimension, whereas carbon chemical shifts were calibrated indirectly.31-33 Homonuclear 2D NMR experiments such as TOCSY,34 DQF-COSY,35 and E.COSY36 were used for 1H chemical shifts assignment. 13C chemical shifts were determined from heteronuclear 2D HSQC37 and HMBC38 experiments. Distance restraints were derived from 2D offset compensated ROESY39 (80 ms mixing time) and NOESY40 experiments with 100 ms NOE mixing time. Data processing and analysis were performed using Bruker XWINNMR software (version 3.2) with standard data processing tools and baseline correction. MD Calculations. Interproton distances were calculated from integration of the offset compensated cross-peaks39 of the ROESY spectra. A tolerance of (10% was applied on these distances to derive lower and upper bounds as distance restraints for structure calculations. All distance geometry calculations were performed with a modified version of the DISGEO program package by Mierke et al. 20,41-44 Molecular Dynamics simulations were carried out using the DISCOVER program package (version 2.9.8)20 with time-averaged distance restraint protocols21-24 in the form of an in-house written extension.25,26 All dynamics simulations were performed in the ESFF force field as implemented in DISCOVER. The program NMRCLUST45 was used to sort the frames of the dynamics trajectories into structural families.

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Acknowledgment. The authors acknowledge financial support from the Fonds der Chemischen Industrie (H.K. and G.G.), the Deutsche Forschungsgemeinschaft (H.K. and G.G.), and the Dr.-Ing. Leonhard LorenzStiftung, Munich (G.G.). H.M. and J.S. acknowledge financial support from the Swiss National Science Foundation. Supporting Information Available: 1H temperature series for YIII-DOTATOC, proton and carbon chemical shift tables of GaIII-DOTATOC (290 K) and YIII-DOTATOC (275 and 290 K), and the NOE tables used for the molecular dynamics calculations. This material is available free of charge via the Internet at http://pubs.acs.org.

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