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Bioconjugate Chem. 2001, 12, 84−91
Isomerism and Solution Dynamics of DTPA-Biomolecule Conjugates
90Y-Labeled
Shuang Liu,* Eric Cheung, Milind Rajopadhye, Neal E. Williams, Kirsten L. Overoye, and D. Scott Edwards Medical Imaging Division, DuPont Pharmaceuticals Company, 331 Treble Cove Road, North Billerica, Massachusetts 01862. Received June 23, 2000; Revised Manuscript Received October 18, 2000
This report describes the synthesis of two DTPA-conjugated cyclic peptides, cyclo(Arg-Gly-Asp-D-PheLys)DTPA (SQ169) and [cyclo(Arg-Gly-Asp-D-Phe-Lys)]2DTPA (SQ170), and a chromatographic study of their 90Y complexes (RP762 and RP763, respectively). The goal is to study the solution structure and the possible isomerism of 90Y-labeled DTPA-biomolecule conjugates at the tracer level (∼10-10 M). RP762 was prepared in high radiochemical purity (RCP > 95%) by reacting 2 µg of SQ169 with 20 mCi of 90YCl3 (corresponding to a SQ169:Y ratio of ∼4:1) in the 0.5 M ammonium acetate buffer (pH 8.0) at room temperature. RP763 was prepared in a similar fashion using SQ170. In both cases, the 90Y-chelation was instantaneous. By a reversed-phase HPLC method, it was found that RP762 exists in solution as a mixture of two detectable isomers (most likely cis and trans isomers), which interconvert at room temperature. The interconversion of different isomeric forms of RP762 involves a rapid exchange of “wrapping isomers” via the “wagging” of the diethylenetriamine backbone, “shuffling” of the two NO2 donor sets, and inversion at the ternimal amine-nitrogen atom. The inversion at a terminal nitrogen atom requires simultaneous dissociation of the NO2 donor set. For RP763, the interconversion of different isomers is much faster than that for RP762 due to the weak bonding of two carbonyl-oxygen donors. Therefore, RP763 shows only one broad radiometric peak in its HPLC chromatogram. The rapid interconversion of different isomers is intramolecular via a partial dissociative mechanism. The results obtained in this study are consistent with the lack of kinetic inertness of 90Y- and 111In-labeled DTPA-biomolecule conjugates. Thus, the design of new BFCs should be focused on those which form lanthanide complexes with high thermodynamic stability and more importantly kinetic inertness.
INTRODUCTION
In the past decade, many acyclic and macrocyclic bifunctional chelators (BFCs) have been synthesized and used for the radiolabeling of antibodies and peptides (124). Although formation of isomers in yttrium and lanthanide complexes of DTPA analogues has been studied by various NMR methods, very little information is available on isomerism of the radiolabeled DTPAbiomolecule conjugates at the tracer level. In many cases, TLC is the only tool for analysis of the final product. Radio-HPLC chromatograms are not presented nor is the HPLC method for the characterization of the radiolabeled biomolecule. It is not surprising that there has been no detailed discussion about isomerism and the impact on the biological properties of the radiolabeled DTPAbiomolecule conjugates. Recently, Caravan and co-workers (25) extensively reviewed both the solid state and solution structures of lanthanide complexes of various acyclic and macrocyclic chelators. Solid state structures provide a wealth of information about the coordination chemistry of a specific chelating system. However, the solution structure may be different from the solid state structure because of possible dissociation of certain donor atom(s) or the coordination of water molecules. The isomerism observed in the solid state structure may not be seen in solution * To whom correspondence should be addressed. Phone: (978) 671-8696.Fax: (978)436-7500.E-mail: shuang.liu@dupontpharma. com.
due to interconversion of isomers or fluxionality of the ligand framework. On other occasions, only one isomeric form is found in the solid state, and more isomers are observed in solution. Thus, it is imperative to study the possible isomerism in the metal chelate, particularly at the tracer level, to understand the biological properties of a new metalloradiopharmaceutical. For radiolabeled antibodies, the attachment of the BFC and the radionuclide normally does not disturb the tertiary structure of the polypeptide; thereby the biological and receptor binding properties are not significantly affected. For small biomolecules, however, the metal chelate (radionuclide and BFC) contributes greatly to the overall size and molecular weight of the radiopharmaceutical. Isomerism may have significant impact on the biological performance (receptor binding and biodistribution) since the distribution of the radiopharmaceutical is dependent on the physical and chemical properties of both the biomolecule and the metal chelate. In this report, we describe the synthesis of two DTPAconjugated cyclic peptides (SQ169 and SQ170) and a chromatographic study of their 90Y complexes (RP762 and RP763 in Figure 1). The purpose of the study is to obtain a sufficient understanding of the solution structure of these complexes, and to explore the possible isomerism in 90Y-labeled DTPA-biomolecule conjugates (DTPAmonoamide and DTPA-bisamide) at the tracer level (∼10-10 M), the kinetics for the formation of isomers, and the dynamics of interconversion between different isomeric forms.
10.1021/bc000071n CCC: $20.00 © 2001 American Chemical Society Published on Web 12/21/2000
90Y-Labeled
DTPA-Biomolecule Conjugates
Figure 1. Structures of yttrium complexes of two DTPA conjugates (SQ169 and SQ170). EXPERIMENTAL SECTION
Materials. Acetic acid (ultrapure), ammonium hydroxide (ultrapure), and diethylenetriaminepentaacetic acid cyclic dianhydride were purchased from either Aldrich or Sigma Chemical Co. and were used as received. 90YCl3 (in 0.05 N HCl) was purchased from New England Nuclear, N. Billerica, MA. The cyclic pentapeptide, cyclo(Arg-Gly-Asp-D-Phe-Lys), as its trifluoroacetic acid (TFA) salt was prepared according to the literature method (26). Synthesis of Cyclo(Arg-Gly-Asp-D-Phe-Lys)DTPA (SQ169) and [Cyclo(Arg-Gly-Asp-D-Phe-Lys)]2DTPA (SQ170). To a solution of cyclo(Arg-Gly-Asp-D-Phe-Lys)‚ 2TFA (0.050 g, 0.060 mmol) in DMF (2 mL) was added triethylamine (41.9 µL, 0.30 mmol). This solution was added dropwise over 4 h to a solution of diethylenetriaminepentaacetic dianhydride (0.107 g, 0.30 mmol) in DMF (2 mL) and dimethyl sulfoxide (2 mL). The reaction mixture was stirred for 16 h, and was then concentrated to give an oily residue, which was purified using a preparative HPLC method, which used a Rainin Rabbit HPLC system, a Knauer VWM UV-vis detector (λ ) 230 nm), and a Vydac C18 preparative column (21.2 mm × 25 cm). The flow rate was 15 mL/min with the mobile phase starting from 98% solvent A (0.1% TFA in H2O) and 2% solvent B (0.1%TFA in ACN/H2O ) 9:1) to 63.2% solvent A and 36.8% solvent B at 16 min. The two peaks at 12 and 15 min were collected separately. LC-MS data showed that the peak at 12 min was from SQ169 and the peak at 15 min was from SQ170. After lyophilization, SQ169 was obtained in 46% yield (30 mg) and SQ170 in 21% yield (21.5 mg). ES-MS for SQ169: m/z 977.5 (MH)- (C41H62N12O16). ES-MS for SQ170: m/z 1562.8 (MH)- (C68H101N21O22). General Procedure for Synthesis of RP762 and RP763. To a lead-shielded clean 5 mL vial containing 2-100 µg of SQ169 or SQ170 in 1 mL of 0.5 M ammonium acetate buffer (pH 8.0) was added 10-20 µL of 90YCl stock solution (∼20 mCi) in 0.05 N HCl. The 3 reaction mixture was allowed to stand at room temperature for 20 min. A sample of the resulting solution was diluted 20-fold with saline and then analyzed by HPLC (Gradient A for RP762 and Gradient B for RP763) and by ITLC.
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The HPLC method used a HP-1100 HPLC system with a UV-vis detector (λ ) 215 nm), an IN-US radiodetector, and a Zorbax C18 column (4.6 mm × 250 mm, 80 Å pore size). The flow rate was 1 mL/min with a gradient mobile phase. For RP762, the gradient was 100% solvent A (95:5 mixture of 25 mM ammonium acetate buffer, pH 6.8, and acetonitrile) to 95% A and 5% solvent B (50:50 mixture of 25 mM ammonium acetate buffer, pH 6.8, and acetonitrile) at 8 min and to 90% of solvent A and 10% solvent B at 24 min. For RP763, the gradient was 85% solvent A and 15% solvent B to 80% of solvent A and 20% solvent B at 18 min. The ITLC method used Gelman Sciences silica gel ITLC paper strips and 1:1 mixture of acetone and saline as eluant. By this method, RP762 and RP763 migrate to the solvent front while [90Y]colloid and [90Y]acetate remain at the origin. Interconversion of the Two Isomers of RP762. To a shielded clean 5 mL vial containing 20 µg of SQ169 or SQ170 in 1 mL of 0.5 M ammonium acetate buffer (pH 8.0) was added 20 µL of 90YCl3 stock solution (∼20 mCi) in 0.05 N HCl. The reaction mixture was allowed to stand at room temperature for 20 min. The two peaks at 16.8 and 20.9 min were separated by HPLC, and were collected into separate 10 mL round-bottom flasks. The activity concentration of two collections (peak A and peak B) was about 0.5 mCi/mL. Each collected fraction was dispensed into two separate vials: one was kept at room temperature while the other was kept in a 54 °C water bath. Samples of these solutions were analyzed by HPLC every 30 min for 6 h. 90Y-Chelation Efficiency of SQ169. To a leadshielded clean 5 mL vial containing 2, 25, 50, and 100 µg of SQ169 in 1 mL of 0.5 M ammonium acetate buffer (pH 8.0) was added 20 µL of 90YCl3 stock solution (∼20 mCi) in 0.05 N HCl. The reaction mixture was allowed to stand at room temperature for 30 min. A sample of the resulting solution was diluted 20-fold with saline, and then analyzed by HPLC (Gradient A) and by ITLC. The RCP was corrected by subtracting the percentage of [90Y]colloid and [90Y]acetate obtained by ITLC from the yield obtained by HPLC. Synthesis of [89Y]RP762 for LC-MS and HPLC Concordance Experiment. To a clean 5 mL vial containing 0.5 mg of SQ169 in 0.5 mL of 0.5 M ammonium acetate buffer (pH 8.0) was added 0.2 mL of the Y(NO3)3‚6H2O solution (10 mg/mL in 0.5 M ammonium acetate buffer, pH 8.0). The reaction mixture was allowed to stand at room temperature for 30 min. A sample of the resulting solution was analyzed by LC-MS. No further purification was performed for the LC-MS analysis and the HPLC concordance experiment. The LC-MS spectra were collected using a HP1100 LC/MSD system with API-electrospray interface. The LC-MS method used a Zorbax C18 column (4.6 mm × 150 mm, 3.5 µm particle size) at a flow rate of 1 mL/min and a gradient mobile phase starting from 92% Solvent A (10 mM ammonium acetate buffer, pH 7.0) and 8% Solvent B (methanol) to 100% B at 23 min. The MSD parameters are found in Table 1. RESULTS AND DISCUSSION
The pyramidal environment at coordinated aminenitrogen atoms has been conclusively established by X-ray methods (27). Rapid inversion (Chart 1) at the amine nitrogen atoms is common. However, once the nitrogen atom in a polydentate chelator is bonded to the metal ion, the inversion becomes more difficult and
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Table 1 detection mode
positive
gain gas temperature nebullizer pressure UV-vis detector mass range fragmentor drying gas flow V capillary
1.0 350 °C 60 psig (max) λ ) 220 nm 0-2000 30 V 13 L/min 4000 V
Chart 1. Inversion at the Coordinated and Uncoordinated Amine-Nitrogen.
involves dissociation and reformation of the coordination bond (Chart 1). Therefore, coordination of a polydentate chelator containing amine-nitrogen donor atoms often results in formation of isomers in its yttrium and lanthanide complexes. For example, DTPA, DTPA-MA (MA ) monoamide), and DTPA-BA (BA ) bisamide) contain three amine-nitrogen donors. In principle, this will result in formation of eight possible isomers once they are coordinated to a metal ion. In the solid state, only some of them were observed by X-ray crystallography (27-31). In solution, however, all eight isomers of lanthanide complexes have been observed by NMR at low temperature (32-39). Some of them interconvert via a partial dissociation at elevated temperatures. For example, the proton NMR spectra of the Pr3+ and Eu3+ complexes of DTPA show that at room temperature there are only two main isomers (Figure 2), which undergo rapid exchange in solution at temperatures higher that 95 °C (32-34). For the Lu3+ chelate of DTPA-BMA, however, the temperature for the rapid exchange is lower (85 °C), reflecting the weak bonding of carbonyl-oxygen as compared to carboxylate-oxygen donors (35-39). The major feature of the exchange process involves the shuffling of coordinated acetate accompanied by a flip of the backbone ethylene bridges between staggered conformations. This results in a change in helicity of the complex and leads to the equilibration of acetate arms. Inversion at the coordinated nitrogen atoms has been excluded based on 1H NMR data (35). In all the cases, the concentration of the lanthanide complexes studied by NMR ranged from 0.05 to 0.20 M. 90 Y-labeled DTPA-biomolecule conjugates (DTPA-MA and DTPA-BA) are often prepared at low concentration (2-20 mCi/mL corresponding to 4 × 10-9 to 4 × 10-8 M). For HPLC analysis, the amount of injected radioactivity is even lower (1.0 µL corresponding to about 1 µCi). The actual complex concentration in the mobile phase is only ∼10-15 M. Because of this concentration difference, these lanthanide complexes may have different solution characteristics at the tracer level. The purpose of the study is to obtain a sufficient understanding of the solution structure and isomerism of the 90Y-labeled DTPA-
peptide conjugates at the tracer level, and the dynamics of interconversion between different isomeric forms. Synthesis of SQ169 and SQ170. The DTPA conjugates (SQ169 and SQ170) were prepared according to Chart 2 by reacting DTPA with the cyclic pentapeptide, cyclo(Arg-Gly-Asp-D-Phe-Lys)‚2TFA, in a mixture of DMF and DMSO. SQ169 and SQ170 were isolated as their TFA salts, and were repurified by HPLC before being used for radiolabeling. Synthesis of RP762 and RP763. RP762 was prepared (Chart 2) by reacting SQ169 with 90YCl3 in the 0.5 M ammonium acetate buffer (pH 8.0) at room temperature. The pH is very important for high yield radiolabeling. A higher pH usually gives better radiolabeling yield. Using 0.5 M ammonium acetate buffer (pH 8.0), RP762 could be prepared in high yield (RCP > 95%) by reacting 2 µg of SQ169 with 20 mCi of 90YCl3 corresponding to a SQ169:Y ratio of ∼4:1. RP763 was prepared in a similar fashion using SQ170 substituting for SQ169. In both cases, the 90Y-chelation was instantaneous. This is consistent with the results reported in the literature (40, 41). HPLC Characterization of RP762. For the past decade, HPLC has become a routine technique for quality control of various radiopharmaceuticals. The advantage of radio-HPLC is its capability to determine the RCP for a specific radiopharmaceutical, and to separate different radiolabeled species in the reaction mixture. It is also a very powerful tool for separation of different isomers such as diastereomers. In this study, we used a reversed-phase HPLC method to analyze reaction mixtures containing RP762. Figure 3 shows typical radio-HPLC chromatogram of RP762. Two radiometric peaks at 16.8 and 20.9 min were detected and there are no other radio-impurities. We also tried several other reversed-phase HPLC methods. In all the cases, RP762 shows only two radiometric peaks, suggesting that the HPLC method is specific. The presence of the two peaks in the HPLC chromatogram also suggests that RP762 exists as two detectable isomers in solution. It is interesting to note that the peak ratio of peak A and peak B changes over time until the equilibrium is reached. For example, the peak ratio at t ) 0 h was 50: 50 while it was about 48:52 at 12 h and remained relatively constant from that time. This observation suggests that there is a slow conversion from peak A to peak B at room temperature, and the formation of the different isomeric forms is probably kinetically controlled. Since the 90Y-chelation was instantaneous under the conditions described in the experimental, it is not surprising that these isomers were formed at the same time with equal population at the early stage. We also studied the conversion rate of HPLC purified peak A and peak B. First, RP762 was prepared using 20 mCi of 90Y, and the isomers at 16.8 and 20.9 min were collected into two separate 5 mL vials. The two vials were either kept at room temperature or placed in a water bath at 54 °C. Samples of the collected fractions (activity concentration ) 0.5 mCi/mL) were analyzed by the HPLC over 6 h. It was found that the rate of interconversion between these two peaks is very slow at room temperature (Figure 4). That may explain why it takes almost 24 h for these isomers to reach the equilibrium. At a higher temperature (54 °C), the rate of interconversion between different isomers of RP762 is accelerated (Figure 5), and it took about 6.5 h to for peak A to reach the 48: 52 (peak A/peak B) ratio. However, it took a much longer time (>7 h) for peak B to reach the equilibrium, suggesting that peak B is thermodynamically more stable. It is
90Y-Labeled
DTPA-Biomolecule Conjugates
Bioconjugate Chem., Vol. 12, No. 1, 2001 87
Figure 2. Proposed mechanism for the interconversion between the two wrapping isomers of lanthanide DTPA complexes. Chart 2. DTPA Conjugation and
90Y-Chelation.
very important to note that both peak A and peak B remained stable in the HPLC mobile phase (containing 10% acetonitrile) at 54 °C for more than 6 h. There is no significant decomposition of the metal chelate, suggesting that interconversion between these isomeric forms is intramolecular. HPLC Concordance Experiment. We performed a HPLC concordance experiment using RP762 and [89Y]RP762. [89Y]RP762 was prepared by reacting SQ169 with yttrium(III) nitrate in 0.5 M ammonium acetate buffer (pH 8.0), and was analyzed without further purification using identical chromatographic conditions to those for RP762. Figure 6 shows HPLC chromatograms of RP762 (top) and [89Y]RP762 (bottom). It is quite clear that the same complexes were prepared at the tracer (90Y) and macroscopic (89Y) levels. We used LC-MS to determine the composition of [89Y]RP762. The two peaks of [89Y]RP762 show identical fragmentation patterns and the same molecular ions at m/z 1065.2 for (M + H)+ and 533.3 for (M + 2H)2+ (Figure 7). This provides direct evidence to support the conclusion that the two peaks in the radio-
Figure 3. Typical radio-HPLC chromatograms for RP762 (top) and RP763 (bottom).
HPLC chromatogram of RP762 are indeed due to the resolution of two isomers. HPLC Characterization of RP763. We used a slightly different mobile phase when analyzing RP763 by radio-HPLC due to its higher lipophilicity. Figure 3 shows a typical radio-HPLC chromatogram for RP763. In contrast to RP762, RP763 shows only one broad peak with the half-width of ∼3 min. There are no other radioimpurities in the HPLC chromatogram. In general, there are two possibilities for the large half-width of the peak.
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Figure 4. Interconversion kinetics of the two isomers of RP762 at room temperature.
Figure 5. Interconversion kinetics of the two isomers of RP762 at 55 °C.
The first possibility is that the peak at ∼15 min is a combination of several unresolved peaks from different isomers. If that were true, it should have been possible to separate them using long gradient or isocratic chromatographic conditions. We tried several different gradients, and found that RP763 always shows one broad peak with different half-widths depending upon the HPLC chromatographic conditions. The second possibility is that there is a rapid interconversion between different isomers at room temperature under the conditions used in this study. In general, the lanthanide complexes of DTPA analogues assume distorted tricapped trigonal prism (TTP) geometries. In the TTP arrangement, the neutral donor atoms with longer bond lengths will prefer to occupy face capping positions rather than a prismatic corner. However, since it is not possible for all three amine-nitrogen donors to occupy capping positions, the central nitrogen atom tends to occupy a prismatic corner (Figure 2) while the two terminal amine-nitrogen atoms occupy capping positions. Because of distortion, the actual coordination geometry is, in most cases, between TTP and monocapped square antiprism (CSAP). SQ169 is a DTPA-monoamide and bonds to yttrium(III) with one carbonyl-oxygen, three amine-nitrogen, and four acetate-oxygen donor atoms. Theoretically, there are eight possible isomers (four pairs of enantionmers in Chart 3) due to the chirality of three coordinated nitrogen donors. If the biomolecule in the DTPA-peptide conjugate is stereochemically pure, it will form an yttrium
complex with the same number of isomers as that of the Y-DTPA complex. Previously, 1H and 13C NMR studies of lanthanide complexes of DTPA and DTPA-bisamide derivatives have revealed a rapid interconversion of two wrapping isomers (26, 32-39). It has been proposed that the rapid exchange process involves “wagging” of the diethylenetriamine backbone and shuffling of coordinated acetate groups. Inversion at the coordinated nitrogen atoms has been excluded due to the fact that the 1H signals of methylene-hydrogens of the acetate and acetamide groups remain as AB quartets even at 85 °C (34). This strongly suggests that the energy barries for the inversion at the coordinated amine-nitrogen donors is much higher than that for the rapid interconversion of “wrapping isomers”. It should be noted that the concentration of the lanthanide complexes for NMR studies ranged from 0.05 to 0.2 M. At this concentration, the dissociation of acetate-oxygen and amine-nitrogen donors is not significant. At the tracer level, however, the concentration of RP762 in the HPLC mobile phase is only ∼10-10 M. At this concentration, it is quite possible for acetate-oxygen, carbonyl-oxygen and amine-nitrogen donors to become dissociated due to the competition from acetate anions in the mobile phase. Partial dissociation of these donor atoms makes the interconversion between “wrapping isomers” much easier. That may account for the different solution characteristics of lanthanide complexes of DTPAMA and DTPA-BA at different concentration levels.
90Y-Labeled
DTPA-Biomolecule Conjugates
Bioconjugate Chem., Vol. 12, No. 1, 2001 89 Chart 3. Eight Isomers of the Complex Y(DTPA-MA).
Figure 6. HPLC concordance of RP762 (top) and [89Y]RP762 (bottom).
Figure 7. LC-MS spectra of the two isomers of RP762.
RP762 has four possible pairs of enantiomers, which are not separable by the HPLC methods described in this
study. If one assumes that the rapid interconversion occurs between “wrapping isomers” at room temperature, isomer 1 is readily converted to 1′ via “shuffling” of the two NO2 donor sets and “wagging” of the diethylenetriamine backbone. The same is true for 2 to 2′. Since 1′/ 2 and 2′/1 are the two enantiomer pairs, these four isomers are not separable by radio-HPLC, and are expected to appear as single peak while the remaining four isomers (3, 3′, 4, and 4′) as another peak in the HPLC chromatogram. This is completely consistent with the presence of two radiometric peaks in the HPLC chromatogram of RP762 (Figure 3). It should be noted that the relative orientation of the acetamide group to the acetate arm at the central nitrogen atom is different (cis and trans, respectively) in isomers 1 and 4 (Chart 3). The same is true for isomers 1′ and 4′, 2 and 3, 2′ and 3′. For cis and trans isomers to interconvert, inversion at the terminal amine-nitrogen atoms has to occur. This suggests that the NO2 donor set have to be dissociated and accompanied by a rapid “umbrella-type” inversion at the nitrogen atom. That explains the fact that both radiometric peaks of RP762 interconvert and the rate of interconversion is temperature dependent. Since RP762 remains stable for at least 6 h at 54 °C without a significant decomposition, it is reasonable to assume that the remaining part of the DTPA-monoamide conjugate remains firmly bonded to Y(III) when one of the two NO2 donor sets undergoes a rapid “umbrella-type” inversion at the terminal nitrogen atom. SQ170 is a DTPA-BA and bonds to yttrium(III) as an octadentate chelator using two carbonyl-oxygen, three amine-nitrogen, and three acetate-oxygen donor atoms. Like DTPA-MA, DTPA-BA forms a Y(III) complex with
90 Bioconjugate Chem., Vol. 12, No. 1, 2001 Chart 4. Eight Isomers of the Complex Y(DTPA-BA).
Liu et al.
some of which undergo rapid interconversion at room temperature. Therefore, the presence of different isomeric forms may not impose a significant problem with respect to the difference in biological properties of these isomers. The most critical issue using DTPA as a BFC is its low kinetic inertness. When the two isomers of RP762 were separated by HPLC, the HPLC mobile phase did not contain any significant amount of trace metal ion. The rapid interconversion of different isomers is purely intramolecular. Once the 90Y-labeled DTPA-biomolecule conjugate is injected into a biological system, the presence of biologically important metal ions such as Ca2+, Cu2+, Fe2+, and Zn2+, is expected to have a significant impact on the biological fate of the 90Y-labeled DTPA-biomolecule conjugate. These metal ions can compete with 90Y for the DTPA-biomolecule conjugate due to their high concentration. Once 90Y is dissociated from the DTPAbiomolecule conjugate, it will most likely deposit in bone. It is not surprising that many 90Y- and 111In-labeled DTPA-biomolecule conjugates often show high bone activity. Thus, the design of new BFCs should be focused on those which form lanthanide complexes with high thermodynamic stability and more importantly kinetic inertness. LITERATURE CITED
eight isomers (Chart 4) due to the chirality of the three coordinated amine-nitrogen atoms. Compared to acetateoxygen, the carbonyl-oxygen is a weak donor for yttrium(III). Thus, it is easier for the two NO2 donor sets to be dissociated in solution, which results in faster interconversion between “wrapping isomers” and inversion at coordinated amine-nitrogen atoms. As a result, RP763 shows only one broad radiometric peak in its HPLC chromatogram (Figure 3). CONCLUSION
In this study, we presented synthesis and characterization of two DTPA-peptide conjugates (SQ169 and SQ170) and the corresponding 90Y complexes (RP762 and RP763, respectively). By a reversed-phase HPLC method, we found that the 90Y-labeled DTPA-monoamide conjugate exists in solution as a mixture of two detectable isomers (cis and trans isomers), which interconvert at room temperature under the chromatographic conditions described in this study. The interconversion of different isomeric forms of RP762 involves a rapid exchange of “wrapping isomers” via the “wagging” of the diethylenetriamine backbone, “shuffling” of the two NO2 donor sets, and inversion at the ternimal amine-nitrogen atom. For the 90Y-labeled DTPA-BA conjugate (RP763), the interconversion of different isomers (particularly cis-cis, cis-trans, trans-cis, and trans-trans isomers) is much faster than that for RP762 due to the weak bonding of two carbonyl-oxygen donors; thereby RP763 shows one broad radiometric peak in its HPLC chromatogram. The results obtained from this study have significant implications for 90Y-labeled DTPA-biomolecule conjugates. The 90Y-labeled DTPA-biomolecule conjugate exists in solution as a mixture of eight possible isomers,
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90Y-Labeled
DTPA-Biomolecule Conjugates
(11) Williams, M. A., and Rapoport, H. (1993) Synthesis of enantiometrically pure diethylenetriaminepentaacetic acid analogues. L-Phenylalanine as the educt for substitution at the central acetic acid. J. Org. Chem. 58, 1151-1158. (12) Camera, L., Kinuya, S., Garmestani, K., Wu, C., Brechbiel, M. W., Pai, L. H., McMurry, T. J., Gansow, O. A., Pastan, I., Paik, C. H., and Carrasquillo, J. A. (1994) Evaluation of the serum stability and in vivo biodistribution of CHX-DTPA and other ligands for yttrium labeling of monoclonal antibodies. J. Nucl. Med. 35, 882-889. (13) Wu, C., Brechbiel, M. W., Gansow, O. A., Kobayashi, H., Carrasquillo, J., and Pastan, I. (1997) Stability of the four 2-(p-nitrobenzyl)-trans-CyDTPA 88Y complexes. Radiochim. Acta 79, 123-136. (14) Wu, C., Kobayashi, H., Sun, B., Yoo, T. M., Paik, C. H., Gansow, O. A., Carrasquillo, J. A., Pastan, I., and Brechbiel, M. W. (1997) Stereochemical influence on the stability of radio-metal complexes in vivo. Synthesis and evaluation of the four stereoisomers of 2-(p-nitrobenzyl)-trans-CyDTPA. Bioorg. Med. Chem. 5, 1925-1934. (15) McMurray, T. J., Pippin, C. G., Wu, C., Deal, K. A., Brechbiel, M. W., Mirzadeh, S., and Gansow, O. A. (1998) Physical parameters and biological stability of yttrium(III) diethylenetriaminepentaacetic acid derivative conjugates. J. Med. Chem. 41, 3546-3549. (16) Smith-Jones, P. M., Stolz, B., Albert, R., Ruser, G., Briner, U., Macke, H. R., and Bruns, C. (1998) Synthesis and characterization of [90Y]-Bz-DTPA-oct: a yttrium-90-labeled oactreotide analogue for radiotherapy of somatostatin receptor-positive tumors. Nucl. Med. Biol. 25, 181-188. (17) Corson, D. T., and Meares, C. F. (2000) Efficient multigram synthesis of the bifunctional chelating agent: (S)-1-p-Isothiocyanatobenzyl-diethylenetriaminepentaacetic acid. Bioconjugate Chem. 11, 292-299. (18) Achilefu, A., Wilhelm, R. R., Jimenez, H. N., Schmidt, M. A., and Srunivasan, A. (2000) A new method for the synthesis of tri-tert-butyl diethylenetriaminepenta-acetic acid and its derivatives. J. Org. Chem. 65, 1562-1565. (19) De Jong, M., Bakker, W. H., Breeman, W. A. P., Bernard, B. F., Hofland, L. J., Visser, T. J., Srinivasan, A., Schmidt, M., Be´he´, M., Ma¨cke, H., and Krenning, E. P. (1998) Preclinical comparison of [DTPAo]octreotide, [DTPAo, Tyr3]octreotide, and [DOTAo, Tyr3]octreotide as carriers for somatostatin receptor-targeted scintigraphy and radionuclide therapy. Int. J. Cancer 75, 406-411. (20) De Jong, M., Bernard, B. F., De Bruin, E., Van Gameren, A., Bakker, W. H., Visser, T. J., Ma¨cke, H., and Krenning, E. P. (1998) Internalization of radiolabeled [DTPAo]octreotide and [DOTAo, Tyr3]octreotide: peptides for somatostatin receptor-targeted scintigraphy and radionuclide therapy. Nucl. Med. Commun. 19, 283-288. (21) De Jong, M., Breeman, W. A. P., Bakker, W. H., Kooij, P. P. M., Bernard, B. F., Hofland, L. J., Visser, T. J., Srinivasan, A., Schmidt, M. A., Erion, J. L., Bugaj, J. E., Ma¨cke, H., and Krenning, E. P. (1998) Comparison of 111In-labeled somatostatin analogues for tumor scintigraphy and radionuclide therapy. Anticancer Res. 58, 437-441. (22) Reubi, J. C., Waser, B., Schaer, J. C., Laederach, U., Erion, J., Srinivasam, A., Schmidt, M. A., and Bugaj, J. E. (1998) Unsulfated DTPA- and DOTA-CCK analogues as specific high affinity ligands for CCK-B receptor-expressing human and rat tissues in vitro and in vivo. Eur. J. Nucl. Med. 25, 481490. (23) Behr, T. M., Jenner, N., Be´he´, M., Angerstein, C., Gratz, S., Raue, F., and Becker, W. (1999) Radiolabeled peptides for targeting cholecystokinin-B-gastrin receptor-expressing tumors. J. Nucl. Med. 40, 1029-1040. (24) Behr, T. M., Jenner, N., Be´he´, M., Angerstein, C., Gratz, S., Raue, F., and Becker, W. (1999) Cholecystokinin-B-gastrin receptor binding peptides: preclinical development and evaluation of their diagnostic and therapeutic potential. Clin. Cancer Res. 5, 3124s-3138s. (25) Caravan, P., Ellison, J. J., McMurry, T. J., and Lauffer, R. B. (1999) Gadolinium(III) chelates as MRI contrast agents: structure, dynamics, and applications. Chem. Rev. 99, 2293-2352, and references therein.
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