Phosphine-Containing HYNIC Derivatives as Potential Bifunctional

Jun 25, 2003 - Ajay Purohit, Shuang Liu,*,† Dave Casebier, and D. Scott Edwards. Discovery Chemistry, Bristol-Myers Squibb Medical Imaging, 331 Treb...
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Bioconjugate Chem. 2003, 14, 720−727

ARTICLES Phosphine-Containing HYNIC Derivatives as Potential Bifunctional Chelators for 99mTc-Labeling of Small Biomolecules Ajay Purohit, Shuang Liu,*,† Dave Casebier, and D. Scott Edwards Discovery Chemistry, Bristol-Myers Squibb Medical Imaging, 331 Treble Cove Road, North Billerica, Massachusetts 01862. Received April 18, 2003

Two prototype phosphine-containing HYNIC chelators, HYNIC-Kp-DPPB and HYNIC-Ko-DPPB (HYNIC ) 6-hydrazinonicotinamide; K ) lysine; and DPPB ) diphenylphosphine-benzoic acid), have been synthesized and characterized by NMR (1H, 13C, and 31P) and LC-MS. Macrocyclic 99mTc complexes, [99mTc(HYNIC-Ko-TPPB)(tricine)] and [99mTc(HYNIC-Kp-DPPB)(tricine)], were prepared by reacting the phosphine-containing HYNIC chelator with 99mTcO4- in the presence of excess tricine and stannous chloride. Results from this study clearly demonstrated that both HYNIC-Kp-DPPB and HYNIC-KoDPPB are able to form highly stable macrocyclic 99mTc complexes, [99mTc(HYNIC-Ko-TPPB)(tricine)] and [99mTc(HYNIC-Kp-DPPB)(tricine)], when tricine is used as the coligand. Radio-HPLC data suggest that the complex [99mTc(HYNIC-Kp-DPPB)(tricine)] exists as only one detectable isomer in solution while the complex [99mTc(HYNIC-Ko-DPPB)(tricine)] has three isomers. It was also found that three isomers of [99mTc(HYNIC-Ko-DPPB)(tricine)] interconvert at elevated temperatures, suggesting that the presence of these isomers might be due conformational changes in the macrocyclic Tc chelate. The LC-MS data for both macrocyclic 99mTc complexes are completely consistent with the proposed composition. The phosphine-containing HYNIC chelators described in this study may have the potential as bifunctional chelators for 99mTc labeling of small biomolecules.

INTRODUCTION

Abrams and co-workers (1, 2) first reported the use of organic hydrazines, including 6-hydrazinonictinamide (HYNIC), as bifunctional coupling agents (BFCs) for the 99m Tc-labeling of polyclonal IgG. Since then, HYNIC has successfully been used for the 99mTc-labeling of antibodies (3, 4) and small biomolecules, including chemotactic peptides (5-8), somatostatin analogues (9-14), liposomes (15), antisense oligonucleotides (16, 17), a folate receptor ligand (18), and polypeptides (19, 20). The use of HYNIC for 99mTc-labeling of small biomolecules has recently been reviewed (21-24). The advantage of HYNIC as a BFC is its high labeling efficiency (rapid and high yield radiolabeling) and the high in vivo stability of resulting 99mTc complexes. Since HYNIC can only occupy one coordination site (Figure 1), a coligand such as tricine is needed to complete the coordination sphere of the Tc. The choice of coligands allows easy modification of the hydrophilicity and biological properties of the 99mTc-labeled small biomolecules. For the last several years, we have been using a ternary ligand system (Figure 1: HYNIC-BM, tricine, and TPPTS) for 99mTc-labeling of small biomolecules. These include chemotactic peptides (25) and LTB4 receptor antagonists (26, 27) for imaging infection and inflam* To whom correspondence should be addressed. Phone: 765494-0236; fax 765-496-3367; e-mail: [email protected]. † Present address: Department of Industrial and Physical Pharmacy, School of Pharmacy, Purdue University, 575 Stadium Dr., West Lafayette, IN 47907-2051.

mation, a vitronectin receptor antagonist for tumor imaging (28), and a GPIIb/IIIa receptor antagonist for imaging thrombosis (29-34). The combination of HYNICBM, tricine, and TPPTS results in a ternary ligand system that forms technetium complexes, [99mTc(HYNICBM)(tricine)(TPPTS)] (BM ) peptide or nonpeptide receptor ligands) with extremely high specific activity. These ternary ligand 99mTc complexes are stable in solution for >6 h and are formed as equal mixtures of two detectable isomers if the biomolecule has a chiral center. The composition of these 99mTc complexes has been determined to be 1:1:1:1 for Tc:HYNIC-BM:tricine: TPPTS through a series of mixed ligand experiments and confirmed by LC-MS at both the macroscopic and tracer (99mTc) levels (27, 28, 35, 36). Like phosphines, pyridine analogues (Figure 1) have also been used as coligands for the 99mTc-labeling of small biomolecules (34). Compared to water-soluble phosphines, pyridine analogues are much smaller and more amenable for further derivatization. In principle, the ternary ligand system (HYNIC-BM, tricine, and TPPTS) can be used for 99mTc-labeling of any HYNIC-conjugated biomolecules. However, problem may arise when it is used for 99mTc-labeling of small biomolecules containing one or more disulfide linkages. The use of a large amount of TPPTS in combination with high temperature heating may destroy the S-S disulfide bonds, which are often vital to keep the rigid cyclic conformation of the small biomolecule and to maintain the high receptor binding affinity, and cause adverse effect on the biological properties of the 99mTc-labeled biomolecule. Therefore, there is a continuing need for a

10.1021/bc034059h CCC: $25.00 © 2003 American Chemical Society Published on Web 06/25/2003

Phosphine-Containing HYNIC Derivatives

Figure 1. Structures of coligands and their ternary ligand

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99mTc

complexes.

biomolecules for the development of target-specific radiopharmaceuticals. As a continuation of our interest in developing new 99m Tc chelating systems, we now report the synthesis of two prototype phosphine-containing HYNIC chelators (Figure 2: HYNIC-Ko-DPPB and HYNIC-Kp-DPPB). The combination of a phosphine-containing HYNIC chelator with tricine results in a unique chelating system that forms highly stable macrocyclic 99mTc complexes with high specific activity. The phosphine-containing HYNIC chelators described in this study have the potential as BFCs for 99mTc-labeling of small biomolecules. EXPERIMENTAL SECTION

Figure 2. Phosphine-containing HYNIC chelators and their macrocyclic Tc complexes.

better chelating system, which does not require the use of large amount of phosphine coligand. One approach to minimize the use of large amount of phosphine coligand is to attach the phosphine coligand onto HYNIC via a linker to form the phosphine-containing HYNIC chelator. Figure 2 shows two prototype phosphine-containing HYNIC chelators, which are designed in such a way that when HYNIC binds to the Tc center, the “effective concentration” of the attached phosphine-P donor in the vicinity of the Tc will be increased dramatically. This makes it much easier for the phosphine-P donor to bond to the Tc and form a macrocyclic 99mTc chelate. The linker between HYNIC and the triphenylphosphine moiety may be a polymethylene chain or a small peptide sequence. The attachment site for the linker may vary for the triphenylphosphine moiety. Lysine is selected as a linker to connect HYNIC with the triphenylphosphine moiety while the carboxylic group of the lysine linker can be used for conjugation of

N--(tert-Butoxycarbonyl)lysine methyl ester hydrochloride, 2-(diphenylphosphino)benzoic acid (o-DPPB), 4-(diphenylphosphino)benzoic acid (p-DPPB), ethylenediamine-N,N′-diacetic acid (EDDA), 1-hydroxybenzotriazole, 1-hydroxyazabenzotriazole, N-(2-hydroxyethyl)iminediacetic acid (HIDA), 4 M HCl in dioxane, 1-O(N,N′,N′′,N′′′-tetramethyluronium)benzotriazoloxy hexafluorophosphate, and 1-O-(N,N′,N′′,N′′′-tetramethyluronium)azabenzotriazoloxy hexafluorophosphate were purchased from Aldrich or Sigma and were used as received. Na99mTcO4 was obtained from a Technelite 99Mo/ 99m Tc generator, Bristol-Myers Squibb Medical Imaging Inc., North Billerica, MA. Sodium succinimidyl 6-(2-(2sulfonatobenzaldehyde)hydrazono)nicotinate (HYNICOSu) was prepared according to published procedure (37). Instruments. NMR spectral data (1H and 13C) were recorded on a 600 MHz Bruker DRX FT NMR spectrometer. The 1H NMR data were reported as δ (ppm) relative to TMS. 31P NMR were proton decoupled and with phosphoric acid as an internal standard. Electrospray MS analyses were performed using an IonSpec Ultima FT Mass Spectrometer in the positive ion mode. LC-MS spectral data were collected using a HP1100 LC/MSD system with an API-electrospray interface. The LC-MS method used a Zorbax C18 column (4.6 mm × 150 mm, 3.5 µm particle size). The flow rate was 1 mL/min with a gradient mobile phase starting 80% solvent A (25 mM ammonium acetate buffer, pH 6.8) and 20% solvent B (acetonitrile) to 75% A and 25% solvent B at 20 min. The HPLC method 1 used a Varian semiprep HPLC system equipped with an UV/visible detector (λ ) 220 nm) and a Varian Prep Star on the Jupiter C18 column

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(15 µm, 300 Å, 41.4 × 250 mm). The flow rate was 80 mL/min with the mobile phase starting with 20% solvent B (0.1%TFA in 90% acetonitrile) and 80% solvent A (0.1%TFA in water) to 90% solvent B and 10% solvent A at 40 min. The radio-HPLC method 2 used a HP-1100 HPLC system with an UV/visible detector (λ ) 215 nm), an INUS radiodetector and a Zorbax C18 column (4.6 mm × 250 mm, 3.5 µm pore size). The flow rate was 1 mL/min with a gradient mobile phase starting 80% solvent A (25 mM ammonium acetate buffer, pH 6.8) and 20% solvent B (acetonitrile) to 75% A and 25% solvent B at 20 min. The ITLC method used Gelman Sciences silica gel paper strips and a 1:1 mixture of acetone and saline as eluant. Synthesis of N-E-(tert-Butoxycarbonyl)-N-r-(6-(2(2-sulfonatobenzaldehyde)hydrazono)nicotinyl)lysine Methyl Ester. To a 25 mL round-bottom flask was added HYNIC-OSu (250 mg, 0.567 mmol) followed by 3 mL of DMF. The solid was allowed to dissolve, and to the solution was then added N--(tert-butoxycarbonyl)lysine methyl ester hydrochloride (184 mg, 0.623 mmol) followed by diisopropylethylamine (0.32 mg, 0.434 mL). The resulting solution was stirred under a nitrogen atmosphere for 15 h. DMF was removed in vacuo and the crude oil subjected to HPLC purification (Method 1). The collected fractions were combined and were lyophilized to give a fluffy white powder. The yield was 239 mg (∼75%). High-resolution MS (ESI): m/z ) 564.2244 for C25H33N5O8S ([M + H]+). 1H NMR (600 MHz, DMSOd6 + DCl): 9.376 (s, 1H), 8.617 (s, 1H), 8.466 (dd, 1H, J ) 2.4 Hz), 7.8 (m, 1H), 7.4 (m, 2H), 7.25 (bd, 1H), 4.38 (t, 1H, J ) 7.2 Hz), 3.64 (s, 3H), 2.89 (m, 2H), 1.8 (q, 2H, J ) 7.2 Hz), 1.34-1.38 (13H). 13C NMR (DMSO-d6 + DCl): 172.4, 162.5, 155.5, 149.8, 147.5, 129.5, 120.2, 77.43, 52.84, 51.97, 31.25, 30.07, 29.6, 29, 28.2, 26.5, and 23. Synthesis of N-r-(6-(2-(2-Sulfonatobenzaldehyde)hydrazono)nicotinyl)lysine Methyl Ester Hydrochloride Salt. To a 25 mL round-bottom flask was added N--(tert-butoxycarbonyl)-N-R-(6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinoyl)lysine methyl ester (200 mg, 0.355 mmol). The flask cooled to 0 °C. Upon addition of 2.0 mL of 4 M HCl in dioxane, the fluffy white starting material immediately turned to a pale yellow solid. The reaction mixture was allowed to stand at 0 °C for 5 min and then at room temperature for 25 min with occasional shaking. The pale yellow solid was separated by filtration and dried under vacuum overnight. The product was used for the next reaction without further purification. The yield was 164 mg (∼100%). High-resolution MS (ESI): m/z ) 464.1526 for C25H33N5O8S ([M + H]+). 1H NMR (DMSO-d6 + D2O): 9.23 (s, 1H) 8.55 (d, 1H, J ) 1.8 Hz), 8.33 (br d, 1H), 8.23 (br d, 1H), 7.8 (d of d, 1H, J ) 7.2, 1.8 Hz), 7.4 (m, 2H), 7.2 (d, 2H, J ) 9.6 Hz), 4.4 (m, 1H), 3.65 (s, 3H), 2.7 (t, 2 H, J ) 7.2 Hz), 1.8 (m, 2H), 1.5 (m, 2H), 1.38-1.42 (m, 2H). Synthesis of N-E-(4-(Diphenylphosphino)benzoyl)N-r-(6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinyl)lysine Methyl Ester (HYNIC-Kp-DPPB). To a round-bottom flask were added 4-(diphenylphosphino)benzoic acid (22.94 mg, 0.074 mmol) and 3 mL of DMF. To the solution were added 1-O-(N,N′,N′′,N′′′tetramethyluronium)benzotriazoloxy hexafluorophosphate (31.24 mg, 0.082 mmol), 1-hydroxybenzotriazole (11.11 mg, 0.082 mmol), and diisopropylethylamine (48.4 mg, 0.065 mL). After the reaction mixture was stirred for 7 min, N-R-(6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinyl)lysine methyl ester hydrochloride (45 mg, 0.089 mmol) in 4 mL of DMF was added to the above

Purohit et al.

mixture. The reaction mixture was stirred for 2 h at room temperature. The solvent was removed in vacuo, and the resulting crude oil was subjected to HPLC purification (method 1). The collected fractions were combined and were lyophilized to give a pale yellow solid. The yield was 33 mg (∼60%). High-resolution MS (ESI): m/z ) 752.2302 for C39H38N5O7PS ([M + H]+). 1H NMR (CD3OD): 9.32 (s, 1H), 8.56 (s, 1H), 8.4 (d of d, 1H), 8.3 (m, 1H), 8.0 (m, 1H), 7.7 (d of d, 2H, J ) 1.2, 7.2 Hz), 7.5 (m, 2H), 7.387.28 (m, 12H), 4.6 (m, 1H), 3.76 (s, 3H), 3.4 (m, 2H), 2.0 & 1.9 (m, 2H), 1.7 (m, 2H), 1.55 (m, 2H). 31P NMR (CD3OD): -4.57. Synthesis of N-E-(2-(Diphenylphosphino)benzoyl)N-r-(6-(2-(2-Sulfonatobenzaldehyde)hydrazono)nicotinyl)lysine Methyl Ester (HYNIC-Ko-DPPB). To a round-bottom flask were added 2-(diphenylphosphino)benzoic acid (17.94 mg, 0.058 mmol) and 3 mL of DMF. To the solution were added 1-O-(N,N′,N′′,N′′′tetramethyluronium)azabenzotriazoloxy hexafluorophosphate (26.73 mg, 0.070 mmol), 1-hydroxyazabenzotriazole (9.56 mg, 0.070 mmol), and diisopropylethylamine (45.4 mg, 0.061 mL). After the reaction mixture was stirred for 7 min, N-R-(6-(2-(2-sulfonatobenzaldehyde)hydrazono)nicotinyl)lysine methyl ester hydrochloride (35.2 mg, 0.070 mmol) dissolved in 4 mL of DMF was added to the above mixture. The reaction mixture was stirred for 2 h. The solvent was removed in vacuo, and the resulting crude oil was subjected to HPLC purification using the method described above. The collected fractions were combined and were lyophilized to give the product as a pale yellow solid. The yield was 29 mg (66%) after HPLC purification. High-resolution MS (ESI): m/z ) 752.2302 for C39H38N5O7PS ([M + H]+). 1H NMR (CD3OD): 9.37 (s, 1H), 8.6 (s, 1H), 8.4 (dd, 2H, J ) 1.8, 7.2 Hz), 7.8 (m, 1H), 7.68-7.41 (m, 16H), 7.17 (bd, 1H), 4.3 (m, 1H), 3.66 (s, 3H), 2.7 (m, 2H), 1.8-1.75 (m, 2H), 1.35-1.26 (m, 4H). 31 P (CD3OD) δ: -8.23. General Procedure for Preparation of Macrocyclic 99mTc Complexes. To a sealed clean 5.0 mL vial were added the phosphine-containing HYNIC chelator (5-40 µg), 0.4 mL of in 0.25 M succinate buffer (pH ) 5.0), 0.2 mL of ethanol, and 0.4 mL of tricine solution (25-100 mg/mL in 0.25 M succinate buffer, pH ) 5.0). The mixture was immediately degassed under vacuum (85% for the complex [99mTc(HYNIC-Kp-DPPB)(tricine)] and >95% for [99mTc(HYNIC-Ko-DPPB)(tricine)]. In both cases, formation of [99mTc]colloid was minimal. The 99mTc-labeling efficiency is extremely high for HYNIC-Kp-DPPB. The minimum amount of HYNICKp-DPPB to achieve high yield 99mTc-labeling is about 5 µg for 20 mCi of 99mTcO4-, corresponding to a molar ratio of ∼1:50 for Tc:HYNIC-Kp-DPPB. We also tried to use other polydentate aminocarboxylates, such as nitrilotriacetic acid (NTA), N-(2-hydroxyethyl)iminodiacetic acid (HIDA), N-(2-hydroxyethyl)glycine (monocine), N,N-bis(2-hydroxymethyl)glycine (Bicine), ethylenediamine-N,N′-diacetic acid (EDDA), and N-(hydroxyethyl)ethylenediamine triacetic acid (HEDTA), as coligands to replace tricine. The yields for the their 99mTc complexes are very low (6 h. We also examined the solution stability of HPLCpurified macrocyclic 99mTc complexes [99mTc(HYNIC-KpDPPB)(tricine)] and [99mTc(HYNIC-Ko-DPPB)(tricine)]. For the complex [99mTc(HYNIC-Kp-DPPB)(tricine)], the peak at 15 min was collected. Volatiles were removed

Figure 5. Solution stability data for [99mTc(HYNIC-Kp-TPPMC)(tricine)] and [99mTc(HYNIC-Kp-TPPMC)(tricine)].

Phosphine-Containing HYNIC Derivatives

Figure 6. Solution stability for the HPLC-purified [99mTc(HYNIC-Ko-DPPB)(tricine)].

under vacuum. The residue was redissolved in saline to give a concentration of ∼2 mCi/mL. Since the HPLC mobile phase contains ammonium acetate buffer (pH ) 6.8), no additional buffer was used for dilution. The solution was monitored by radio-HPLC (method 2). It was found that the macrocyclic complex [99mTc(HYNIC-KpDPPB)(tricine)] remains stable for >6 h at room temperature after HPLC purification. There is no indication of any significant decomposition during this period of time. Addition of TPPTS (1 mg/mL: ∼2 × 10-3 M) into the solution above caused no significant change in the radioHPLC chromatogram. The fact that the macrocyclic complex [99mTc(HYNIC-Kp-DPPB)(tricine)] remains intact in the presence of a large excess of TPPTS is significant. This suggests that the ligand exchange reaction is not dependent on the concentration of the entering ligand, and dissociation of the Tc-P bond is the rate-limiting step. Therefore, the high solution stability

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of [99mTc(HYNIC-Kp-DPPB)(tricine)] is probably due to its kinetic inertness. For the macrocyclic complex [99mTc(HYNIC-Ko-DPPB)(tricine)], only the peak at ∼15 min was collected. Volatiles were removed under reduced pressure. The residue was redissolved in saline to give a concentration of ∼2 mCi/mL. Figure 6 shows staggered HPLC chromatograms for the HPLC-purified complex [99mTc(HYNICKo-DPPB)(tricine)]. Obviously, the main species ∼15 min remains unchanged over 4 h at room temperature. At 50 °C, however, it is slowly converted to the two minor components until it reaches equilibrium over ∼6 h. There is no indication of any significant decomposition during this period of time. These results clearly demonstrate the high solution stability of the complex [99mTc(HYNIC-KoDPPB)(tricine)]. LC-MS Data of Macrocyclic 99mTc Complexes. One of the important aspects of radiochemistry is to know the chemical composition of the radiopharmaceutical prepared in the radiolabeled kit. Since the total Tc (99mTc and 99Tc) concentration in the generator eluant is very low (10-8 to 10-6 M), it is impossible to use spectroscopic (IR, UV/vis, and NMR) methods to characterize 99mTc radiopharmaceuticals at the tracer level. Recently, we reported the use of LC-MS for characterization of small molecule 99mTc radiopharmaceuticals (36). It was found that the LC-MS is particularly useful for determining the composition of 99mTc radiopharmaceuticals at the tracer (99mTc) level. In this study, we used a decayed generator eluant to prepare macrocyclic 99mTc complexes [99mTc(HYNIC-KpDPPB)(tricine)] and [99mTc(HYNIC-Ko-DPPB)(tricine)]. We used LC-MS to analyze solutions containing macrocyclic 99mTc complexes [99mTc(HYNIC-Kp-DPPB)(tricine)] and [99mTc(HYNIC-Ko-DPPB)(tricine)]. Figure 7 shows LC-MS spectra for [99mTc(HYNIC-Kp-DPPB)(tricine)] (top) and [99mTc(HYNIC-Ko-DPPB)(tricine)] (bottom). The LC-MS data is completely consistent with the proposed composition. The LC-MS data (Table 1) of [99Tc(HYNICKp-TPPMC)(tricine)] also show that all three peaks have the same molecular weight with m/z ) 857.3 for [M + H]+. These results clearly show that (1) the phosphine-P is indeed bonded to Tc in the macrocyclic complex [99mTc(HYNIC-Ko-DPPB)(tricine)], (2) the presence of three radiometric peaks in its radio-HPLC chromatogram is due to resolution of three different isomers; and (3)

Figure 7. Mass spectra of the peaks at ∼15 min for macrocyclic 99mTc complexes [99mTc(HYNIC-Kp-DPPB)(tricine)] (left) and [99mTc(HYNIC-Kp-DPPB)(tricine)] (right) prepared using decayed [99mTc]pertechnetate eluant.

726 Bioconjugate Chem., Vol. 14, No. 4, 2003 Table 1. LC-MS Data for Macrocyclic 99mTc

99mTc

Purohit et al. Complexes

complex

[99mTc(HYNIC-Kp-DPPB)(tricine)] [99mTc(HYNIC-Ko-DPPB)(tricine)], peak A [99mTc(HYNIC-Ko-DPPB)(tricine)], peak B [99mTc(HYNIC-Ko-DPPB)(tricine)], peak C

Figure 8. Proposed structures for macrocyclic

99mTc

formula ([M + H]+)

formula weight

found ([M + H]+)

C38H24N6PO9 C38H24N6PO9 C38H24N6PO9 C38H24N6PO9

857.25 857.25 857.25 857.25

857.2 857.3 857.3 857.3

complexes.

the attachment site of the linker has a significant impact on the coordination chemistry of the phosphine-containing HYNIC chelator.

planation remains a speculation. Studies on structures of macrocyclic 99Tc complexes will definitely help us to understand the fundamental coordination chemistry of HYNIC chelators in their macrocyclic 99mTc complexes.

CONCLUSIONS

Our preliminary results clearly demonstrate that the phosphine-containing HYNIC chelators (HYNIC-KpDPPB and HYNIC-Ko-DPPB) described in this study are able to form highly stable macrocyclic 99mTc complexes when tricine is used as the coligand. The complex [99mTc(HYNIC-Kp-TPPB)(tricine)] shows only one detectable isomer in solution while the complex [99mTc(HYNICKo-TPPB)(tricine)] has three isomers. The composition of the two macrocyclic 99mTc complexes has been determined by LC-MS at the tracer level. The LC-MS data is completely consistent with the proposed composition of the two macrocyclic 99mTc complexes. We also found that the triphenylphosphine moiety is sensitive to oxidation by the oxygen in the reaction mixture and free radicals produced during radiolabeling due to radiolysis. Several questions remain unanswered. These include (1) the bonding modality of the HYNIC chelator and the coordinated tricine, (2) the identity of three isomers in the macrocyclic 99mTc complex [99mTc(HYNIC-Ko-TPPB)(tricine)], (3) the reason that there are three isomers for the complex [99mTc(HYNIC-Ko-TPPB)(tricine)] while only one is detectable for the complex [99mTc(HYNIC-KpTPPB)(tricine)], and (4) the impact of linker length and attachment site on the coordination chemistry of phosphine-containing HYNIC chelators. In ternary ligand 99mTc complexes, [Tc(HYNIC-BM)(tricine)(TPPTS)], the HYNIC group is monodentate due to the presence of the tetradentate tricine and monodentate phosphine coligands. The HYNIC group in macrocyclic 99mTc complexes, [Tc(HYNIC-L)(tricine)] (L ) oTPPB and p-TPPB), may be forced to become bidentate (Figure 8) due to the bonding of the attached phosphineP. The tridentate tricine are expected to result in formation of different isomers due to its asymmetric nature in bonding to the Tc. If HYNIC is monodentate in the macrocyclic 99mTc complex, conformational isomers may form due to the limited freedom of rotation in the macrocyclic Tc chelate. This may explain the presence of three different isomers, which interconvert in solution at elevated temperatures, for [99mTc(HYNIC-Ko-TPPB)(tricine)]. However, in the absence of solid-state structure and variable temperature NMR studies, this ex-

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