A New Strategy for the Preparation of Peptide-Targeted Technetium

Chemistry, Syracuse University, Syracuse, New York 13244-4100, and Molecular Insight Pharmaceuticals Inc.,. Cambridge, Massachusetts 02142. Received ...
1 downloads 0 Views 170KB Size
Download PDF
Bioconjugate Chem. 2005, 16, 1189−1195

1189

A New Strategy for the Preparation of Peptide-Targeted Technetium and Rhenium Radiopharmaceuticals. The Automated Solid-Phase Synthesis, Characterization, Labeling, and Screening of a Peptide-Ligand Library Targeted at the Formyl Peptide Receptor Karin A. Stephenson,† Sangeeta Ray Banerjee,‡ Oyebola O. Sogbein, Murali K. Levadala,‡ Nicole McFarlane,† Douglas R. Boreham,† Kevin P. Maresca,§ John W. Babich,§ Jon Zubieta,‡ and John F. Valliant*,† Department of Chemistry, McMaster University, Hamilton, ON, Canada L8S 4M1, The Medical Physics and Applied Radiation Sciences Unit, McMaster University, Hamilton, ON, Canada, L8S 4M1, Department of Chemistry, Syracuse University, Syracuse, New York 13244-4100, and Molecular Insight Pharmaceuticals Inc., Cambridge, Massachusetts 02142. Received March 3, 2005; Revised Manuscript Received June 16, 2005

A new solid-phase synthetic methodology was developed that enables libraries of peptide-based Tc(I)/Re(I) radiopharmaceuticals to be prepared using a conventional automated peptide synthesizer. Through the use of a tridentate ligand derived from N-R-Fmoc-L-lysine, which we refer to as a single amino acid chelate (SAAC), a series of 12 novel bioconjugates [R-NH(CO)ZLF(SAAC)G, R ) ethyl, isopropyl, n-propyl, tert-butyl, n-butyl, benzyl; Z ) Met, Nle] that are designed to target the formyl peptide receptor (FPR) were prepared. Construction of the library was carried out in a multiwell format on an Advanced ChemTech 348 peptide synthesizer where multi-milligram quantities of each peptide were isolated in high purity without HPLC purification. After characterization, the library components were screened for their affinity for the FPR receptor using flow cytometry where the Kd values were found to be in the low micromolar range (0.5-3.0 µM). Compound 5j was subsequently labeled with 99mTc(I) and the product isolated in high radiochemical yield using a simple Sep-Pak purification procedure. The retention time of the labeled compound matched that of the fully characterized Reanalogue which was prepared through the use of the same solid-phase synthesis methodology that was used to construct the library. The work reported here is a rare example of a method by which libraries of peptide-ligand conjugates and their rhenium complexes can be prepared.

INTRODUCTION

Short peptide sequences are one of the most effective vectors for targeting radionuclides to specific receptor systems (1). As a result, there are a number of peptidebased imaging agents that are currently used clinically or that are undergoing clinical trials (2). The archetypal example is 111In-labeled Octreotide, which is a somatostatin analogue used to image neuroendocrine tumors (3). One of the attractive features of peptides is that they can be easily modified to optimize receptor binding affinity, pharmacokinetics, and clearance rates and pathways. The sequence of the amino acids, the addition of prosthetic groups, and, in the case of metalloradionuclides, the nature of the chelating group can all be changed to obtain the desired biodistribution profile. Because of this flexibility, the opportunity presents itself to prepare libraries of peptide-ligand conjugates in parallel or in combination as a means of rapidly identifying the ideal choice of factors. Despite recent reports on the development of new solid-phase synthetic procedures (47), the majority of radiopharmaceuticals continue to be * To whom correspondence should be addressed. Tel: 905-525-9140 ext. 22840; FAX: 905-522-2509; E-mail: valliant@ mcmaster.ca. † McMaster University. ‡ Syracuse University. § Molecular Insight Pharmaceuticals Inc.

prepared using linear discovery strategies which are significantly less efficient than contemporary drug discovery methodologies. Recently we described a novel solid-phase synthetic strategy for incorporating a Tc(I)/Re(I) binding ligand, which we refer to as a single amino acid chelate (SAAC) (1a, Figure 1), into peptides (8, 9). The SAAC, which is derived from lysine, can be introduced into a peptide as if it were a natural amino acid making the chelate a convenient and versatile synthon for preparing peptidetargeted radiopharmaceuticals. The current objective was to develop the means to use the SAAC system to prepare libraries of chelate-derived peptides as a new strategy for discovering novel radiopharmaceuticals. As a model system, we selected the fMLF peptide which has been used to direct 99mTc to sites of infection and inflammation by targeting the formal peptide receptor (FPR) on leukocytes (10-24). Unfortunately, fMLF is an agonist which causes unwanted side effects (neutropenia) even when the apparent specific activity of the tracer is very high (23). One approach to overcoming this issue is to develop an analogue of fMLF which behaves as an antagonist rather than an agonist (16). By changing the nature of the formyl group in fMLF to substituted ureas, a series of antagonists for the FPR that have reasonably high binding affinities were discovered (25). A 99mTc-labeled SAAC analogue of the ureaderived peptides could therefore be used to image sites of infection and inflammation without causing neutro-

10.1021/bc0500591 CCC: $30.25 © 2005 American Chemical Society Published on Web 08/26/2005

1190 Bioconjugate Chem., Vol. 16, No. 5, 2005

Figure 1. A Single Amino Acid Chelate (SAAC) and its complex with the [Re(CO)3]+ core.

penia. To obtain a viable agent, the peptide-SAAC derivative must maintain reasonable affinity for the FPR while also possessing the appropriate distribution kinetics and clearance mechanism (16, 23). As a means of identifying compounds that meet these criteria, a parallel array of RNHC(O)ZLF(SAAC)G derivatives were prepared in which factors that can change receptor binding affinity and/or the distribution/mode of clearance (i.e. the substituents off the urea (R) and the nature of the first amino acid (Z)) were varied. EXPERIMENTAL PROCEDURES

Materials. Unless otherwise stated, all reagents and solvents were ACS grade or higher and used without further purification from commercial suppliers. Polystyrene-based N-R-9-fluorenylmethoxycarbonyl (Fmoc)-glycine loaded SASRIN resin (0.69 mmol g-1, 1% divinylbenzene, 200-400 mesh) was obtained from Bachem Inc. Fmoc-protected amino acids were purchased from NovaBiochem Inc., Bachem Inc., and Advanced ChemTech Inc. Fluorescein-labeled fNLFNTK was purchased from Molecular Probes Inc. (product # F-1314). The isocyanates were purchased shortly before use from Sigma-Aldrich Co. Instrumentation. All peptides were prepared on an Advanced ChemTech 348 Ω Peptide Synthesizer using a 40-well reaction block. Peptides were analyzed by positive ion electrospray mass spectrometry on a Micromass Quattro Ultima instrument. Samples were dissolved in 50/50 CH3CN/H2O prior to analysis. FTIR spectra were acquired on a Bio-Rad FTS-40 FTIR spectrometer. NMR experiments were performed using a Bruker AV 600 NMR spectrometer. In terms of signals, s refers to a singlet, d refers to doublet, dd refers to doublet of doublets, and m refers to multiplet. Analytical HPLC was performed using a Varian Pro Star model 330 PDA detector and model 230 solvent delivery system and a C-18 Microsorb column (4.6 × 250 mm, 300 Å/5 µm). For the peptides and the analysis of the Re complex, the elution protocol was a linear gradient of 100% water containing 0.1% TFA to 100% acetonitrile containing 0.1% TFA over 20 min. The flow rate was 1.0 mL min-1, and all runs were monitored at λ ) 254 and 214 nm. For the reactions involving 99mTc, HPLC experiments were performed on a Varian Prostar Model 230 HPLC (high performance liquid chromatography) instrument coupled to a Bio-Rad IN/US γ-detector using a Nucleosil (L x ID ) 250 × 4.6 mm), analytical column (300 Å/5 µm, RP-C18). The mobile phase consisted of Solvent A ) triethylammonium phosphate buffer (pH ) 2-2.5), Solvent B ) CH3OH. Gradient: 0-3 min, 100% A; 3-6 min, 100% A to 75% A; 6-9 min, 75% A to 67% A; 9-20 min, 67% A to 0% A; 20-22 min, 0% A; 22-25 min, 0% A to 100% A; 25-30 min, 100% A.

Stephenson et al.

Solid-Phase Peptide Synthesis. Fmoc-glycine-loaded SASRIN resin (150 mg, 0.69 mmol/g) was added to 12 wells of the reaction block, suspended in DMF (2 mL/ well), and shaken at 900 rpm for 1 min. The wells were subsequently filtered, suspended in THF (2 mL/well), shaken at 900 rpm for 1 min, and drained for 90 s. The THF wash was repeated two more times. The DMF wash was then repeated a final two times to complete the general wash cycle. This procedure was used between every deprotection and coupling step. Fmoc deprotection was brought about through the addition of 25% v/v piperidine-DMF solution to the active vessels (2 mL/ well) and shaking for 5 min at 900 rpm. Following filtration, the process was repeated, shaking for 10 min. The deprotected resin-bound amino acid was washed using the general wash procedure and subsequently coupled to the next Fmoc-protected amino acid using a standard HBTU coupling technique. Coupling reactions initially involved adding DMF (200 µL) to the active vessels followed by the addition of a 4-fold excess of the protected amino acid as a 0.5 M solution in DMF. Four equivalents of HBTU as a 0.5 M solution in DMF was then added followed by a 8-fold excess of DIPEA as 2.0 M solution in DMF. The reaction block was subsequently shaken for 80 min at 900 rpm. Following filtration, the resin was washed using the general washing procedure prior to the start of the next cycle. Once the final amino acid had been added and the Fmoc group removed, a pause was introduced into the peptide program so that 10 equiv of the isocyanates in DMF (0.5 M solution) could be added. The reaction block was then shaken at 900 rpm under nitrogen for 12 h. The reaction vessels were emptied and the resins washed using the general wash cycle. Peptides were cleaved from the resin support using a TFA cocktail containing EDT (2%), water (2%), and TIS (2%). The cleavage cocktail, cooled to 0 °C, was added to the resin and the mixture allowed to warm to room temperature over 60 min. The suspension was filtered into cold diethyl ether and the resulting heterogeneous solution centrifuged at 3000 rpm and -5 °C for 10 min. The resulting pellet was washed with cold diethyl ether (3 × 25 mL), dissolved in distilled-deionized water, and lyophilized, yielding a white solid for all compounds. Preparation of [99mTc(CO)3(OH2)3]+. A 10 mL penicillin vial containing K2[BH3‚CO2], (8.5 mg, 63 µmol), Na2B4O7‚10H2O (2.9 mg, 8.0 µmol), Na/K-tartrate (15.0 mg, 53 µmol), and Na2CO3 (4.0 mg, 38 µmol) was fitted with a rubber septum and the vial flushed with N2(g) for 15 min. 99mTc-generator eluate (370 MBq, 10 mCi) in 500 µL of saline was added by a syringe, and the solution was heated to 95 °C for 30 min. After cooling on an icebath, the alkaline solution was neutralized by the addition of 28 µL of HCl (12 M HCl in 100 µL of H2O). Quality control was performed by gradient HPLC (yields g 95%). Radiolabeling 5j. An aliquot (250 µL) of 5j (0.5 mg, 0.58 µmol) dissolved in 200 µL of 100 mM phosphate buffer (pH ) 7.2) and 300 µL of CH3CN was added to the solution containing [99mTc(CO)3(OH2)3]+ by syringe and the mixture heated to 70 °C for 45 min. The reaction mixture was cooled in an ice bath and the product isolated using a Sep-Pak (Waters, C18). The Sep-Pak was conditioned prior to use with absolute ethanol (10 mL), acetonitrile (10 mL), 1:1 acetonitrile/10 mM HCl (10 mL), and 10 mM HCl (10 mL). After loading the reaction solution onto the conditioned Sep-Pak, the column was washed with 10 mM HCl (7 × 1 mL), 1:4 acetonitrile/10 mM HCl (2 × 1 mL), 1:1 acetonitrile/10 mM HCl (2 × 1 mL), 4:1 acetonitrile/10 mM HCl (2 × 1 mL), and finally

Bioconjugate Chem., Vol. 16, No. 5, 2005 1191

Peptide-Targeted Te and Re Radiopharmaceuticals Scheme 1

acetonitrile (5 × 1 mL). Compound 6b eluted in fractions 12-17 (radiochemical yield ) 90%, radiochemical purity g 98%). Ligand Challenge Experiments. Cysteine (0.13 µmol in 5 mL of ddH2O) and histidine (0.13 µmol in 5 mL of ddH2O) were added to two separate vials each containing 11.1 MBq to 18.5 MBq (300-500 µCi) of purified 6b in 250 µL of 100 mM phosphate buffer. Both vials were incubated at 37 °C for 24 h. Aliquots were taken periodically and analyzed by HPLC. Leukocyte Preparation. Whole human blood (1015 mL) was collected in vacutainer tubes (Becton Dickinson) containing sodium heparin. The red blood cells were lysed using a modified ammonium chloride method (1 part whole blood to 23 parts 0.145 M ammonium chloride solution containing 1.5 mM potassium bicarbonate and 0.1 mM EDTA). Tubes were incubated at room temperature for 15 min and then centrifuged in a Beckman Coulter Allegra 6R centrifuge at 900 rpm (400 g) for 7 min at 5 °C. The supernatant was removed and the pellet of leukocytes subsequently resuspended in HBSS containing 0.1% BSA, 10 mM HEPES, and 1.5 mM CaCl2 (HBSS+). The cells were spun for 7 min at 400 g at 5 °C, the supernatant was removed, and the pellet was resuspended in HBSS+. Cell counts were performed using a hemacytometer and a Beckman Coulter Z2 Coulter Particle Count and Size Analyzer. Cell concentrations were adjusted to 2 × 106 mL-1. Flow Cytometry. Samples were run on a Beckman Coulter EPICS XL, equipped with an argon laser (488 nm excitation wavelength). Fluorescence was measured using a 525 ( 15 nm band-pass filter. The flow cytometer was calibrated with fluorescein isothiocyanate (FITC) labeled beads (Quantum 26 Beads, Bangs Laboratories, Inc, Fishers, IN) prior to sample analysis. The fluorescence intensity of the standard was provided by the manufacturer in units of Molecules of Equivalent Soluble Fluorochrome (MESF) and ranged from 5300 to 468 800. A linear fit of MESF versus mean channel number of the beads was used to convert all subsequent mean channel values to MESF values. Equilibrium Binding Assay. The equilibrium-binding assay was used to determine the number of formyl peptide receptors (FPRs) on the surface of the neutrophils and the dissociation constant (Kd) of the fluoresceinlabeled fNLFNTK (Molecular Probes) (1.0 nM) by preparing a saturated binding curve of peptide concentrations verses fluorescence intensity. Peptide stock solutions

were made by dissolving the peptide derivatives in DMSO and then diluting with HBSS+ to the desired concentrations, ensuring the concentration of DMSO was less than 0.1%. Cells at 1 × 106 mL-1 were equilibrated with 0.25, 0.50. 1.0, 3.0, 5.0, 10.0, and 20.0 nM solutions of the peptide derivatives (U) in duplicate. The samples were incubated for 2 h at 0 °C in the dark and then run on the flow cytometer. Neutrophils were gated on the basis of forward and side scatter parameters using Expo 32 ADC software (Beckman Coulter). All samples were repeated in the presence of 30 µM fMLF to account for nonspecific binding. The number of receptor-ligand complexes per cell (B) was determined using the following equation, where Q is a normalization factor used by Waller et al. (28) and F is the total fluorescence per cell:

×Q (MESF cell )

B(in ligand bound per cell) ) F

The level of nonspecific binding did not vary significantly with the concentration of labeled peptide; thus, an average value of nonspecific binding was subtracted from the total fluorescence per cell, F, when determining the number of receptor-ligand complexes. The total number of N-formyl peptide receptors, Rtot, and the equilibrium dissociation constant, Kd, were evaluated by minimizing the squared residual of the equilibrium solution using a one-site binding model (see the following equation). The values of Rtot and Kd for each donor were solved simultaneously.

[LR] )

Rtot[L] [L] + Kd

RESULTS

Peptide Synthesis. Solid-phase synthesis (Scheme 1) was carried out on an advanced Advanced ChemTech 348 Ω synthesizer using all L-amino acids. For each peptide, the synthesis began by coupling Fmoc protected SAAC to SASRIN resin bound glycine using a standard HBTU coupling protocol. After deprotection with 20% piperidine in DMF, the phenylalanine and leucine residues were added. Following removal of the Fmoc group on leucine, Fmoc-methionine or Fmoc-norleucine were added and subsequently deprotected. Conversion of 4 to the corresponding ureas (see Table 4) was carried out by adding different isocyanates to the

1192 Bioconjugate Chem., Vol. 16, No. 5, 2005

Stephenson et al. Table 2. for 5j

1H

1H

Table 1. HPLC Elution Times, Electrospray MS, and Select IR Data compd

found m/z

calcd m/z

[urea C(O)] (cm-1)

N[M(CO)] (cm-1)

5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 6a

12.9 13.5 13.6 13.8 14.6 14.7 13.2 13.7 13.8 14.4 14.2 14.0 16.9

848 862 862 876 910 876 830 844 844 858 858 892 1128

847 861 861 875 909 875 829 843 843 857 857 891 1127

1545 1549 1547 1543 1553 1549 1551 1544 1549 1543 1547 1546 1535

1916, 2031

reaction wells. The duration of the reaction needed to achieve complete conversion to the urea (12 h) was determined in advance of preparing the library by carrying out a model reaction using the least reactive isocyanate (tert-butyl isocyanate). During these experiments, samples of resin were periodically extracted from the reaction well and exposed to ninhydrin (26) until complete conversion to the urea was indicated. Peptides were cleaved from the support under argon or nitrogen using a cocktail consisting of 94% TFA, 2% EDT, 2% TIS, and 2% water (27). It was important to exclude moisture and oxygen during the cleavage reaction to avoid oxidation of the methionine containing peptides. Following precipitation using cold diethyl ether and centrifugation, the peptide conjugates were dissolved in water, lyophilized, and analyzed for purity. The HPLC chromatograms of the peptides, including the tert-butyl derivative 5j (Figure 2), showed that the products had been prepared in very high purity in all cases and that no further purification was needed. The Re complex of 5j was prepared using an identical solid-phase synthetic procedure except that compound 1b was substituted for 1a. Characterization. Peptides were analyzed by electrospray mass spectrometry, and in each case the observed molecular ions were consistent with the mass of the desired compounds (Table 1). IR analysis confirmed the presence of the urea group, in which the CdO stretch appeared between 1535 cm-1 and 1553 cm-1 (Table 1). In the case of the Re complex of 5j, the metal CO stretches appeared at 1916 cm-1 and 2031 cm-1.

chemical shift (ppm)

2-4 6 7 (2H) 8 (2H) 9 (2H) 10 (3H) 12 13 (2H) 14

Figure 2. HPLC chromatogram (UV-detection, λ ) 254 nm) of 5j.

elution time (min)

NMR Assignments and Coupling Constant

coupling constant (Hz)

1.292 (s) 4.042 (m) 1.704 (m) 1.280 (m) 1.280 (m) 0.840 (m) 4.419 (m) 1.279 (m) 1.562 (m)

3J(14, 3J(14,

15 (3H) 16 (3H) 18 19′ 19′′ 21, 25 (2H) 22-24 (3H) 27 28′ 28′′ 29 (2H) 30 (2H) 31 (2H) 32/38 (4H) 34/40 (2H) 35/41 (2H) 36/42 (2H) 37/43 (2H) 45 (2H) Table 3.

13C

0.804 (d) 0.840 (d) 4.695 (m) 3.112 (m) 2.971 (m) 7.221 (m) 7.172 (m) 4.419 (m) 1.562 (m) 1.704 (m) 1.279 (m) 1.562 (m) 2.647 (m) 4.270 (s) 8.009 (d) 8.515 (dd) 7.948 (dd) 8.724 (d) 3.939 (d)

15) ) 6.6 16) ) 6.6

35) ) 7.8 36) ) 7.2 3J(36, 37) ) 6.0 3J(34, 3J(35,

NMR Assignments for 5j

13C

chemical shift (ppm)

13C

chemical shift (ppm)

1 2-4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

54.5 29.5 177.6 57.7 32.7 24.1 29.2 14.4 175.2 55.4 41.4 26.0 21.9 23.3 175.4 56.9 38.9 137.2

21, 25 22-24 26 27 28 29 30 31 32/38 33/39 34/40 35/41 36/42 37/43 44 45 46

130.3 128.6, 130.9 174.5 54.2 32.7 23.4 26.5 56.2 56.8 153.4 129.5 149.6 128.9 143.5 175.3 42.6 173.6

The tert-butyl urea 5j was further characterized using H and 13C NMR spectroscopy (Tables 2 and 3). The 1H NMR of 5j (Figure 3) shows a number of multiplets in the aromatic region which were assigned to the pyridine rings of the SAAC and the aromatic group of phenylalanine. The alpha protons of the amino acids appear between 4 and 5 ppm, which along with the side chain residues, were readily assigned using standard twodimensional experiments (COSY, HSQC, and HMBC). A 1

Bioconjugate Chem., Vol. 16, No. 5, 2005 1193

Peptide-Targeted Te and Re Radiopharmaceuticals

Figure 3.

1H

NMR of 5j (600 MHz, 1:1 CD3CN/TFA-d).

Table 4. Peptides and Binding Affinities (Kd) compd

peptide

Kd (µM)

5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l 5m

ethyl-NH(CO)-Met-Leu-Phe-SAAC-Gly isopropyl-NH(CO)Met-Leu-Phe-SAAC-Gly n-Propyl-NH(CO)-Met-Leu-Phe-SAAC-Gly tert-Butyl-NH(CO)-Met-Leu-Phe-SAAC-Gly benzyl-NH(CO)-Met-Leu-Phe-SAAC-Gly n-Butyl-NH(CO)-Met-Leu-Phe-SAAC-Gly ethyl-NH(CO)-Nle-Leu-Phe-SAAC-Gly isopropyl-NH(CO)-Nle-Leu-Phe-SAAC-Gly n-Propyl-NH(CO)-Nle-Leu-Phe-SAAC-Gly tert-Butyl-NH(CO)-Nle-Leu-phe-SAAC-Gly n-Butyl-NH(CO)-Nle-Leu-Phe-SAAC-Gly benzyl-NH(CO)-Nle-Leu-Phe-SAAC-Gly fMLF

2.9 ( 0.8 3.0 ( 0.3 1.3 ( 0.2 2.3 ( 0.4 1.7 ( 0.2 0.5 ( 0.1 1.2 ( 0.2 1.2 ( 0.3 1.1 ( 0.6 2.6 ( 0.7 0.7 ( 0.2 0.8 ( 0.1 0.032 ( 0.003

singlet corresponding to the tBu group appeared at 1.29 ppm in the 1H NMR while the signal arising from the alpha proton of norleucine was present at 4.04 ppm, which showed a correlation to the urea carbonyl carbon (177.6 ppm) in the HMBC spectrum. The resonances remaining in the carbonyl region of the 13C NMR spectrum were consistent with the number of amide/acid groups in the peptide backbone. The carbonyl groups were assigned using a TOCSY experiment, which in turn confirmed the proposed connectivity of the peptide. In Vitro Screening Studies using Flow Cytometry. Peptides were screened on a flow cytometer using a competitive binding assay where the concentration of the competitor was allowed to vary while maintaining the concentration of the fluorescent peptide (28-30). Competitive binding data was obtained on the same day using the same human donor as for the equilibrium binding data, to minimize variability in the number of receptors. Cells (106 mL-1) were incubated with 1.0 nM fluorescein-labeled fNLFNTK and 0, 1, 15, 100, 300, 700, 1000, and 1400 nM competitor in duplicate for 3 h at 0 °C in the dark. The equilibrium dissociation constant for the competing unlabeled ligand, Kd, was determined following literature procedures by fitting the specific ligand bound data to the same one-site binding model. The peptide, fMLF was used as a reference standard. Its Kd value was in good agreement with the value reported in the literature (21, 28). The Kd values for a reference standard (fMLF) and library constituents are given in Table 4. Scheme 2

Figure 4. γ-Radiochromatograms of [99mTc(CO)3(OH2)3]+ and 5j after 45 min at 70 °C (bottom) and purified 6b (top).

Radiochemistry. The peptide 5j (250 µg) was combined with 307 MBq (8.3 mCi) of [99mTc(CO)3(OH2)]+ in a mixture of PBS buffer and acetonitrile (Scheme 2). After heating the reaction to 70°C for 45 min, the desired product, 6b, was isolated, free from any unlabeled peptide, using a simple Sep-Pak purification method. The radiochemical yield of the isolated product was 90% with a radiochemical purity of greater than 98%. The γ-HPLC trace of the crude reaction mixture and the purified product are shown in Figure 4. Compound 6b was subsequently incubated with a large excess of cysteine and histidine as a preliminary means of evaluating the complexes tendency to undergo transchelation in the presence of a large excess of competing donors. After 24 h, the labeled peptide showed no signs of degradation which is consistent with the low spin d6 electronic configuration of the octahedral metal complex. DISCUSSION

The single amino acid chelate (SAAC) is a lysine derivative where the Fmoc protected form can be readily incorporated into a growing peptide using conventional peptide synthesis protocols. The chelate, which is soluble in water (when completely deprotected) and organic solvents (when protected), was prepared in very high yield on the multigram scale using a one-pot procedure involving relatively inexpensive starting materials. In the work reported here, a convenient solid-phase synthesis strategy was developed for preparing a small 12-member library of N-terminal urea derivatives of MLF(SAAC)G and NleLF(SAAC)G peptides where the nature of the urea substituent was varied. The peptides 5a-l were prepared using a standard coupling/deblocking procedure in very high purities without having to employ preparative HPLC. The fact that the products were produced in such high purity contrasts the synthesis of the N-formyl analogue where it was necessary to purify fMLF(SAAC)G by HPLC because the methionine group rapidly epimerized during synthesis to give two diastereomers. With the approach reported here, there was no evidence of epimerization in the HPLC or the NMR data.

1194 Bioconjugate Chem., Vol. 16, No. 5, 2005

The reported methodology was capable of producing sufficient quantities of each peptide to allow for both detailed characterization of the desired products and in vitro screening studies (vide infra). The electrospray mass spectrometry data agreed in all cases with the calculated spectra, and, as mentioned previously, HPLC confirmed the purity of each sample. Additional characterization of the library constituents can be obtained through 1H and 13C NMR experiments. The spectra, including the data presented for compound 5j, were consistent with the proposed structures of the peptides. In terms of binding to the FPR receptor, all compounds had Kd values in the low micromolar range which is in agreement with the data reported by Higgins et al. (25). These results indicate that the addition of the SAAC group does not have a detrimental influence on the behavior of the peptide in terms of binding to the FPR receptor. This observation in turn is consistent with the data reported by Cavicchioni et al. which suggests that C-terminal variants of fMLF type peptides do not significantly influence the peptides receptor binding affinity (31). The structure of the urea appeared to have a small affect on the Kd values where bulky groups (tBu, iPr) generally reduced the Kd values whereas the peptides containing linear alkyl substituents, particularly the nBu groups, showed somewhat higher affinity. In addition to considering Kd values, it is important to note that changing the nature of the urea and first amino acid will have a significant impact on the distribution and mode of clearance of the agents. Consequently, the 99mTc complexes of the library components will be screened in vivo in the appropriate animal model and the derivative showing the optimal distribution profile evaluated further. We have previously established that the single amino acid chelates form stable complexes with Re(I). To demonstrate that the SAAC-peptide derivatives can also be labeled efficiently with 99mTc, compound 5j was reacted with [99mTc(CO)3(OH2)]+, which was prepared using a formulation that is employed in commercially available carbonyl labeling kits. The labeled peptide, 6b, was isolated in excellent yield using a small amount of ligand (250 µg). To verify the nature of the 99mTc complex, the corresponding Re complex 6a was prepared via the same solid-phase synthetic procedure used to prepare 5j except that compound 1b was employed in place of 1a. The HPLC retention time of a sample of compound 6a, which had been previously characterized by LCMS, matched that for 6b. The ability to verify the nature of a 99mTc complex using a well characterized Re standard is an attractive feature of the SAAC system. With a number of traditional ligand systems (32-36) it is often difficult to prepare the corresponding Re or 99Tc complexes (let alone to use them for solid-phase synthesis) which in turn complicates the process of identifying compounds produced at the tracer level. With the SAAC system, the Re complexes can be prepared after hits from the library are identified or they can be synthesized in concert with the free ligands without having to significantly modify the original solidphase synthetic methodology. CONCLUSIONS

The reported methodology demonstrates the ease with which a library of peptide-ligand derivatives can be developed using the SAAC system. Increasing the size and structural diversity of the library is readily achievable by not only varying the end terminal groups and

Stephenson et al.

the nature of the natural amino acids, but also the position of the SAAC. Furthermore, because the ligand is based on a natural amino acid, either enantiomer of the SAAC can be readily incorporated at any position in a peptide. As such, the SAAC chelating system can be used to prepare libraries of a wide range of peptide-based radiopharmaceuticals using a conventional automated peptide synthesizer. In the case reported here, the library constituents show similar Kd values and will therefore be screened in vivo to determine the impact of the various substitutions on biodistribution profiles. ACKNOWLEDGMENT

We would like to acknowledge the Ontario Research and Development Challenge Fund (ORDCF) and The National Sciences and Engineering Research Council (NSERC) of Canada for funding and for providing a Scholarship for K.A.S. Supporting Information Available: HPLC, ESMS, 1H and 13C NMR, FT-IR, and select competitive binding data. This material is available free of charge via the Internet at http://pubs.acs.org. LITERATURE CITED (1) Liu, S., and Edwards, D. S. (1999) 99mTc-Labeled Small Peptides as Diagnostic Radiopharmaceuticals. Chem. Rev. 99, 2235-2268. (2) Welch, M. J., and Redvanly, C. S. (2002) Handbook of Radiopharmaceuticals: Radiochemistry and Applications, John Wiley and Sons, New York. (3) Kwekkeboom, D., Krenning, E. P., and de Jong, M. (2000) Peptide Receptor Imaging and Therapy. J. Nucl. Med. 41, 1704-1713. (4) Gershell, L. J., and Atkins, J. H. (2003) A Brief History of Novel Drug Discovery Technologies. Nat. Rev. Drug Discovery 2, 321-327. (5) Smith, C. J., Gali, H., Sieckman, G. L., Higginbotham, C., Volkert, W. A., and Hoffman, T. J. (2003) Radiochemical Investigations of 99mTc-N(3)S-X-BBN[7-14]NH2: An in Vitro/ in Vivo Structure-Activity Relationship Study Where X ) 0-, 3-, 5-, 8-, and 11-Carbon Tethering Moieties. Bioconjugate Chem. 14, 93-102. (6) Garie´py, J., Re´my, S., Zhang, X., Ballinger, J. R., BolewskaPedyczak, E., Rauth, M., and Bisland, S. K. (2002) A Simple Two-Step Approach for Introducing a Protected Diaminedithiol Chelator during Solid-Phase Assembly of Peptides. Bioconjugate Chem. 13, 679-684. (7) Greenland, W. E. P., Howland, K., Hardy, J., Fogelman, I., and Blower, P. J. (2003) Solid-Phase Synthesis of Peptide Radiopharmaceuticals Using Fmoc-N--(Hynic-Boc)-Lysine, a Technetium-Binding Amino Acid: Application to Tc-99mLabeled Salmon Calcitonin. J. Med. Chem. 46, 1751-1757. (8) Stephenson, K. A., Zubieta, J., Banerjee, S. R., Levadala, M. K., Taggart, L., Ryan, L.; McFarlane, N., Boreham, D. R.; Maresca, K. P., Babich, J. W., and Valliant, J. F. (2004) A New Strategy For The Preparation of Peptide-Targeted Radiopharmaceuticals Based on an Fmoc-Lysine-Derived Single Amino Acid Chelate (SAAC). Automated Solid-Phase Synthesis, NMR Characterization, and in Vitro Screening of fMLF(SAAC)G and fMLF[(SAAC)-Re(CO)3)+]G. Bioconjugate Chem. 15, 128-136. (9) Banerjee, S. R., Levadala, M. K., Lazarova, N., Wei, L.; Valliant, J. F., Stephenson, K. A., Babich, J. W., Maresca, K. P., and Zubieta, J. (2002) Bifunctional Single Amino Acid Chelates (SAAC) for Labeling of Biomolecules with {Tc(CO)3}+1 and {Re(CO)3}+1 cores. The crystal and molecular structures of [ReBr(CO)3(H2NCH2C5H4N)], [Re(CO)3{X(Y)NCH2CO2CH2CH3}]Br, (X ) Y ) 2-pyridylmethyl; X ) 2-pyridylmethyl, Y ) 2-(1-methylimidazolyl)methyl; X ) Y) 2-(1-methylimidazolyl)methyl, [ReBr(CO)3{(C5H4NCH2)NH(CH2C4H3S)] and [Re(CO)3{(C5H4NCH2)N(CH2C4H3S)CH2CO2)]. Inorg. Chem. 41, 6417-6425.

Bioconjugate Chem., Vol. 16, No. 5, 2005 1195

Peptide-Targeted Te and Re Radiopharmaceuticals (10) Babich, J. W., Coco, W. G., Barrow, S., Fischman, A. J., Femia, F. L., and Zubieta, J. (2000) 99mTc-Labeled Chemotactic Peptides: Influence of Coligand on Distribution of Molecular Species and Infection Imaging Properties. Synthesis and Structural Characterization of Model Complexes with the {Re(η2-HNNC5H4N)(η1-NNC5H4N)}core. Inorg. Chim. Acta 309, 123-136. (11) Babich, J. W., Graham, W., Barrow, S. A., Dragotakes, S. C., Tompkins, R. G., Rubin, R. H., and Fischman, A. J. (1993) Technetium-99m-Labeled Chemotactic Peptides: Comparison with Indium-111-Labeled White Blood Cells for Localizing Acute Bacterial Infection in the Rabbit. J. Nucl. Med. 34, 2176-2181. (12) Babich, J. W., Graham, W., Barrow, S. A., and Fischman, A. J. (1995) Comparison of the Infection Imaging Properties of a 99mTc Labeled Chemotactic Peptide with 111In IgG. Nucl. Med. Biol. 22, 643-648. (13) Babich, J. W., Solomon, H., Pike, M. C., Kroon, D., Graham, W., Abrams, M. J., Tompkins, R. G., Rubin, R. H., and Fischman, A. J. (1993) Technetium-99m-Labeled Hydrazino Nicotinamide Derivatized Chemotactic Peptide Analogues for Imaging Focal Sites of Bacterial Infection. J. Nucl. Med. 34, 1964-1974. (14) Baidoo, K. E., Scheffel, U., Stathis, M., Finley, P., Lever, S. Z., Zhan, Y., and Wagner, H. N., Jr. (1998) High-Affinity No-Carrier-Added 99mTc-Labeled Chemotactic Peptides for Studies of Inflammation In Vivo. Bioconjugate Chem. 9, 208217. (15) Edwards, D. S., Liu, S., Ziegler, M. C., Harris, A. R., Crocker, A. C., Heminway, S. J., Barrett, J. A., Bridger, G. J., Abrams, M. J., and Higgins, J. D., III (1999) RP463: A Stabilized Technetium-99m Complex of a Hydrazino Nicotinamide Derivatized Chemotactic Peptide for Infection Imaging. Bioconjugate Chem. 10, 884-891. (16) Pollak, A.; Goodbody, A. E.; Ballinger, J. R.; Duncan, G. S.; Tran, L. L.; Dunn-Dufault, R.; Meghji, K.; Lau, F.; Andrey, T. W.; Boxen, I.; and Sumner-Smith, M. Imaging Inflammation with 99mTc-Labelled Chemotactic Peptides: Analogues with Reduced Neutropenia. Nucl. Med. Commun. 1996, 17, 132-139. (17) Van Der Laken, C. J.; Boerman, O. C.; Oyen, W. J. G.; Van De Ven, M. T. P.; Edwards, D. S.; Barrett, J. A.; Van Der Meer, J. W. M.; and Corstens, F. H. M. Technetium-99mLabeled Chemotactic Peptides in Acute Infection and Sterile Inflammation. J. Nucl. Med. 1997, 38, 1310-1315. (18) Verbeke, K., Verbeke, A., Vanbilloen, H.; and Verbruggen, A. (2002) Preparation and Preliminary Evaluation of 99mTcEC-For-MLFK. Nucl. Med. Biol. 29, 585-592. (19) Verbeke, K., Snauwaert, K., Cleynhens, B., Scheers, W., and Verbruggen, A. (2000) Influence of the Bifunctional Chelate on the Biological Behavior of 99mTc-Labeled Chemotactic Peptide Conjugates. Nucl. Med. Biol. 27, 769-779. (20) Rennen, H. J., Boerman, O. C., Oyen, W. J., and Corstens, F. H. (2001) Imaging Infection/Inflammation in the New Millenium. Eur. J. Nucl. Med. 28, 241-252. (21) Rennen, H. J., Boerman, O. C., Oyen, W. J. G., and Corstens, F. H. M. (2002) Scintigraphic Imaging of Inflammatory Processes. Curr. Med. Chem.: Antiinflammatory & Anti-Allergy Agents 1, 63-75. (22) Rennen, H. J., Corstens, F. H., Oyen, W. J., and Boerman, O. C. (2001) New Concepts in Infection/Inflammation Imaging. Quart. J. Nucl. Med. 45, 167-173.

(23) Fischman, A. J., Rauh, D., Solomon, H., Babich, J. W., Tompkins, R. G., Kroon, D., Strauss, H. W., and Rubin, R. H. (1993) In Vivo Bioactivity and Biodistribution of Chemotactic Peptide Analogues in Nonhuman Primates. J. Nucl. Med. 34, 2130-2134. (24) Edwards, D. S., and Liu, S. (2001) Tc-99m-Labeled Small Biomolecules as Potential Radiopharmaceuticals for Infection and Inflammation Imaging. Drugs Future 26, 375-382. (25) Higgins, J. D., III, Bridger, G. J., Derian, C. K., Beblavy, M. J., Hernandez, P. E., Gaul, F. E., Abrams, M. J., Pike, M. C., and Soloman, H. F. (1996) N-terminus Urea-Substituted Chemotactic Peptides: New Potent Agonists and Antagonists Toward the Neutrophil fMLF Receptor. J. Med. Chem. 39, 1013-1015. (26) Sarin, V. K., Kent, S. B., Tam, J. P., and Merrifield, R. B. (1981) Quantitative Monitoring of Solid-Phase Peptide Synthesis by the Ninhydrin Reaction. Anal. Biochem. 117, 147157. (27) Chan, W. C., and White, P. D. (2000) FMOC Solid-Phase Peptide Synthesis, A Practical Approach, Oxford University Press, New York. (28) Waller, A., Pipkorn, D., Sutton, K. L., Linderman, J. J., and Omann, G. M. (2001) Validation of Flow Cytometric Competitive Binding Protocols and Characterization of Fluorescently Labeled Ligands. Cytometry 45, 102-114. (29) Fay, S. P., Posner, R. G., Swann, W. N., and Sklar, L. A. (1991) Real-Time Analysis of the Assembly of Ligand, Receptor, and G Protein by Quantitative Fluorescence Flow Cytometry. Biochemistry 30, 5066-5075. (30) Hoffman, J. F., Keil, M. L., Riccobene, T. A., Omann, G. M., and Linderman, J. J. (1996) Interconverting Receptor States at 4 °C for the Neutrophil N-formyl Peptide Receptor. Biochemistry 35, 13047-13055. (31) Cavicchioni, G., Varani, K., Niccoli, S., Rizzuti, O., and Spisani, S. (1999) An Evaluation of fMLF Pocket Dimensions and Features using formyltetrapeptides. J. Pept. Res. 54, 336-343. (32) Su, Z. i.-F., He, J., Rusckowski, M., and Hnatowich, D. J. (2003) In Vitro Cell Studies of Technetium-99m Labeled RGDHYNIC Peptide, A Comparison of Tricine and EDDA as Coligands. Nucl. Med. Biol. 30, 141-149. (33) Ono, M., Arano, Y., Mukai, T., Saga, T., Fujioka, Y., Ogawa, K., Kawashima, H., Konishi, J., and Saji, H. (2002) Control of Radioactivity Pharmacokinetics of 99mTc-HYNIC-Labeled Polypeptides Derivatized with Ternary Ligand Complexes. Bioconjugate Chem. 13, 491-501. (34) Qu, T., Wang, Y., Zhu, Z., Rusckowski, M., and Hnatowich, D. J. (2001) Different Chelators and Different Peptides Together Influence the In Vitro and Mouse In Vivo Properties of 99mTc. Nucl. Med. Commun. 22, 203-215. (35) Rennen, H. J., Oyen, W. J., Cain, S. A., Monk, P. N., Corstens, F. H., and Boerman, O. C. (2003) Tc-99m-Labeled C5a and C5a des Arg74 for Infection Imaging. Nucl. Med. Biol. 30, 267-272. (36) Francesconi, L., Zheng, Y., Bartis, J., Blumenstein, M., Costello, C., and De Rosch, M. A. (2004) Preparation and Characterization of [99TcO] Apcitide: A Technetium Labeled Peptide. Inorg. Chem. 43, 2867-2875.

BC0500591