Synthesis and radioiodination of some daunorubicin and doxorubicin derivatives

Daunorubicin and doxorubicin are efficient agents for cancer treatment. Their clinical efficacy is, however, hampered by their indiscriminant toxicity. This problem may be circumvented by encapsulating the drugs in liposomes and selectively targeting the tumor cells using tumor targeting agents. Furthermore, the antitumor effect could be enhanced by attaching the Auger electron emitter, (125)I, to daunorubicin and doxorubicin derivatives. In this context a number of ester, amide, and amine derivatives of daunorubicin and doxorubicin were synthesized. Benzoic acid ester derivatives of daunorubicin were synthesized by nucleophilic esterification of the 14-bromodaunorubicin with the potassium salt of the corresponding benzoic acid, resulting in good yields. Nicotinic acids and benzoic acids, activated with a succinimidyl group, were coupled to the amino group of daunorubicin to give the corresponding amide derivatives. Amine derivatives were obtained by the reductive amination of aromatic aldehydes with daunorubicin hydrochloride. The stannylated ester and amide derivatives were used as precursors for radioiodination. Radiolabeling with (125)I was performed using chloramine-T as an oxidant. The optimized labeling resulted in high radiolabeling yields (85-95%) of the radioiodinated daunorubicin and doxorubicin derivatives. Radioiodination of the amines was conducted at the ortho position of the activated phenyl rings providing moderate radiochemical yields (55-75%).     

Synthesis and evaluation of potent and selective human V1a receptor antagonists as potential ligands for PET or SPECT imaging

SRX246 is a potent, highly selective human vasopressin V1a antagonist that crosses the blood-brain barrier in rats. CNS penetration makes SRX246 an ideal candidate for potential radiolabeling and use in visualization and characterization of the role of the V1a receptor in multiple stress-related disorders. Before radiolabeling studies, cold reference analogs of SRX246 were prepared. This study describes the synthesis and in vitro screening for human V1a receptor binding and permeability of fluoro, iodo, and methyl reference compounds for SRX246 and the preparation of a tin precursor. For each compound, the potential utility of corresponding radiolabeled analogs for PET and SPECT imaging is discussed.     

Development of a new thiol-reactive prosthetic group for site-specific labeling of biomolecules with radioactive iodine

In this report, we describe the radiosynthesis of a new thiol-targeting prosthetic group for efficient radioactive iodine labeling of biomolecules. Radioiodination using the precursor 3 was performed to obtain ¹²⁵I-labeled tetrazole 4b with high radiochemical yield (73%) and radiochemical purity. Using the radiolabeled 4b, a single free cysteine containing peptide and human serum albumin were labeled with ¹²⁵I in modest-to-good radiochemical yields (65–99%) under mildly reactive conditions. A biodistribution study of [¹²⁵I]7 in normal ICR mice exhibited lower thyroid uptake values than those of ¹²⁵I-labeled human serum albumin prepared via a traditional radiolabeling method. Thus, [¹²⁵I]7 could be employed as an effective radiotracer for molecular imaging and biodistribution studies. The results clearly demonstrate that 4b has the potential to be effectively implemented as a prosthetic group in the preparation of radiolabeled biomolecules.     

Detection of human neutrophil elastase with peptide-bound cross-linked ethoxylate acrylate resin analogs.

An assessment of elastase-substrate kinetics and adsorption at the solid-liquid interface of peptide-bound resin was made in an approach to the solid-phase detection of human neutrophil elastase (HNE), which is found in high concentration in chronic wound fluid. N-succinyl-alanine-alanine-proline-valine-p-nitroanilide (suc-Ala-Ala-Pro-Val-pNA), a chromogenic HNE substrate, was attached to glycine-cross-linked ethoxylate acrylate resins (Gly-CLEAR) by a carbodiimide reaction. To assess the enzyme-substrate reaction in a two-phase system, the kinetic profile of resin-bound peptide substrate hydrolysis by HNE was obtained. A glycine and di-glycine spacer was placed between the resin polymer and substrate to assess the steric and spatial requirements of resin to substrate with enzyme hydrolysis. The enzymatic activities of suc-Ala-Ala-Pro-Val-pNA and suc-Ala-Ala-Pro-Ala-pNA on the solid-phase resin were compared with similar analogs in solution. An increase in visible wavelength absorbance was observed with increasing amounts of substrate-resin and enzyme concentration. Enzyme hydrolysis of the resin-bound substrate was also demonstrated on a polypropylene surface, which was employed for visible absorbance of released chromophore. A soluble active substrate analog was released from the resin through saponification of the ethoxylate ester linkages in the resin polymer. The resin-released conjugate of the HNE substrate demonstrated an increased dose response with increasing enzyme concentration. The synthesis and assay of elastase substrates bound to CLEAR resin gives an understanding of substrate-elastase adsorption and activity at the resin's solid-liquid interface for HNE detection with a solid-phase peptide.     

Rapid and simple one-step F-18 labeling of peptides

Labeling biomolecules with 1?F is usually done through coupling with prosthetic groups, which requires several time-consuming radiosynthesis steps and therefore in low labeling yield. In this study, we designed a simple one-step 1?F-labeling strategy to replace the conventional complex and the long process of multiple-step radiolabeling procedure. Both monomeric and dimeric cyclic RGD peptides were modified to contain 4-NO?-3-CF? arene as precursors for direct 1?F labeling. Binding of the two functionalized peptides to integrin α(v)β? was tested in vitro using the MDA-MB-435 human breast cell line. The most promising functionalized peptide, the dimeric cyclic RGD, was further evaluated in vivo in an orthotopic MDA-MB-435 tumor xenograft model. The use of relatively low amount of precursor (~0.5 μmol) gave reasonable yield, ranging from 7 to 23% (decay corrected, calculated from the start of synthesis) after HPLC purification. Overall reaction time was 40 min, and the specific activity of the labeled peptide was high. Modification of RGD peptides did not significantly change the biological binding affinities of the modified peptides. Small animal PET and biodistribution studies revealed integrin specific tumor uptake and favorable biokinetics. We have developed a novel one-step 1?F radiolabeling strategy for peptides that contain a specific arene group, which shortens reaction time and labor significantly, requires low amount of precursor, and results in specific activity of 79 ± 13 GBq/μmol. Successful introduction of 4-fluoro-3-trifluoromethylbenzamide into RGD peptides may be a general strategy applicable to other biologically active peptides and proteins.     

Highly efficient method for 125I-radiolabeling of biomolecules using inverse-electron-demand Diels–Alder reaction

In this report, we present a rapid and highly efficient method for radioactive iodine labeling of trans-cyclooctene group conjugated biomolecules using inverse-electron-demand Diels–Alder reaction. Radioiodination reaction of the tetrazine structure was carried out using the stannylated precursor 2 to give ¹²⁵I-labeled product ([¹²⁵I]1) with high radiochemical yield (65 ± 8%) and radiochemical purity (>99%). For radiolabeling application of [¹²⁵I]1, trans-cyclooctene derived cRGD peptide and human serum albumin were prepared. These substrates were reacted with [¹²⁵I]1 under mild condition to provide the radiolabeled products [¹²⁵I]6 and [¹²⁵I]8, respectively, with excellent radiochemical yields. The biodistribution study of [¹²⁵I]8 in normal ICR mice showed significantly lower thyroid uptake values than that of ¹²⁵I-labeled human serum albumin prepared by a traditional radiolabeling method. Therefore [¹²⁵I]8 will be a useful radiolabeled tracer in various molecular imaging and biological studies. Those results clearly demonstrate that [¹²⁵I]1 will be used as a valuable prosthetic group for radiolabeling of biomolecules.     

Enhanced target-specific accumulation of radiolabeled antibodies by conjugating arginine-rich peptides as anchoring molecules

We have devised and estimated a new strategy to prolong the residence time of radiolabeled antibodies in tumor in which an octaarginine peptide (R?) was used as an anchoring molecule to fix antibodies against CD20 (NuB2; IgG2a) on tumor cells. Conjugation of R? with antibodies was performed by maleimide-thiol chemistry using thiol groups generated by reducing the disulfide bonds of the antibody. The R?-conjugated NuB2 was then reacted with succinimidyl meta-[12?I]iodobenzoate to prepare [12?I]SIB-NuB2(I) (0.92 R?/NuB2) and [12?I]SIB-NuB2(III) (3.38 R?/NuB2). Both SIB-NuB2(I) and SIB-NuB2(III) exhibited size-exclusion HPLC elution profiles and immunoreactivity to CD20-positive cells similar to those of NuB2. NuB2(I) also possessed isoelectric focusing (IEF) profile similar to NuB2. However, NuB2(III) registered a broad IEF band toward higher pI. When incubated with CD20-positive cells, [12?I]SIB-NuB2(I) and [12?I]SIB-NuB2(III) exhibited 1.4 and 4.0 times higher cell-associated radioactivity than [12?I]SIB-NuB2. After the cells were washed and reincubated in a fresh medium for 3 h, [12?I]SIB-NuB2(I) and [12?I]SIB-NuB2(III) exhibited significantly higher cell-associated radioactivity than [12?I]SIB-NuB2. In biodistribution studies in normal mice, while both [12?I]SIB-NuB2(I) and [12?I]SIB-NuB2 exhibited similar biodistribution profiles, [12?I]SIB-NuB2(III) showed faster clearance from the blood and higher hepatic radioactivity levels than [12?I]SIB-NuB2. In SCID mice bearing CD20-positive xenografts, [131I]SIB-NuB2(I) exhibited significantly higher radioactivity in xenografts than those of [12?I]SIB-NuB2 with no significant increase being observed in other tissues. The findings indicate that appropriate R? modification of antibodies satisfies both specific targeting ability of antibody and strong cell-association property of R?, which was reflected in the increased radioactivity levels in tumor. These findings supported the applicability of this approach to enhance target-specific accumulation of radiolabeled antibodies.     

Application of Mitsunobu reaction to solid-phase peptide nucleic acid (PNA) monomer synthesis

PNA type I monomer backbone with a reduced peptide bond was synthesized on a Merrifield resin in Mitsunobu reaction of Boc-aminoethanol with resin-bound o-nitrobenzenesulfonylglycine. The pseudodipeptide secondary amine group was deprotected by thiolysis and acylated with thymin-1-ylacetic acid. The monomer was released as a methyl ester. The procedure seems to be of general applicability and allows various modifications of PNA structure by using diverse alcohols and amino acid esters.     

A novel 99mTc-labeled testosterone derivative as a potential agent for targeting androgen receptors

With an insight that ligands possessing a N2S2 tetradentate array of donor atoms serve as ideal bifunctional chelating agents (BFCA) in the radiolabeling of target-specific agents, 5-hydroxy-3,7-diazanonan-1,9-dithiol (DAHPES) with a derivatizable substituent in the form of a hydroxyl group in the backbone was synthesized. The preparation of a steroid conjugate via coupling of this BFCA with testosterone-3-(O-carboxymethyl) oxime and the subsequent radiolabeling of the conjugate under optimized conditions with 99mTc, the ideal diagnostic radionuclide in nuclear medicine procedures, are reported. The immunoreactivity of the radiolabeled conjugate was demonstrated in a study using anti-testosterone antibodies, wherein the radiolabeled conjugate exhibited significant binding with antiserum to testosterone. Cell-uptake studies in DU145 prostate carcinoma cell line bearing androgen receptors (ARs) and comparison with AR non-bearing breast carcinoma cell line revealed the specific binding of the steroidal moiety with the testosterone receptor.     

Stimulation of cartilage macromolecule synthesis by adenosine 3',5'-monophosphate.

The role of cyclic AMP in the regulation of cartilage macromolecule synthesis in vitro was studied in pelvic cartilage from 10-12 day chick embryos. Incubation of cartilages in medium containing 0.5 mM cyclic AMP resulted in a 30% inhibition of 35SO4-2, [3H]leucine and [3H]uridine incorporation into proteoglycan, total protein and RNA, respectively. Higher concentrations of cyclic AMP had no greater effects. In contrast, butyrylated cyclic AMP derivatives (0.5-5.0 mM) added to the incubation medium stimulated (50-100%) the incorporation of these radiolabeled precursors into cartilage macromolecules. Theophylline, in concentrations (0.1-0.5 mM) which raise intracellular cyclic AMP, also increases the incorporation of radiolabeled precursors into macromolecules. The data indicate that exogenous cyclic AMP and butyrylated cyclic AMP derivatives have paradoxical effects on cartilage macromolecule synthesis. Butyrylated cyclic AMP derivatives, not exogenous cyclic AMP, mimic the effects of intracellular cyclic AMP. Incubation of embryonic chicken cartilage with exogenous cyclic AMP results in the extracellular degradation of the cyclic AMP to adenosine. Adenosine (0.125 mM) inhibits precursor incorporation into cartilage macromolecules. The metabolism of exogenous cyclic AMP generates sufficient adenosine to account for the observed inhibitory effects of exogenous cyclic AMP on cartilage macromolecule synthesis. Butyrylated cyclic AMP derivatives are not degraded during incubation with cartilage. The data indicate that cartilage is a tissue in which the effect of cyclic AMP is to stimulate anabolic processes.     

Synthetic resin-bound truncated Candida antarctica lipase B for production of fatty acid alkyl esters by transesterification of corn and soybean oils with ethanol or butanol

A gene encoding a synthetic truncated Candida antarctica lipase B (CALB) was generated via automated PCR and expressed in Saccharomyces cerevisiae. Western blot analysis detected five truncated CALB variants, suggesting multiple translation starts from the six in-frame ATG codons. The longest open reading frame, which corresponds to amino acids 35-317 of the mature lipase, appeared to be expressed in the greatest amount. The truncated CALB was immobilized on Sepabeads? EC-EP resin and used to produce ethyl and butyl esters from crude corn oil and refined soybean oil. The yield of ethyl esters was 4-fold greater from corn oil than from soybean oil and was 36% and 50% higher, respectively, when compared to a commercially available lipase resin (Novozym 435) using the same substrates. A 5:1 (v/v) ratio of ethanol to corn oil produced 3.7-fold and 8.4-fold greater yields than ratios of 15:1 and 30:1, respectively. With corn oil, butyl ester production was 56% higher than ethyl ester production. Addition of an ionic catalytic resin step prior to the CALB resin increased yields of ethyl esters from corn oil by 53% compared to CALB resin followed by ionic resin. The results suggest resin-bound truncated CALB has potential application in biodiesel production using biocatalysts.     

APPLICATION OF MITSUNOBU REACTION TO SOLID-PHASE PEPTIDE NUCLEIC ACID (PNA) MONOMER SYNTHESIS

ABSTRACT

PNA type I monomer backbone with a reduced peptide bond was synthesized on a Merrifield resin in Mitsunobu reaction of Boc-amino ethanol with resin-bound o-nitrobenzenesulfonylglycine. The pseudo dipeptide secondary amine group was deprotected by thiolysis and acylated with thymin-1-ylacetic acid. The monomer was released as a methyl ester. The procedure seems to be of general applicability and allows various modifications of PNA structure by using diverse alcohols and amino acid esters.     

A novel prosthetic group for site-selective labeling of peptides for positron emission tomography

Efficient methodologies for the radiolabeling of peptides with [(18)F]fluoride are a prerequisite to enabling commercialization of peptide-containing radiotracers for positron emission tomography (PET) imaging. It was the purpose of this study to investigate a novel chemoselective ligation reaction comprising conjugation of an [(18)F]-N-methylaminooxy-containing prosthetic group to a functionalized peptide. Twelve derivatives of general formula R1-CO-NH-Lys-Gly-Phe-Gly-Lys-OH were synthesized where R1 was selected from a short list of moieties anticipated to be reactive toward the N-methylaminooxy group. Conjugation reactions were initially carried out with nonradioactive precursors to assess, in a qualitative manner, their general suitability for PET chemistry with only the most promising pairings progressing to full radiochemical assessment. Best results were obtained for the ligation of O-[2-(2-[(18)F]fluoroethoxy)ethyl]-N-methyl-N-hydroxylamine 18 to the maleimidopropionyl-Lys-Gly-Phe-Gly-Lys-OH precursor 10 in acetate buffer (pH 5) after 1 h at 70 degrees C. The non-decay-corrected isolated yield was calculated to be 8.5%. The most encouraging result was observed with the combination 18 and 4-(2-nitrovinyl)benzoyl-Lys-Gly-Phe-Gly-Lys-OH, 9, where the conjugation reaction proceeded rapidly to completion at 30 degrees C after only 5 min. The corresponding non-decay-corrected radiochemical yield for the isolated (18)F-labeled product 27 was 12%. The preliminary results from this study demonstrate the considerable potential of this novel strategy for the radiolabeling of peptides.     

14C-methylamine-glutaraldehyde conjugation as an alternative to iodination for protein labeling

Radiolabeling of native proteins conventionally has required iodination using 125Iodine (125I). Although radioiodination can result in high specific activity, there are several drawbacks in the use of 125I (e.g., radiological hazards and short half-life). 14C-Methylamine-glutaraldehyde conjugation to proteins offers an alternative for radiolabeling of proteins that is safer and longer-lived alpha-2-Macroglobulin was radiolabeled by conjugation to a 14C-methylamine-glutaraldehyde conjugate. Analysis of the labeling procedure was performed using scintillation counting, gel filtration chromatography, and protein assays. The radiolabeled alpha-2-macroglobulin was activated using established protocols and tested for functional integrity using competitive binding assays in the presence of recombinant receptor associated protein, an alternative ligand for the alpha-2-macroglobulin cellular receptor. The function of alpha-2-macroglobulin was unaffected by the labeling procedure. Comparison of 14C-methylamine-labeling and iodination by Scatchard analysis yielded nonlinear plots that suggested the presence of two sets of receptors with different binding affinities but that do not show cooperativity. This technique offers an alternative to radioiodination for the sensitive labeling of proteins.     

Stoichiometric model of Escherichia coli metabolism: incorporation of growth-rate dependent biomass composition and mechanistic energy requirements

A stoichiometric model of metabolism was developed to describe the balance of metabolic reactions during steady-state growth of Escherichia coli on glucose (or metabolic intermediates) and mineral salts. The model incorporates 153 reversible and 147 irreversible reactions and 289 metabolites from several metabolic data bases for the biosynthesis of the macromolecular precursors, coenzymes, and prosthetic groups necessary for synthesis of all cellular macromolecules. Correlations describing how the cellular composition changes with growth rate were developed from experimental data and were used to calculate the drain of precursors to macromolecules, coenzymes, and prosthetic groups from the metabolic network for the synthesis of those macromolecules at a specific growth rate. Energy requirements for macromolecular polymerization and proofreading, transport of metabolites, and maintenance of transmembrane gradients were included in the model rather than a lumped maintenance energy term. The underdetermined set of equations was solved using the Simplex algorithm, employing realistic objective functions and constraints; the drain of precursors, coenzymes, and prosthetic groups and the energy requirements for the synthesis of macromolecules served as the primary set of constraints. The model accurately predicted experimentally determined metabolic fluxes for aerobic growth on acetate or acetate plus glucose. In addition, the model predicted the genetic and metabolic regulation that must occur for growth under different conditions, such as the opening of the glyoxylate shunt during growth on acetate and the branching of the tricarboxylic acid cycle under anaerobic growth. Sensitivity analyses were performed to determine the flexibility of pathways and the effects of different rates and growth conditions on the distribution of fluxes. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 398-421, 1997.     

Preparation of thiol-reactive Cy5 derivatives from commercial Cy5 succinimidyl ester

The present study offers reliable protocols for the preparation of new thiol-reactive Cy5 derivatives which are urgently needed for single molecule fluorescence microscopy. In a systematic approach, two alternate strategies were found for the extension of commercial amine-reactive Cy5 with thiol-reactive end groups. In the two-step method, Cy5 succinimidyl ester was first reacted with ethylenediamine under conditions which gave approximately 99% asymmetric "Cy5-amine" and only approximately 1% symmetric product with two Cy5 residues. Subsequently, "Cy5-amine" was derivatized with commercial heterobifunctional cross-linkers to introduce thiol-reactive end groups (maleimide or pyridyldithio). Alternatively, commercial Cy5 succinimidyl ester was reacted with a primary amine (MTSEA, methanethiosulfonylethylamine, or PDEA, pyridyldithioethylamine) or a secondary amine (PEM, piperazinylethylmaleimide) to give the corresponding thiol-reactive derivatives in a single step. Results were good for MTSEA, moderate for PEM, and poor for PDEA. An additional drawback of the one-step method was the need for rigorous removal of unreacted Cy5 succinimidyl ester, which would label lysine residues on probe molecules. It is concluded that, except for the Cy5-MTSEA conjugate, the two-step method is much more general, reliable, and easier to follow by the typical biophysicist, biologist, etc., for whose benefit, these procedures are being published. All thiol-reactive Cy5 derivatives showed similar absorption and fluorescence properties as Cy5 succinimidyl ester, and fluorescence was fully retained after binding to thiols on proteins. The kinetics of protein labeling was also examined in order to get an idea of proper labeling conditions.     

Synthesis of diketopiperazines with on-resin N-methylation and cyclative release.

Enantiomerically pure N-methylated diketopiperazines (DKP) can be obtained by treating a N-methylated resin-bound dipeptide with 20% piperidine in dimethylformamide via a process known as cyclative release. N-methylated resin-bound dipeptides can be formed from N-methylated precursors or N-methylation can be selectively performed on the resin. When on-resin N-methylation was performed on the C-terminal side of the dipeptide, diastereomers were formed. Yet the cyclative release is shown to be a stereoselective process, as seen using preformed N-methylated amino acids. The procedure was also applied to synthesize the pseudodiketopiperazine cyclo(Phepsi[CH2NH]Leu). When comparing nonmethylated, monomethylated and bismethylated derivatives, we find that N-methylation results in a dramatic increase in solubility.     

In vitro and in vivo evaluation of Alexa Fluor 680-bombesin[7-14]NH2 peptide conjugate, a high-affinity fluorescent probe with high selectivity for the gastrin-releasing peptide receptor

Gastrin-releasing peptide (GRP) receptors are overexpressed on several types of human cancer cells, including breast, prostate, small cell lung, and pancreatic cancers. Bombesin (BBN), a 14-amino acid peptide that is an analogue of human GRP, binds to GRP receptors with very high affinity and specificity. The aim of this study was to develop a new fluorescent probe based on BBN having high tumor uptake and optimal pharmacokinetics for specific targeting and optical imaging of human breast cancer tissue. In this study, solid-phase peptide synthesis was used to produce H(2)N-glycylglycylglycine-BBN[7-14]NH(2) peptide with the following general sequence: H(2)N-G-G-G-Q-W-A-V-G-H-L-M-(NH(2)). This conjugate was purified by reversed-phase high-performance liquid chromatography and characterized by electrospray-ionization mass spectra. The fluorescent probe Alexa Fluor 680-G-G-G-BBN[7-14]NH(2) conjugate was prepared by reaction of Alexa Fluor 680 succinimidyl ester to H(2)N-G-G-G-BBN[7-14]NH(2) in dimethylformamide (DMF). In vitro competitive binding assays, using (125)I-Tyr(4)-BBN as the radiolabeling gold standard, demonstrated an inhibitory concentration 50% value of 7.7 +/- 1.4 nM in human T-47D breast cancer cells. Confocal fluorescence microscopy images of Alexa Fluor 680-G-G-G-BBN[7-14]NH(2) in human T-47D breast cancer cells indicated specific uptake, internalization, and receptor blocking of the fluorescent bioprobe in vitro. In vivo investigations in SCID mice bearing xenografted T-47D breast cancer lesions demonstrated the ability of this new conjugate to specifically target tumor tissue with high selectivity and affinity.     

Radiolabeled biomolecules with 186Re: Potential for radioimmunotherapy

Rhenium-186 has been theoretically determined to be among the best therapy radiolabels due to its unique half-life, paniculate and γ emissions and chelation properties. Traditionally, rhenium chelates have been synthesized by the tin reduction method at low pH which frequently produces denaturation of acid labile proteins. A comparative study has been carried out to assess three techniques of reducing ¹⁸⁶ReO⁻₄ in order to label biologically active macromolecules (i.e. human serum albumin (HSA), anti-human serum albumin antibody (HSA-Ab) and a monoclonal antibody to T-cells [T-101]). These experiments showed that stannous and dithionite reduction methods provide for an overall labeling yield of between 5 and 18% with an associated immunoreactivity of 12–40%. The hypophosphorous acid (H₃PO₂) reduction method, however, yielded no usable radiolabeled product. The preparation of ¹⁸⁶Re-DTPA-HSA produced an 18.2% radiochemical yield (7.4 × 10⁶ Bq/mg) and 40% retention of binding affinity. Using the stannous or dithionite reduction methods for the radiolabeling of HSA-Ab and T-101 resulted in a relatively low yield (9%), but the labeled product retained binding affinity of 12–25% with Protein A.     

Site-selective radiolabeling of peptides by 18F-fluorobenzoylation with [18F]SFB in solution and on solid phase: a comparative study

Peptides labeled with short-lived positron-emitting radionuclides are of outstanding interest as probes for molecular imaging by positron emission tomography (PET). Herein, the site-selective incorporation of fluorine-18 into lysine-containing peptides using the prosthetic labeling agent N-succinimidyl 4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB) is described. The reaction of [¹⁸F]SFB with four biologically relevant resin-bound peptides was studied and optimized. For comparison, each peptide was ¹⁸F-fluorobenzoylated in solution under different conditions and the product distribution was analyzed confirming the advantages of the solid-phase approach. The method’s feasibility for selective radiolabeling either at the N-terminus or at the lysine side chain was demonstrated. Labeling on solid phase with [¹⁸F]SFB resulted in crude ¹⁸F-fluorobenzoylpeptides whose radiochemical purities were typically greater than 90% and that could be prepared in synthesis times from 65 to 76?min.