Synthesis of radioiodinated N-succinimidyl iodobenzoate: Optimization for use in antibody labelling

doi:10.1016/0883-2889(89)90131-7

International Journal of Radiation Applications and Instrumentation. Part A. Applied Radiation and Isotopes

Volume 40, Issue 6, 1989, Pages 485-490

https://doi.org/10.1016/0883-2889(89)90131-7 Get rights and content

Abstract

N-succinimidyl-3-(tri-n-butylstannyl)benzoate (m-BuATE), N-succinimidyl-3-(tri-methylstannyl)benzoate (m-MeATE) and N-succinimidyl-4-(tri-n-butylstannyl)benzoate (p-BuATE) were synthesized and radioiodinated using either N-chlorosuccinimide (NCS) or t-butylhydroperoxide (TBHP) as the oxidant. Radiohalogenation of m-MeATE proceeded more rapidly than m-BuATE. NCS was the more efficient oxidant at reaction times <15 min; use of both TBHP and NCS resulted in nearly quantitative yields after 15 min when m-MeATE was used. Using NCS, achieving optimal antibody coupling and specific binding required purification of the active ester by HPLC; in contrast, with TBHP, only Sep-Pak purification was needed.

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Radiopharmaceutical chemistry of targeted radiotherapeutics, part 4: Strategies for 211At labeling at high activities and radiation doses of 211At α-particles
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Citation Excerpt :
Gradient system (II): 90:10 (A/B) held for 10 min to 72:28 (A/B) over 10 min then held for another 10 min, followed by 0:100 (A/B) over 20 min followed by 100% solvent B for the remainder of the HPLC run. SAB synthesis was performed by astatodestannylation of BuSTB, which was prepared as described previously [10]. The purity of the tin precursor was confirmed before each set of experiments by thin layer chromatography.
Alpha particles are radiation of high energy and short range, properties that can lead to radiolysis-mediated complications in labeling chemistry at the high radioactivity levels required for clinical application. In previous papers in this series, we have shown that radiation dose has a profound effect on the astatine species that are present in the labeling reaction and their suitability for the synthesis of N-succinimidyl 3-[²¹¹At]astatobenzoate. The purpose of this study was to evaluate the effects of adding N-chlorosuccinimide (NCS) to the methanol solution used for initial isolation of ²¹¹At after distillation, a process referred to as ²¹¹At stabilization, on ²¹¹At chemistry after exposure to high radiation doses.
High performance liquid chromatography was used to evaluate the distribution of ²¹¹At species present in methanol in the 500–65,000 Gy radiation dose range and the synthesis of SAB from N-succinimidyl 3-(tri-n-butylstannyl)benzoate in the 500–120,000 Gy radiation dose range using different ²¹¹At timeactivity combinations under conditions with/without ²¹¹At stabilization.
In the absence of NCS stabilization, a reduced form of astatine, At(2), increased with increasing radiation dose, accounting for about half the total activity by about 15,000 Gy, while with stabilization, At(2) accounted for <10% of ²¹¹At activity even at doses >60,000 Gy. SAB yields without stabilization rapidly declined with increasing dose, falling to ~20% at about 5000 Gy while with stabilization, yields >80% were obtained with ²¹¹At solutions stored for more than 23 h and receiving radiation doses >100,000 Gy.
Adding NCS to the methanol solution used for initial isolation of ²¹¹At is a promising strategy for countering the deleterious effects of radiolysis on ²¹¹At chemistry.
This strategy could facilitate the ability to perform ²¹¹At labeling at sites remote from its production and at the high activity levels required for clinical applications.
N-Succinimidyl guanidinomethyl iodobenzoate protein radiohalogenation agents: Influence of isomeric substitution on radiolabeling and target cell residualization
2014, Nuclear Medicine and Biology
Citation Excerpt :
When we designed the SGMIB reagent, we opted to use the 1,3,4- over 1,3,5-isomer simply due to the ready availability of the starting material for the synthesis of both its standard and tin precursor. However, the yields for the synthesis of Boc2-[131I]SGMIB from its tin precursor were only 60–65%, significantly lower than that obtained with similar compounds [10,20]. Hypothesizing that the lower radiochemical yield might be due to steric hindrance imparted by the bulky Boc2-guanidinomethyl group present at the ortho position of the tin moiety in the precursor, in the current study, we have developed an isomeric agent, N-succinimidyl 3-guanidinomethyl-5-[131I]iodobenzoate (iso-[131I]SGMIB) wherein the guanidinomethyl moiety has been moved to the 3-position to give a 1,3,5-substitution.
N-succinimidyl 4-guanidinomethyl-3-[^⁎I]iodobenzoate ([^⁎I]SGMIB) has shown promise for the radioiodination of monoclonal antibodies (mAbs) and other proteins that undergo extensive internalization after receptor binding, enhancing tumor targeting compared to direct electrophilic radioiodination. However, radiochemical yields for [¹³¹I]SGMIB synthesis are low, which we hypothesize is due to steric hindrance from the Boc-protected guanidinomethyl group ortho to the tin moiety. To overcome this, we developed the isomeric compound, N-succinimidyl 3-guanidinomethyl-5-[¹³¹I]iodobenzoate (iso-[¹³¹I]SGMIB) wherein this bulky group was moved from ortho to meta position.
Boc₂-iso-SGMIB standard and its tin precursor, N-succinimidyl 3-((1,2-bis(tert-butoxycarbonyl)guanidino)methyl)-5-(trimethylstannyl)benzoate (Boc₂-iso-SGMTB), were synthesized using two disparate routes, and iso-[*I]SGMIB synthesized from the tin precursor. Two HER2-targeted vectors — trastuzumab (Tras) and a nanobody 5 F7 (Nb) — were labeled using iso-[^⁎I]SGMIB and [^⁎I]SGMIB. Paired-label internalization assays in vitro with both proteins, and biodistribution in vivo with trastuzumab, labeled using the two isomeric prosthetic agents were performed.
When the reactions were performed under identical conditions, radioiodination yields for the synthesis of Boc₂-iso-[¹³¹I]SGMIB were significantly higher than those for Boc₂-[¹³¹I]SGMIB (70.7 ± 2.0% vs 56.5 ± 5.5%). With both Nb and trastuzumab, conjugation efficiency also was higher with iso-[¹³¹I]SGMIB than with [¹³¹I]SGMIB (Nb, 33.1 ± 7.1% vs 28.9 ± 13.0%; Tras, 45.1 ± 4.5% vs 34.8 ± 10.3%); however, the differences were not statistically significant. Internalization assays performed on BT474 cells with 5 F7 Nb indicated similar residualizing capacity over 6 h; however, at 24 h, radioactivity retained intracellularly for iso-[¹³¹I]SGMIB-Nb was lower than for [¹²⁵I]SGMIB-Nb (46.4 ± 1.3% vs 56.5 ± 2.5%); similar results were obtained using Tras. Likewise, a paired-label biodistribution of Tras labeled using iso-[¹²⁵I]SGMIB and [¹³¹I]SGMIB indicated an up to 22% tumor uptake advantage at later time points for [¹³¹I]SGMIB-Tras.
Given the higher labeling efficiency obtained with iso-SGMIB, this residualizing agent might be of value for use with shorter half-life radiohalogens.
Development and preclinical evaluation of new 124I-folate conjugates for PET imaging of folate receptor-positive tumors
2014, Nuclear Medicine and Biology
Citation Excerpt :
The synthetic approaches for preparation of [124I]SIB- and [124I]SIP-folate conjugates entailed a straightforward and simple two-step reactions (Scheme 2). The key precursors N-hydroxysuccinimide 3-tri-n-butylstannyl-benzoate and pyridine carboxylate were treated using classical iodogen no-carrier-added radioiodine described previously [25,26]. The purified intermediates [124I]-SIB and [124I]-SIB were reacted with hydrazide-folate (3) in DMSO at pH 9.
In an attempt to develop new folate radiotracers with favorable biochemical properties for detecting folate receptor-positive cancers, we have synthesized [¹²⁴I]-SIB- and [¹²⁴I]-SIP-folate conjugates using a straightforward and two-step simple reactions. Radiochemical yields for [¹²⁴I]-SIB- and [¹²⁴I]-SIP-folate conjugates were greater than 90 and 60% respectively, with total synthesis time of 30–40 min. Radiochemical purities were always greater than 98% without HPLC purification. These synthetic approaches hold considerable promise as rapid and simple method for ¹²⁴I-folate conjugate preparation with high radiochemical yield in short synthesis time. In vitro tests on KB cell line showed that the significant amounts of the radioconjugates were associated with cell fractions. In vivo characterization in normal Balb/c mice revealed rapid blood clearance of these radioconjugates and favorable biodistribution profile for [¹²⁴I]-SIP-folate conjugate over [¹²⁴I]-SIB-folate conjugate. Biodistribution studies of [¹²⁴I]-SIP-folate conjugate in nude mice bearing human KB cell line xenografts, demonstrated significant tumor uptake. The uptake in the tumors was blocked by excess injection of folic acid, suggesting a receptor-mediated process. These results demonstrate that [¹²⁴I]-SIP-folate conjugate may be useful as a molecular probe for detecting and staging of folate receptor-positive cancers, such as ovarian cancer and their metastasis as well as monitoring tumor response to treatment.
SIB-DOTA: A trifunctional prosthetic group potentially amenable for multi-modal labeling that enhances tumor uptake of internalizing monoclonal antibodies
2012, Bioorganic and Medicinal Chemistry
Citation Excerpt :
Conjugation of [131I]10 with mAb L8A4 was achieved in 27 ± 6% (n = 2) yields. The lower conjugation yields, compared with SIB and substituted SIB analogues,13,37,38 might be due to partial hydrolysis of [131I]10 leaving less active ester available for conjugation. The integrity of the labeled mAb was evaluated by several methods.
A major drawback of internalizing monoclonal antibodies (mAbs) radioiodinated with direct electrophilic approaches is that tumor retention of radioactivity is compromised by the rapid washout of iodo-tyrosine, the primary labeled catabolite for mAbs labeled via this strategy. In our continuing efforts to develop more versatile residualizing labels that could overcome this problem, we have designed SIB-DOTA, a prosthetic labeling template that combines the features of the prototypical, dehalogenation-resistant N-succinimidyl 3-iodobenzoate (SIB) with DOTA, a useful macrocyclic chelator for labeling with radiometals. Herein we describe the synthesis of the unlabeled standard of this prosthetic moiety, its protected tin precursor, and radioiodinated SIB-DOTA. An anti-EGFRvIII-reactive mAb, L8A4 was radiolabeled with [¹³¹I]SIB-DOTA in 27.1 ± 6.2% (n = 2) conjugation yields and its targeting properties to the same mAb labeled with [¹²⁵I]SGMIB both in vitro and in vivo using U87MG·ΔEGFR cells and xenografts were compared. In vitro paired-label internalization assays showed that the intracellular radioactivity from [¹³¹I]SIB-DOTA-L8A4 was 21.4 ± 0.5% and 26.2 ± 1.1% of initially bound radioactivity at 16 and 24 h, respectively. In comparison, these values for [¹²⁵I]SGMIB-L8A4 were 16.7 ± 0.5% and 14.9 ± 1.1%. Similarly, the SIB-DOTA prosthetic group provided better tumor targeting in vivo than SGMIB over 8 d period. These results suggest that SIB-DOTA warrants further evaluation as a residualizing agent for labeling internalizing mAbs including those targeted to EGFRvIII.
Radiolabelled proteins for positron emission tomography: Pros and cons of labelling methods
2010, Biochimica et Biophysica Acta - General Subjects
Dynamic biomedical research is currently yielding a wealth of information about disease-associated molecular alterations on cell surfaces and in the extracellular space. The ability to visualize and quantify these alterations in vivo could provide important diagnostic information and be used to guide individually-optimized therapy. Biotechnology can provide proteinaceous molecular probes with highly specific target recognitions. Suitably labelled, these may be used as tracers for radionuclide-based imaging of molecular disease signatures. If the labels are positron-emitting radionuclides, the superior resolution, sensitivity and quantification capability of positron emission tomography (PET) can be exploited.
This article discusses different approaches to labelling proteins with positron-emitting nuclides with suggestions made depending on the biological features of the tracers.
Factors such as matching biological and physical half-lives, availability of the nuclide, labelling yields, and influences of labelling on targeting properties (affinity, charge and lipophilicity, cellular processing and retention of catabolites) should be considered when selecting a labelling strategy for each proteinaceous tracer.
The labelling strategy used can make all the difference between success and failure in a tracer application. This review emphasises chemical, biological and pharmacological considerations in labelling proteins with positron-emitting radionuclides.
Influence of prosthetic radioiodination on the chemical and biological behavior of chemotactic peptides labeled at high specific activity
2006, Applied Radiation and Isotopes
The influence of radioiodination made through prosthetic group $N$ -succinimidyl-3-[¹³¹I]iodo-benzoate ([¹³¹I]SIB) on the behavior of small peptides was investigated using as model the chemotactic hexapeptide $N α$ -for-Nle-Leu-Phe-Nle-Tyr-Lys. No carrier added labeled peptide was isolated by reverse-phase HPLC (RP-HPLC) with coupling efficiencies up to 59–75%. Biodistribution in normal and infected C57 mice showed mainly a hepatobiliary clearance, a very low thyroid uptake and the highest uptake at the infection site was within $1 h$ of injection. Superoxide production and competitive binding assays studies in human polymorphonuclear leukocytes showed a preserved biological activity and high-affinity specific binding. However, the results indicated that the changes observed in the receptor-binding properties with an IC $_{50}$ almost twice than the unlabeled peptide and the increasing in the hepatobiliary excretion could be the consequence of the increased lipophicity observed due to the presence of the prosthetic group together with a strong influence of the radioisotope per se.

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^☆: Supported by National Cancer Institute Grants CA 42324, CA 11898, CA 43638, 5-P50-NS 20023, and 5-P30-CA 14236.

^∗: Supported by the National Cancer Institute Grants CA 42324, CA 11898, CA 43638, 5-P50-NS 20023, and 5-P30-CA 14236.

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International Journal of Radiation Applications and Instrumentation. Part A. Applied Radiation and Isotopes

Abstract

References (18)

Advantages of radioiodine over radioindium labeled monoclonal antibodies for imaging solid tumors

Nucl. Med. Biol.

Position-specific radioiodination utilizing an artyltributylstannyl intermediate. Synthesis of ¹²⁵I-iodotamoxifen

Int. J. Appl. Radiat. Isot.

A method for the radiohalogenation of proteins resulting in decreased thyroid uptake of radioiodine

Appl. Radiat. Isot.

A study on the electrophilic iodination of chloroquine

J. Labelled Compd. Radiopharm.

Chlorination as a side reaction in radioiodination with chloramine-T

J. Radioanal. Chem.

Innovations in labeling techniques for radioimmunoassays

Immunochemical and biochemical characterization of a glioma-associated extracellular matrix glycoprotein

J. Cell Biochem.

Substitution electrophile aromatique. IV. Effets steriques dans la reaction d'iodo-demetallation de derives organostanniques aromatiques

Helv. Chim. Acta

Practically carrier-free labelling of aromatic compounds with bromine-77 via n-chloro-tetrafluorosuccinimide

J. Labelled Compd. Radiopharm.

Cited by (65)

Radiopharmaceutical chemistry of targeted radiotherapeutics, part 4: Strategies for <sup>211</sup>At labeling at high activities and radiation doses of <sup>211</sup>At α-particles

N-Succinimidyl guanidinomethyl iodobenzoate protein radiohalogenation agents: Influence of isomeric substitution on radiolabeling and target cell residualization

Development and preclinical evaluation of new <sup>124</sup>I-folate conjugates for PET imaging of folate receptor-positive tumors

SIB-DOTA: A trifunctional prosthetic group potentially amenable for multi-modal labeling that enhances tumor uptake of internalizing monoclonal antibodies

Radiolabelled proteins for positron emission tomography: Pros and cons of labelling methods

Influence of prosthetic radioiodination on the chemical and biological behavior of chemotactic peptides labeled at high specific activity

Synthesis of radioiodinated N-succinimidyl iodobenzoate: Optimization for use in antibody labelling☆

Abstract

Nucl. Med. Biol.

Int. J. Appl. Radiat. Isot.

Appl. Radiat. Isot.

A study on the electrophilic iodination of chloroquine

J. Labelled Compd. Radiopharm.

Chlorination as a side reaction in radioiodination with chloramine-T

J. Radioanal. Chem.

Innovations in labeling techniques for radioimmunoassays

Immunochemical and biochemical characterization of a glioma-associated extracellular matrix glycoprotein

J. Cell Biochem.

Substitution electrophile aromatique. IV. Effets steriques dans la reaction d'iodo-demetallation de derives organostanniques aromatiques

Helv. Chim. Acta

Practically carrier-free labelling of aromatic compounds with bromine-77 via n-chloro-tetrafluorosuccinimide

J. Labelled Compd. Radiopharm.