Efficient and Site-Specific ¹²⁵I-Radioiodination of Bioactive Molecules Using Oxidative Condensation Reaction

Abstract

In this report, the novel and site-specific radioiodination of biomolecules by using aryl diamine and alkyl aldehyde condensation reaction in the presence of a Cu²⁺ catalyst under ambient conditions was reported. ¹²⁵I-labeled alkyl aldehyde was synthesized using a tin precursor with a high radiochemical yield (72 ± 6%, n = 5) and radiochemical purity (>99%). The utility of the radioiodinated precursor was demonstrated through aryl diamine-installed c[RGDfK(C)] peptide and human serum albumin (HSA). Radioiodinated c[RGDfK(C)] peptide and HSA protein were synthesized with high radiochemical yields and purity. ¹²⁵I-HSA protein showed excellent in vivo stability and negligible thyroid uptake as compared with directly radioiodinated HSA by using the tyrosine group. Excellent reaction kinetics and the in vitro and in vivo stabilities of ¹²⁵I-labeled alkyl aldehyde have suggested the usefulness of the strategy for the radioiodination of bioactive molecules.

Introduction

Among the various available radioisotopes, radioiodine is the most extensively used for radiolabeling of biologically active macro and micromolecules.¹⁻³ Radioiodine can be used for positron emission tomography (PET) imaging (¹²⁴I),⁴ singlephoton emission computed tomography (SPECT) imaging (^123/125I),^5,6 treatment of various cancers (¹³¹I),⁷ and pharmacokinetics and biodistribution studies of newly designed therapeutic drugs (¹²⁵I).⁸⁻¹⁰ Several methodologies have been reported in the literature for the efficient and chemoselective radioiodination of biomolecules.^11,12 The most frequently used strategy is direct radioiodination via the tyrosine group in the selected peptides and proteins by using some strong oxidizing agents.¹³ The use of an electrophilic aromatic substitution reaction on the tyrosine group provides a high radiochemical yield within a short time.¹⁴ However, radioiodine attached to biomolecules are usually unstable inside a living body. In vivo deiodination and the accumulation of free radioiodine in the blood, thyroid, and other organs results in a high background signal and poor image quality.¹⁵ Moreover, strong oxidizing agents can damage and/or alter the bioactivity of radiolabeled biomolecules.¹⁶ To address these issues, many radioiodinated maleimide and N-succinimidyl ester-based prosthetic groups have been synthesized for thiol and amino group coupling reactions, respectively (Scheme 1).¹⁷⁻¹⁹ Unfortunately, these prosthetic groups cannot provide chemoselective, fast, and high-yield radiolabeling.¹⁶ Recently, conventional bioconjugation methods including radioiodinated azide–alkyne cycloaddition,²⁰ Staudinger–Bertozzi ligation,²¹ and inverse electron-demand Diels–Alder ligation between trans-cyclooctene (TCO)-conjugated biomolecules and radioiodinated tetrazine were utilized for radioiodination.²² The bioconjugation reactions based on azide–alkyne cycloaddition and Staudinger–Bertozzi ligation demonstrated slow reaction kinetics. The reaction between TCO and radioiodinated tetrazine is fast but it provides a mixture of radioiodinated products, which may not be acceptable to regulatory authorities for clinical application.²² Therefore, the search for more efficient, easy to apply, and clean bioconjugation reactions is still in demand to satisfy diverse applications. Moreover, expansion can provide additional and better choices when multiple reactions are used to modify the biomolecules.

Scheme 1. Synthesis of maleimide-based aryl diamine linker 4.

Reagents and conditions: (i) Zn/HCOOH, MeOH, 1 h, (ii) (Boc)₂O, H₂O, rt, 2 d, (iii) methyl 5-bromovalerate, K₂CO₃, dimethylformamide (DMF), rt, 24 h, (iv) LiOH, dioxane/H₂O, rt, 2 h, (v) 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), N,N-diisopropylethylamine (DIPEA), rt, N-(2-aminoethyl)-maleimidetrifluoromethylacetate, and (vi) HCl, Et₂O, 2 h.

For several decades, benzimidazole molecules have been widely applied in organic synthesis, and various strategies have been developed for the construction of benzimidazole.²³⁻²⁶ Previously published reports have suggested that o-phenylenediamine and alkyl aldehyde can react quickly in the presence of oxygen and a catalytic amount of copper to provide highly stable benzimidazole with a high yield at room temperature (rt).^27,28 In this report, a new radioiodination strategy using an aryl diamine and alkyl aldehyde condensation reaction has been demonstrated. The condensation reactions between aryl diamine and radioiodinated alkyl aldehyde have been tested. The cRGD peptide and human serum albumin (HSA) protein were used as the model substrates.²² The biodistribution data for radioiodinated HSA have been acquired to confirm the in vivo stability against deiodination. Among the various available radioiodine, ¹²⁵I has been selected because of its easy commercial availability and suitable half-life (59.4 days), which allows repetitive experiments to optimize the radiolabeling procedure.²⁹

Results and Discussion

Radiosynthesis of ¹²⁵I-Labeled Aldehyde [¹²⁵I] 5

The radiosynthesis of ¹²⁵I-labeled aldehyde [¹²⁵I] 5 is shown in Scheme 3. An alkyl chain containing aldehyde has been synthesized by considering its importance for the efficient production of the corresponding benzimidazole. For the identification and characterization of radioiodinated [¹²⁵I] 5, nonradioactive analogue 4-iodo-N-(4-oxobutyl)benzamide 5 has been synthesized by the coupling of 4-aminobutyraldehyde diethyl acetal and 4-iodobenzoic acid under basic conditions and then subsequent reduction under acidic conditions in the next step (Scheme 2). Radioiodination of 6 was accomplished using [¹²⁵I]NaI and chloramine-T as an oxidizing agent (Scheme 3). The radioiodination reaction was terminated by adding aqueous sodium metabisulfite. After high performance liquid chromatography (HPLC) purification of the crude product, [¹²⁵I] 5 was obtained with a high radiochemical yield (72 ± 6%, n = 5). The specific activity was 45 GBq/μmol and chemical purity was more than 99%. A HPLC chromatogram of the crude mixture clearly showed the major product [¹²⁵I] 5 at 22.6 min (Figure S16). Radiolabeling reaction was performed by using varying amounts of radioactivity (100 μCi to 1 mCi), however, the radiochemical results were consistent. The radioiodinated compound [¹²⁵I] 5 was found to be stable in the refrigerator (4 °C) for more than 6 months and did not undergo hydrolysis, unlike already used active ester-based prosthetic groups (Bolton–Hunter-reagent).⁸ Compound [¹²⁵I] 5 was found to be stable in different media, including phosphate-buffered saline (PBS), saline, and mouse serum at 37 °C for more than 24 h, as determined using radio-HPLC (Figure S17).

Scheme 3. Radiosynthesis of linker [¹²⁵I] 5 and standard compound [¹²⁵I] 7.

Reagents and conditions: (i) [¹²⁵I]NaI, chloramine-T, dimethyl sulfoxide (DMSO), rt, 10 min and (ii) Cu²⁺, pH 7.5, rt, air.

Scheme 2. Synthesis of reference compound 5 and 7.

Reagents and conditions: (i) HBTU, DIPEA, 4-aminobutyraldehyde diethyl acetal, rt, 2 h, (ii) acid hydrolysis, (iii) Pd(Ph₃P)₄, bis(tributyltin), 1,4-dioxane, reflux, and (iv) Cu²⁺, pH 7.5, rt, 2 h, air.

Radiosynthesis of Compound [¹²⁵I] 7

Aldehyde diamine coupling reactions were carried out using radioiodinated compound [¹²⁵I] 5 and diamine group containing compound 4. An aryl diamine ring supplemented with the electron-withdrawing group (−OCH₂−) has been selected to accelerate the coupling reaction (Scheme 1). Before the radiosynthesis of the target compound [¹²⁵I] 7, a nonradioactive analogue 7 was synthesized for the HPLC identification and characterization of [¹²⁵I] 7. Compound 7 was synthesized by stirring compounds 4 and 5 in the presence of oxygen and a catalytic amount of copper sulfate at rt (Scheme 2). The radiolabeling reaction was performed by mixing various concentrations of 5-(3,4-diaminophenoxy)-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)pentanamide 4 (5, 25, and 50 μM) with 100 μCi of [¹²⁵I] 5 at rt. The reaction was monitored at different time points by using radio-HPLC, and radiochemical yields observed are summarized in Table 1. More than 99 and 91% of [¹²⁵I] 7 were obtained using 50 and 25 μM of 4, respectively, within 30 min and at rt (entries 2 and 3). In addition, more than 80% of [¹²⁵I] 5 was converted into [¹²⁵I] 7 using 5 μM of substrate 4 in 30 min (entry 4). The reaction was slow at 5 min although we used 50 μM of precursor 4 (entry 1), however, a high radiochemical yield was observed 30 min post incubation (entry 3). The radiochemical yields and reaction kinetics were found to be comparable to many bioconjugate reactions given in the literature.²⁸ These results encouraged us to label the model protein using a diamine and aldehyde coupling reaction.

Table 1. In vitro radiolabeling results of [¹²⁵I] 7 and [¹²⁵I] 8.

entrya	concentrationb (μmol)	substrate	time (min)	product	% RCYc
1	50	4	5	[125I] 7	26
2	50	4	30	[125I] 7	>99
3	25	4	30	[125I] 7	91
4	5	4	30	[125I] 7	80
5	50	8	30	[125I] 8	>99
6	25	8	30	[125I] 8	93
7	5	8	30	[125I] 8	69

^aSolvent: DMSO/PBS, Cu²⁺ (0.1 equiv), 25 °C.

^bFinal concentration in the reaction mixture.

^cRadiochemical yield determined by radio-HPLC.

Radiosynthesis of Compound [¹²⁵I] 8

Radiolabeled cRGD peptides have a high binding affinity and selectivity for α_vβ₃ receptors and are extensively used to detect metastatic diseases and rapidly growing cancers in preclinical studies. Many radiolabeled cRGD peptides have been developed for SPECT or PET-based studies of tumor models.³⁰ We also selected cRGD as the model peptide to test the efficiency of our radiolabeling strategy. Radiosynthesis of the titled compound [¹²⁵I] 8 is shown in Scheme 4. The radiolabeling reaction was performed by mixing the different concentrations of aryl diamine-installed cRGD 8 (5, 25, and 50 μM) with 100 μCi of [¹²⁵I] 5 at rt in the presence of a catalyst. The radioiodination reaction was monitored using radio-HPLC, and the observed radiochemical yields are summarized in Table 1. More than 99% of [¹²⁵I] 8 were obtained using 50 μM of 8 within 30 min and at rt (entry 5). In addition, 93% and 69% of [¹²⁵I] 8 were obtained for 25 and 5 μM of 8, respectively. The radiolabeling reaction was neat, and only one radioiodinated product was observed in the radio-HPLC chromatogram. In our previously reported cRGD peptide radioiodination using TCO and radioiodinated tetrazine-based bioconjugation reaction, a mixture of isomers was observed.²² These results can support preclinical studies, however, they can impede with getting an approval from regulatory authorities for clinical application of the bioconjugtion reaction. In this regard, it is a clear advantage for aryl diamine alkyl aldehyde coupling reaction to radiolabeled small molecules with high radiochemical purity.

Scheme 4. Radiosynthesis of standard compound [¹²⁵I] 8.

Reagents and conditions: (i) [¹²⁵I] 5, Cu²⁺, rt, air.

Radioiodination of HSA [¹²⁵I] 9

To determine the radiolabeling efficiency of an aryl diamine alkyl aldehyde coupling reaction, [¹²⁵I] 5 was treated with aryl diamine containing HSA protein 9 (Scheme 5). The HSA protein not only serves as a drug-delivery carrier but also enhances the blood circulation time of rapidly clearing drugs.³¹⁻³⁴ It is readily available against chemical modifications and can be stable at different pH levels (4–9) and at high temperature (60 °C for 10 h).³⁵ For successful noninvasive sentinel lymph node imaging, ⁶⁸Ga, ^99mTc, and ¹⁸F-labeled HSA have been synthesized and used.³⁶⁻⁴⁰ In this experiment, we used maleimide–cysteine-34 conjugation instead of N-succinimidyl ester–lysine based coupling on HSA because it offers a better diamine installed HSA protein with minimum alteration of the protein structure and a very high purity product. In our previous study, for the synthesis of TCO-modified HSA, TCO succinimidyl ester was used, which caused changes in the structure and biological activity of modified HSA.²² This is because lysine groups are abundant in the HSA protein, and TCO succinimidyl ester group addition can go in an uncontrolled fashion. The maleimide-cysteine reaction used in this study showed clear advantage over previous results.

Scheme 5. Radioiodination of aryl diamine-functionalized HSA [¹²⁵I] 9.

Reagents and conditions: (i) PBS, rt, pH 7.5, 10 h and (ii) [¹²⁵I] 5, Cu²⁺, air, rt.

For the chemical installation of the aryl diamine group to the HSA, a 4 molar excess amount of maleimide containing aryl diamine 4 was incubated with HSA in PBS of pH 7.5 at 25 °C for 10 h (Scheme 5). At the end of the reaction, the mixture was passed through a PD-10 (size exclusion) column to get chemically installed aryl diamine HSA. The matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis confirmed the purity and characterization of the desired product. The results showed that 1.3 aryl diamine groups were conjugated with one HSA protein at rt (Figure S14). To determine the efficiency of an alkyl aldehyde aryl diamine condensation reaction, various concentrations of aryl diamine-installed HSA were incubated with 100 μCi of ¹²⁵I-labeled alkyl aldehyde [¹²⁵I] 5 in the presence of a catalytic amount of CuSO₄. The radiochemical yields were determined using radio thin-layer chromatography (TLC) and are summarized in Table 2. The conversion yield was concentration-dependent and more than a 94% radiochemical yield was obtained for 50 μM aryl diamine-modified HSA 9 within 2 h (entry 3). Under the same reaction conditions, 89 and 67% radiochemical yields were obtained for 25 and 5 μM of aryl diamine-modified HSA, respectively (entries 4 and 5). In the control experiment, [¹²⁵I] 5 was incubated with nonmodified pure HSA under the same conditions, but no nonspecific interactions were observed (Figure S15). Next, we prepared [¹²⁵I] 9 for a biodistribution study through incubation of 50 μM aryl diamine-modified HSA 9 with 1.0 mCi of ¹²⁵I-labeled alkyl-aldehyde [¹²⁵I] 5 for 2 h in the presence of a catalyst. The crude mixture was purified using a PD-10 desalting column to provide [¹²⁵I] 9 with 90% isolated radiochemical yield and more than 99% radiochemical purity. To compare the in vivo behavior, HSA was also radioiodinated via a tyrosine ring using [¹²⁵I] NaI and chloramine-T as the oxidizing agent. ¹²⁵I-HSA ([¹²⁵I] 10) was synthesized with an 83% radiochemical yield and more than 99% radiochemical purity after purification through the PD-10 column.

Table 2. In vitro radiolabeling results of aryl diamine-conjugated HSA 9 using [¹²⁵I] 5.

entrya	concentration of 9b (μmol)	time (h)	% RCYc
1	50	0.5	54
2	50	1	63
3	50	2	94
4	25	2	89
5	5	2	67

^aSolvent: PBS, Cu⁺², (0.1 equiv), 25 °C.

^bFinal concentration in the reaction mixture.

^cRadiochemical yield determined by radio-TLC.

Biodistribution of Radioiodinated HSA

Biodistribution studies were performed using [¹²⁵I] 9 and ICR male mice. Each mouse was intravenously injected with 1 μCi of a radiolabeled product, and the biodistribution data were acquired at 0.5, 3, 6, 24, and 36 h post injection. A large amount of radioactivity (24.07 ± 1.28% ID/g) was found in the blood pool during the initial time point; the level of activity was quite high even after 24 h (8.32 ± 0.72% ID/g) and 36 h (6.73 ± 0.48% ID/g) post injection (Figure 1). These observations suggest that [¹²⁵I] 9 has a long blood circulation time, which can be used to increase the blood half-life of the fast clearing drugs after conjugation with [¹²⁵I] 9.⁴¹ The radioactivity uptake in other organs including spleen, liver, small intestine, large intestine, lungs, heart, and stomach was significantly high at the initial time point but decreased with the passage of time. Slightly high kidney uptake suggests renal clearance of the labeled compound via urine. The accumulation of radioactivity in the thyroid, which normally indicates in vivo deiodination, was 5.20 ± 1.61% ID/g and 4.86 ± 1.01% ID/g at 24 and 36 h post iv injection, respectively. We performed the biodistribution study of directly radioiodinated HSA ([¹²⁵I] 10), and the data are summarized in Table S1. The accumulation of radioactivity from [¹²⁵I] 10 in the thyroid was normal during the first time point but increased with the passage of time. The thyroid uptake was found to be 255.1 ± 66.21% ID/g and 395.2 ± 59.98% ID/g at 24 and 36 h post iv injection, respectively. These results suggest the high instability of radioiodine on the tyrosine ring in vivo. Moreover, the [¹²⁵I] 5 structure is proved to be stable and altogether different from iodotyrosine, which can undergo easy deiodination in vivo. The overall isolated radiochemical yield and in vivo stability of alkyl aldehyde aryl diamine condensation reaction is far better as compared with the direct radioiodination of tyrosine using some harsh oxidizing agents such as chloramine-T, which can damage the protein and decrease the bioactivity of the radiolabeled protein. The aryl diamine and alkyl aldehyde condensation reaction can be used for the radioiodination of many other small and large bioactive molecules. Moreover, the in vitro and in vivo stabilities of precursor [¹²⁵I] 5 are quite high and it is normally unreacted toward all functional groups in biologically active proteins.

Figure 1.

Biodistribution results of [¹²⁵I] 9 in normal ICR male mice (n = 5).

Conclusions

In the present study, we demonstrated an efficient and novel ¹²⁵I-labeling strategy for biologically active molecules (proteins and peptides) by using an alkyl aldehyde and aryl diamine condensation reaction. The rapid condensation reaction using [¹²⁵I] 5 was applied on HSA and cRGD peptide, which provided ¹²⁵I-labeled HSA ([¹²⁵I] 9) and cRGD peptide ([¹²⁵I] 8) in an excellent radiochemical yield and radiochemical purity. The biodistribution study suggested that ¹²⁵I-labeled HSA ([¹²⁵I] 9) is highly stable against in vivo deiodination and can be used for the SPECT imaging study. Moreover, the present methodology is potent; it proceeds in aqueous medium under ambient conditions, and the coupling reaction has excellent conversion, favorable reaction kinetics, and excellent biocompatibility, as compared with previously reported ligation. The investigation of the novel alkyl aldehyde and aryl diamine condensation reaction by using different therapeutic and diagnostic radioisotopes as well as engineered proteins such as repebody protein is in progress.

Experimental Section

Materials and Instruments

[¹²⁵I]NaI with a 10 mCi activity in an aqueous solution of 0.1 M NaOH was supplied by PerkinElmer Republic of Korea. All chemicals including 4-amino-3-nitrophenol, methyl 5-bromovalerate, zinc dust (<10 μm) N-(2-aminoethyl)maleimide trifluoroacetate, chloramine-T trihydrate, tris(2-carboxyethyl)phosphine hydrochloride (TCEP), DIPEA, and HSA were purchased from Sigma-Aldrich. Cysteine containing the cRGD peptide was purchased from Peptide International. All chemicals were pure and used without further purification step. HPLC experiments were performed using an Agilent Technologies 1290 infinite analytical HPLC system (Eclipse XDB-C18, 4.6 × 250 mm, 5 μm) and 1260 infinite preparative HPLC system (Eclipse XDB-C18, 21.2 × 150 mm, 7 μm). Solvent A (0.1% formic acid in deionized water) and solvent B (0.1% formic acid in acetonitrile) were used for the HPLC analysis and purification. All nuclear magnetic resonance (¹³C NMR and ¹H NMR) spectra were acquired using a JEOL 500 MHz spectrometer with DMSO-d₆, acetone-d₆, or chloroform-d (CDCl₃) as a solvent. Chemical shifts are given as δ (ppm) relative to tetramethylsilane (0.0 ppm) as an internal standard; multiplicities are given as singlet (s), doublet (d), doublet-of-doubles (dd), or multiplet (m). Agilent ESI-TOF analyzer and 4800 MALDI TOF/TOF Analyzer-(AB SCIEX) were used for mass spectroscopy.

Synthesis of Di-tert-butyl-(4-hydroxy-1,2-phenylene)-dicarbamate 1

To the mixture of HCl-activated Zn dust (4.2 g, 61.0 mmol) in methanol (30 mL), 4-amino-3-nitrophenol (2 g, 12.9 mmol) was added at 0 °C. To start a reaction, 10 mL of formic acid was added drop-wise. The reaction mixture was carried out at rt for 1 h before filtration. The excess amount of solvent was removed under reduced pressure. The solid residue was redissolved in methanol (15 mL), and di-tert-butyl dicarbonate (7.0 g, 32.0 mmol) was added to the reaction mixture. The reaction was carried out at rt for 3 days. At the end of the reaction, methanol was removed under reduced pressure, and the compound was dissolved in chloroform and washed with deionized water. The combined organic layer was dried using anhydrous sodium sulfate (Na₂SO₄) and concentrated. The crude product was purified using a silica gel column (ethyl acetate/hexane = 1:2) to give di-tert-butyl-(4-hydroxy-1,2-phenylene)dicarbamate (2.2 g, 6.79 mmol, 52%) as a white solid. ¹H NMR (500 MHz, acetone-d₆): δ 8.217 (br, 1H), 7.727 (br, 1H), 7.236 (s, 1H), 7.129 (d, J = 8.5 Hz, 1H), 6.525 (dd, J₁ = 8.5 Hz, J₂ = 2.5 Hz, 1H), 1.460 (s, 9H) and 1.448 (s, 9H); ¹³C NMR (125 MHz, acetone-d₆): 155.246, 154.502, 153.167, 133.670, 126.641, 121.146, 110.702, 109.214, 79.502, 79.092, 29.083, 27.671; HRMS: [M + Na]⁺ found, 347.1677; calcd, 347.1585.

Synthesis of Methyl 5-(3,4-Bis((tert-butoxycarbonyl)amino)phenoxy)pentanoate 2

To the mixture of intermediate precursor 1 (2.0 g, 6.17 mmol) and methyl 5-bromovalerate (1.7 g, 9.2 mmol) in 20 mL of DMF, potassium carbonate (K₂CO₃) (2.5 g, 18.51 mmol) was added gradually. The reaction was carried out at 25 °C for 1 day. After the consumption of the starting material, the crude mixture was dissolved in chloroform and washed with water to remove the excess amount of DMF and other side-products. The crude product was then dried using anhydrous sodium sulfate (Na₂SO₄), concentrated under a reduced pressure, and purified by using a silica gel column (ethyl acetate/hexane = 1:4) to give 2 as a brown oil (1.9 g, 4.33 mmol, 70%). ¹H NMR (500 MHz, CDCl₃): δ 7.137 (br, 1H), 6.904 (br, 1H), 6.574 (dd, J₁ = 8.5 Hz, J₂ = 2.5 Hz, 1H), 3.928 (t, J = 6.6 Hz, 2H), 3.65 (s, 3H), 2.369 (t, J = 7.3 Hz, 2H), 1.70–1.78 (m, 6H), 1.495 (s, 9H) and 1.482 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): 174.027, 157.430, 154.740, 153.310, 133.480, 126.688, 120.660, 110.654, 108.346, 80.780, 80.685, 67.627, 51.631, 33.747, 28.672, 28.367, 21.681; HRMS: [M + Na]⁺ found, 461.2166; calcd, 461.2263.

Synthesis of Di-tert-butyl-(4-((5-((2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-5-oxopentyl)oxy)-1,2-phenylene)dicarbamate 3

To the water/dioxane mixture (1:3, 4.8 mL), intermediate precursor 2 (1.5 g, 3.4 mmol) and lithium hydroxide (287 mg, 11.98 mmol) were added at 0 °C. The reaction was carried out for 2.5 h at rt and neutralized with 1 M HCl and extracted with ethyl acetate. The organic phase was dried using anhydrous sodium sulfate (Na₂SO₄), and the solvent was removed under a reduced pressure. The crude product was redissolved in DMF (10 mL), and a coupling agent HBTU (1.3 g, 3.5 mmol), DIPEA (900 mg, 7.0 mmol), and N-(2-aminoethyl)maleimide trifluoroacetate salt (890 mg, 3.5 mmol) were added sequentially. The reaction was performed at rt for 2.5 h and acidified using aqueous 1 M HCl. The crude product was extracted with ethyl acetate, washed with distilled water, dried over Na₂SO₄, and concentrated under reduced pressure. The crude mixture was purified using a silica gel column (ethyl acetate/hexane 2.5:1) to give compound 3 as a yellow oil (1 g, 1.8 mmol, 52.94%). ¹H NMR (500 MHz, CDCl₃): δ 7.138 (br, 1H), 6.929 (br, 1H), 6.687 (s, 2H), 5.951 (dd, J₁ = 8.5 Hz, J₂ = 2.5 Hz, 1H), 3.928 (t, J = 6.6 Hz, 2H), 3.669 (t, J = 6.5 Hz, 2H), 3.446 (m, 2H), 2.169 (t, J = 7.3 Hz, 2H), 1.668–1.862 (m, 6H), 1.498 (s, 9H) and 1.488 (s, 9H); ¹³C NMR (125 MHz, CDCl₃): 173.264, 171.041, 157.459, 154.683, 153.243, 134.319, 126.850, 110.750, 108.689, 80.885, 80.771, 67.780, 60.502, 38.840, 37.658, 36.103, 28.606, 28.386, 22.177; HRMS: [M + Na]⁺ found, 569.7869; calcd, 569.2591.

Synthesis of 5-(3,4-Diaminophenoxy)-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)pentanamide 4

Intermediate compound 3 (100 mg, 0.18 mmol) was stirred in the mixture of trifluoroacetic acid (2 mL) and dichloromethane (2 mL) for 3 h at an ambient temperature. At the end of the reaction, the product was placed under high vacuum. The final product 4 was obtained as an off-white solid (95 mg, 0.27 mmol). ¹H NMR (500 MHz, CDCl₃): δ 8.048 (s, 1H), 6.763 (d, J = 8.0 Hz, 1H), 6.241 (d, J = 8.0 Hz, 1H), 5.254 (s, 1H), 3.952 (t, J = 6.6 Hz, 2H), 3.681 (t, J = 6.5 Hz, 2H), 3.401 (m, 2H), 2.134 (t, J = 7.3 Hz, 2H), 1.805–1.752 (m, 6H), 1.562–1.452 (m, 6H); ¹³C NMR (125 MHz, CDCl₃): 173.310, 171.136, 150.153, 139.956, 134.926, 126.850, 120.126, 107.254, 103.512, 68.218, 60.502, 37.658, 36.235, 28.921, 22.292; HRMS: [M + H]⁺ found, 347.7940; calcd, 347.1641.

Synthesis of 4-Iodo-N-(4-oxobutyl)benzamide 5

To a stirring solution of 4-iodobenzoic acid (0.5 g, 2.02 mmol) in DMSO (10 mL), HBTU coupling agent (0.75 g, 1.88 mmol) and DIPEA (0.75 g, 5.8 mmol) were added at 0 °C. After 20 min, 4-aminobutyraldehyde diethyl acetal (0.35 g, 1.89 mmol) was added, and a reaction was performed at 25 °C for 2.5 h. The reaction was terminated by adding diluted 1 M HCl (6 mL). The crude product was dissolved in dichloromethane and washed with distilled water, dried over Na₂SO₄, and concentrated under reduced pressure. The dried residue was redissolved in tetrahydrofuran/H₂O (3:1, 15 mL) and stirred with concentrated HCl (3.5 mL) for an additional 2.5 h. The crude product was extracted with CH₂Cl₂, dried, and concentrated. A silica gel column and ethyl-acetate/hexane (2:1) were used to purify the crude product, giving titled compound 5 (0.33 g, 1.0 mmol, 53%). ¹H NMR (500 MHz, DMSO-d₆): δ 9.872 (t, J = 6.3 Hz, 2H), 7.772 (d, J = 8.0 Hz, 1H), 7.499 (d, J = 8.0 Hz, 1H), 3.470 (t, J = 7.3 Hz, 2H), 2.629 (t, J = 6.5 Hz, 2H), 1.990–1.925 (m, 6H); ¹³C NMR (125 MHz, DMSO-d₆): 202.423, 167.341, 137.648, 133.623, 129.111, 98.302, 42.017, 23.913, 21.681; HRMS: [M + H]⁺ found, 317.9940; calcd, 317.9913.

Synthesis of N-(4-Oxobutyl)-4-(tributylstannyl)benzamide 6

To a solution of 5 (200 mg, 0.61 mmol) in 15 mL of 1, 4 dioxane, tetrakis(triphenylphosphine)palladium (72 mg, 0.06 mmol) and bis(tributyltin) (730 mg, 1.2 mmol) were added. The reaction was refluxed for 18 h. The reaction mixture was cooled down at rt and filtered through a filter paper. The crude mixture was diluted with diethyl ether and washed with distilled water, dried over anhydrous magnesium sulfate, concentrated under vacuum, and purified using a silica gel column and 3% ethyl acetate in hexane to give titled compound 6 (190 mg, 0.39 mmol, 65%). ¹H NMR (500 MHz, DMSO-d₆): δ 8.496 (t, J = 6.3 Hz, 2H), 7.629 (d, J = 8.0 Hz, 1H), 7.499 (d, J = 8.0 Hz, 1H), 3.35 (t, J = 7.3 Hz, 2H), 2.69 (t, J = 6.5 Hz, 2H), 1.937–1.992 (m, 6H), 1.436–1.557 (m, 6H), 1.226–1.286 (m, 6H), 1.008–1.040 (m, 6H), 0.809 (t, J = 9.3 Hz, 9H); ¹³C NMR (125 MHz, DMSO-d₆): 203.577, 167.341, 146.242, 136.541, 134.710, 126.841, 39.546, 29.111, 27.204, 22.348, 20.899, 14.079, 9.729; HRMS: [M + H]⁺ found, 482.2302; calcd, 482.2120.

Synthesis of N-(3-(5-((5-((2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)amino)-5-oxopentyl)oxy)-1H-benzo[d]imidazole-2-yl)propyl)-4-iodobenzamide 7

To a stirring solution of 5 (50 mg, 0.15 mmol) and 4 (25 mg, 0.075 mmol) in 10 mL of DMSO, 20 μL of copper sulfate (50 mmol) was added to give a final concentration of the CuSO₄ catalyst as 0.10 mmol. The reaction was carried out at rt for 1.5 h. The crude product was diluted in chloroform, washed with distilled water, and dried over anhydrous magnesium sulfate. The crude product was purified using a silica gel column (ethyl acetate/hexane 2:1) to give the titled compound 7 (38 mg, 0.06 mmol, 80%). MALDI-TOF: ([M + H]⁺) found, 644.1321; calculated, 644.1322.

Synthesis of Diamine-Conjugated cRGD 8

To the stirred solution of cysteine containing the cRGD peptide (10 mg, 0.01 mmol) in DMF, TCEP (5.5 mg, 0.02 mmol) and compound 4 (4.0 mg, 0.01 mmol) were added sequentially at pH 7.0. After 2 h of stirring at rt, the crude product was purified using preparative HPLC (solvent flow rate 10 mL/min, eluent gradient: 0–2 min: 95%A/5%B; a linear gradient to 35%A/65%B from 95%A/5%B; 2–25 min; Rt: 14.6 min). MALDI-TOF: ([M + H]⁺) calcd for C₄₇H₆₉N₁₄O₁₂S⁺, 1053.4991; found, 1053.5125.

Synthesis of Diamine-Conjugated HSA 9

HSA (350 μM) was dissolved in 100 μL of phosphate buffer solution of pH 7.5. Intermediate compound 4 (15 mM) in 10 μL of DMSO was added to the above solution. After 10 h, the crude product was diluted in 2.5 mL of deionized water and passed through a preconditioned PD-10 desalting column. The compound was extracted with 3.5 mL of phosphate buffer and centrifuged (5500 rpm, 10 min) using a 30 kDa centrifugal filter to obtain pure diamine-conjugated HSA. Pure diamine-conjugated HSA protein was characterized by MALDI-TOF, and the concentration was determined through a protein analysis and quantification system (Millipore).

Radioiodination of ¹²⁵I-Labeled Alkyl Aldehyde [¹²⁵I] 5

To the solution of N-(4-oxobutyl)-4-(tributylstannyl)benzamide 6 (1 mg) in 100 μL of DMSO, chloramine-T solution (1 mg, 10 μL of H₂O), acetic acid (3 μL), and [¹²⁵I]NaI solution (1.7 mCi, 10 μL) in 0.1 M NaOH was added sequentially. The reaction was performed at 25 °C for 10 min and terminated using a sodium metabisulfite solution (Na₂S₂O₅) (2 mg in 20 μL of H₂O). The mixture was purified using a preparative HPLC system (flow rate: 10 mL/min, eluent gradient: 0–2 min: 95%A/5%B; 2–13 min: a linear gradient to 65%A/35%B from 95%A/5%B; 13–25 min: a linear gradient to 35%A/65%B from 65%A/35%B; 25–30 min: a linear gradient to 0%A/100%B from 35%A/65%B, Rt = 22.6 min) and 1.2 mCi of ¹²⁵I-labeled product ([¹²⁵I] 5) were obtained (72% of radiochemical yield, n = 5). The radiochemical purity was more than 99% as determined using an analytical HPLC (flow rate: 1 mL/min, eluent gradient: 0–2 min: 95%A/5%B; 2–13 min: a linear gradient to 65%A/35%B from 95%A/5%B; 13–25 min: a linear gradient to 35%A/65%B from 65%A/35%B; 25–30 min: a linear gradient to 0%A/100%B from 35%A/65%B, Rt: 22.6 min).

Radiosynthesis of Compound [¹²⁵I] 7

To a mixture of 5-(3,4-diaminophenoxy)-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)pentanamide 4 (50 μL in DMSO), 10 μL of copper sulfate (CuSO₄) (0.1 equiv) and 100 μCi of ¹²⁵I-labeled alkyl aldehyde [¹²⁵I] 5 (40 μL in DMSO) were added sequentially. The final reaction volume was 100 μL, and the actual concentration of 4 was 50, 25, or 5 μM. The labeling reaction was conducted at rt in the presence of oxygen. The reaction was monitored at different time points using a radio-HPLC system. The radiochemical yield of the reaction was measured through integration of a radio-HPLC chromatogram (system flow rate: 1 mL/min, gradient: 0–2 min: 95%A/5%B; 2–13 min: a linear gradient to 65%A/35%B from 95%A/5%B; 13–25 min: a linear gradient to 35%A/65%B from 65%A/35%B; 25–30 min: a linear gradient to 0%A/100%B from 35%A/65%B). The observed radiochemical yields are summarized in Table 1.

Radioiodination of the cRGD Peptide [¹²⁵I] 8

The procedure for radioiodination of the cRGD peptide [¹²⁵I] 8 was the same as that adopted for [¹²⁵I] 7. The radiolabeling reaction was monitored at different time points using a radio-HPLC system (system flow rate: 1 mL/min, gradient: 0–2 min: 95%A/5%B; a linear gradient to 35%A/65%B from 95%A/5%B; 2–25 min; Rt: 17.7 min). The observed radiochemical yields are summarized in Table 1.

Radioiodination of HSA [¹²⁵I] 9

To a mixture of aryl diamine-conjugated HSA 9 (5–50 μM, 70 μL in 1× PBS), 20 μL of copper sulfate (CuSO₄) (0.1 equiv) and 100 μCi of ¹²⁵I-labeled alkyl-aldehyde [¹²⁵I] 5, (10 μL in DMSO) were added. The final reaction volume was 100 μL, and the labeling reaction was conducted at rt in the presence of oxygen. The reaction was monitored at different time points using a radio-TLC system. The observed radiochemical yields are summarized in Table 2. For the tissue distribution study of radioiodinated HSA ([¹²⁵I] 9), aryl diamine-containing HSA 9 (50 μM, in 70 μL of PBS) was treated with ¹²⁵I-labeled alkyl-aldehyde [¹²⁵I] 5 (1.0 mCi, 10 μL in DMSO) in the presence of CuSO₄ (20 μL, 0.1 equiv) at rt. After 2 h, the crude product was dissolved in saline and then purified using a PD-10 (desalting) column to give [¹²⁵I] 9 of 90% radiochemical yield and more than 99% radiochemical purity.

Direct Radioiodination of HSA [¹²⁵I] 10

To a solution of HSA (25 μM) in 100 μL of PBS (1× PBS, pH = 7.4), [¹²⁵I] NaI (800 μCi, 5 μL) and chloramine-T oxidizing agent (1 mg, 10 μL of H₂O) were added at 4 °C. The reaction was carried out for 30 min at 4 °C and terminated using sodium metabisulfite solution (Na₂S₂O₅) (2 mg in 20 μL of H₂O). The product was purified using a PD-10 desalting column by using saline as an eluent to give the titled compound with a 83% radiochemical yield and >99 radiochemical purity.

In Vitro Stability Test for ¹²⁵I-Labeled Alkyl Aldehyde [¹²⁵I] 5

To evaluate the in vitro stability of a radiotracer, purified [¹²⁵I] 5 (100 μL, 100 μCi) was incubated in 900 μL of saline, or 1× PBS, or mouse serum at 37 °C. The stability of [¹²⁵I] 5 in each media was evaluated at the given time points (0.5, 3, 6, 8, and 24 h) using a radio-HPLC system.

Biodistribution of Radioiodinated HSA [¹²⁵I] 9

For a biodistribution study, 25 male ICR mice (6 weeks old) were purchased from Orientbio Co., Ltd (Jeonbuk, Korea Republic). The ICR mice were divided into five groups so that each group contained five animals. Each mouse was injected with an aqueous solution of [¹²⁵I] 9 or [¹²⁵I] 10 (100 μL, 1 μCi) through the tail vein. After 0.5, 3, 6, 24, and 36 h post injection, a group of mice were sacrificed under isoflurane dose; the organs of interest (thyroid, lungs, stomach, heart, liver, kidneys, spleen, large intestines, and small intestine) and blood were collected. The collected blood and organs were weighed and the accumulated radioactivity was determined using a 1480 wizard 3, (PerkinElmer, USA) gamma counter. The final biodistribution data were reported in terms of the percentage injected dose per gram of organ or blood (% ID/g). All animal experimental procedures were approved by the Institutional Animal Ethical Committee and performed according to the guidelines prescribed by the committee.

Glossary

Abbreviations

PET

positron emission tomography

SPECT

single photon emission computed tomography

HSA

human serum albumin

% ID/g

injected dose per gram of organ or blood

Supporting Information Available

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b00416.

• NMR, HRMS, and HPLC data (PDF)

The authors declare no competing financial interest.