Highly efficient and stable strain-release radioiodination for thiol

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Highly efficient and stable strain-release radioiodination for thiol chemoselective bioconjugation Pu Zhang, Rongqiang Zhuang, Xiangyu Wang, Huanhuan Liu, Jindian Li, Xinhui Su, Xiaoyuan Chen, and Xianzhong Zhang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00790 • Publication Date (Web): 27 Jan 2018 Downloaded from http://pubs.acs.org on January 29, 2018

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Bioconjugate Chemistry

Highly Efficient and Stable Strain-Release Radioiodination for Thiol Chemoselective Bioconjugation Pu Zhang,† Rongqiang Zhuang,† Xiangyu Wang,† Huanhuan Liu,† Jindian Li,† Xinhui Su,‡ Xiaoyuan Chen,§ and Xianzhong Zhang*† †

Center for Molecular Imaging and Translational Medicine, State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen 361102, China ‡ Zhongshan Hospital Affiliated of Xiamen University, Xiamen 361004, China §

Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengineering (NIBIB), National Institutes of Health (USA), Bethesda, Maryland 20892, United States ABSTRACT: We report a novel thiol selective radioiodination method based on strain-release reaction. A new heterobifunctional radioiodination agent which has very good thiol selectivity and excellent stability with high radiolabeling yield was synthesized, characterized and applied successfully for thiol-contained peptide labeling.

INTRODUCTION Radioactive iodine isotopes (123I, 124I, 125I and 131I) labeled radiopharmaceuticals are widely used in nuclear medicine for disease diagnosis and therapy.1-4 Among these radiopharmaceuticals, radiolabeled peptides have always played an important role in both research and clinical application.5 Methods used for those radiopharmaceuticals direct radioiodination are usually based on electrophilic substitution reaction with amino acids containing aromatic substituents like tyrosine, histidine and tryptophane or just phenol structure.6 Due to the electrondonating substituent hydroxyl, tyrosine becomes the main peptide and protein radioiodination site, which can be radioiodinated by Chloramine-T,7 iodogen,8 iodobead9 methods. Although these methods are most widely used in radioiodination procedure up to now for their efficiency and ease of handling, the using of oxidation agents as well as the reducing agents to stop the labeling process may cause undesirable side reactions of the biomolecules.10 To avoid the oxidizing conditions, twostep labeling procedure using pre-radioiodinated heterobifunctional agents such as Bolton and Hunter agent11 and SIB12 via amidation reaction with lysine residue is used, however the hydrolysis of the succinimidyl ester group especially at the optimum labeling condition which is often alkaline is also one disadvantage that may decrease the radiochemical yield and purity. Besides these problems, no matter the direct radioiodination methods or using the Bolton and Hunter agent, volatile molecule iodine will be generated during the labeling process which limits the operation to avoid the airborne release. Except for SIB, the in vivo deiodination of radioiodinated phenol structure which may result in low target-to-background ratio of the images and increase the radiation dose to nontarget tissues is another inherent drawback of these traditional radioiodination labeling methods.13-14 Moreover, the chemoselective radiolabeling is also very important especially for pep-

tide, protein and antibody, while it is a special challenge for traditional radioiodination because of the abundant functional groups in these biomolecules. Thiol alkylation is also a powerful method for peptide, protein and nano-biomaterials modification.15-18 As the most widely used alkylating agents which based on Michael addition, maleimides have always played an important role in both research and clinical analysis, of which radioiodinated maleimide derivatives were also studied for antibody labeling.19-22 Although useful, limitations of these agents such as the disruptive cleavage because of undergoing thiol exchange reaction with free cysteine and glutathione residues and the ring opening of the succinimide linkage decrease the stability in vivo.23 Considering these reasons, the development of one new mild, efficient, chemoselective and stable in vitro and in vivo radioiodination method is a subject of much current interest. Small strained ring systems such as bicyclo[1.1.1]pentanes, azetidines or cyclobutanes are increasingly valued for their chemical application.24 Recently, Phil Baran and co-workers reported one new mild and simple operation synthesis method for strain-release amination,25-27 besides these, the remarkable chemoselectivity of thiol and the excellent stability exhibited by strain-release agents motivated us to develop a new kind heterobifunctional radioiodination agent for thiol-contained peptide labeling. To overcome the limitations of conventional radioiodination methods, we recently developed a highly efficient coppermediated radioiodination approach using aryl boronic acids which is technically simple, mild and no volatile molecular iodine generated.28 For these reasons, herein we report a novel stable and thiol chemoselective radioiodination method using a new developed heterobifunctional agent based on strainrelease reaction.

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Scheme 1. Thiol selectively heterobifunctional radioiodination agents: maleimide-based radioiodination agents (A) and bicyclo[1.1.0]butane based strain-release radioiodination agent (B).

RESULTS AND DISCUSSION Synthesis of the heterobifunctional agent 131I-S8. 1-((4bromophenyl)sulfonyl)bicyclo[1.1.0]butane (S6) was synthesized from 4-bromobenzene-1-sulfonyl chloride according to the synthesis route reported by Phil Baran and co-workers.25 Then the product (4-(bicyclo[1.1.0]butan-1ylsulfonyl)phenyl)-boronic acid (S7) which contains phenylboronic acid structure for radioiodine labeling was obtained by photo-induced reaction under ultraviolet irradiation.29 Based on the previous results from our group,28 the radioiodination conditions of S7 were as follows: substrate S7 (2 µmol), Cu2O (0.4 µmol), and 1,10-phenanthroline (0.8 µmol) dissolved in acetonitrile (50 µL), [131I]NaI (about 37 MBq, 5-10 µL in water), reacted for 1 h at 25 °C to give the desired new heterobifunctional agent 131I-S8. O S O Br

O S O

B2 (OH)4 HO

UV, 254 nm S6

B OH

S7 O S O

Na 131 I Cu2 O/1,10-phenanthroline

131

I 131

Scheme 2. Synthesis route of

bling argon through the solution to exclude oxygen. At 25 °C, this approach gave high radiochemistry yield (91%) taking 3 h and at 60 °C it just took 30 min to obtain the excellent yield (95%) which are both acceptable for radioiodine labeling. The only one new radioactive product testified by high performance liquid chromatography (radio-HPLC) indicated the highly chemoselective of thiol (Figure 1b). The excellent stability of 131I-S8 in vitro was confirmed by radio-HPLC analysis after incubation in the mixed solvents for 24 h at room temperature (Figure 1c).

131

I-S8

I-S8.

Chemoselective radiolabeling of peptide. At the start investigation of thiol radioiodine labeling, the short peptide (sequence CAQK) which can target the proteoglycan complex upregulated in acute brain injuries30 was selected as the model substrate to verify the feasibility and the chemoselectivity of this approach for the reason that it has both thiol and amino group. Considering the water solubility of peptide and the lipophilicity of 131I-S8, mixed solvents N, Ndimethylformamide (DMF) and phosphate buffer were chosen as solvents to develop this labeling method. Strain-release reaction with thiol do not proceed under acidic condition has been testified according to the published report. To make sure the pH of the reaction mixture was alkaline, phosphate buffer (pH 8.0) and K2CO3 were employed allowing the deprotonation of the cysteine thiol of CAQK. To keep the thiol in reduced form, the solvents were degassed prior to use by bub-

Figure 1. a HPLC analysis of 131I-S8 and non-radioactive reference with radiometric and UV detection; b HPLC analysis of 131I-CAQK; c Stability analysis of 131I-S8 after incubation in the mixture solvent of DMF (40 µL), K2CO3 in water (0.2 M, 20 µL) and phosphate buffer (0.2 M, pH 8.0, 90 µl) for 24 h; d HPLC analysis of 131I-c(RGDyC). Finally, the optimized labeling conditions of peptide are: 2 µmol substrate in 90 µL phosphate buffer (0.2 M, pH 8.0), 20 µL K2CO3 in water (0.2 M), and 40 µL 131I-S8 in DMF were mixed together and reacted for 30 min at 60 °C. Substrate scope and further thiol chemoselectivity study. To further investigate the substrate scope and the thiol selectivity of this method, several amino acids with or without thiol served as substrates and the labeling results were shown in Table 1, when mixed glycine and N-Acetyl-cysteine together with the molar ratio 1:1, there was still no reaction with gly-

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Bioconjugate Chemistry cine which confirmed the excellent chemoselectivity of thiol again.

Table 1. Reactions of thiol-contained substrates with S8. [a]

131

I-

[b]

Entry

Substrate

RCY

1

L-Cysteine

100

2

L-Cysteine methyl ester hydrochloride

88

3

N-Acetyl-cysteine

100

4

CAQK

95

5

c(RGDyC)

99

6

N-Acetyl-cysteine, glycine

100

[a] All reactions were performed using substrate 2 µmol. [b] RCY determined by radio-HPLC.

Figure 2. SPECT imaging of U87MG tumor bearing mice with 125I-c(RGDyC) obtained from 125I-S8. Stability study and SPECT imaging. To demonstrate the stability and the targeting ability of the peptide labeled with radioiodine using this method, cyclic peptide c(RGDyC) served as substrate and was labeled with 131I and 125I respectively using this strain-release radioiodination approach. The specific activity of the radioiodinated c(RGDyC) was calculated as 1.13×1012 Bq/g based on the standard curve (Figure S8). Good stability in vitro was verified by HPLC analysis after incubated in fetal bovine serum (FBS) for 96 h (Figure S6). Ex vivo biodistribution study in normal mice was performed and compared with the conventional Iodogen method (Table S2), the uptakes of thyroid and stomach indicated that the radioiodinated peptide c(RGDyC) prepared by using this strainrelease method had obviously less deiodination in vivo than that of conventional Iodogen method. Single photon emission computed tomography (SPECT) imaging of tumor-bearing mice with 125I-c(RGDyC) showed moderate tumor uptake (Figure 2) and very low thyroid accumulation at 4 h after injection (Figure S9), which confirmed the targeting ability and excellent in vivo stability of this tracer. In conclusion, we have developed a new mild and efficient thiol selective radioiodination method using strain-release reaction. The excellent stability and thiol chemoselectivity of

this novel heterobifunctional agent may make this method be a universal procedure for peptide, protein antibody and nanobiomaterials radioiodine labeling.

EXPERIMENTAL PROCEDURES Materials and general methods. Commercially available chemicals were obtained from commercial suppliers (Energy Chemical and Alfa Aesar) and used as received without further purification. Silica gel GF254 aluminum plates used for thin layer chromatography (TLC) analysis were purchased from Energy Chemical and preparation silica gel plates used for product purification were purchased from Sinopharm Chemical Reagent Beijing Co., Ltd. TLC plates were visualized by exposure to short wave ultraviolet light (254 nm, 365nm) and/or iodine. Radio-TLC analysis was performed on BioScan Mini-Scan radio-TLC Strip Scanner. HPLC analysis was performed on Thermo Fisher Dionex UltiMate 3000 using the Nucleosil C18 (250 × 4 mm, 10 µm, 100 Å) column and chromatograms were collected at 254 nm wavelength. 1H and 13 C NMR spectra were recorded on Bruker AVANCE III 600 MHz and Bruker AVANCE II 400 MHz spectrometer. Mass spectra were recorded on Waters Xevo G2-XS QTof mass spectrometer. SPECT/CT imaging was performed using nanoScan SC (Mediso Medical Imaging System) equipped with pinhole collimator under standard animal scan procedure. All animal experimental protocols were carried out in accordance with the relevant guidelines of Xiamen University. Procedure for the preparation of S7. 4Bromobenzenesulfonyl chloride (10.0 g, 39.0 mmol) was added to a solution of Na2SO3 (9.8 g, 2.0 equiv) and NaHCO3 (6.5 g, 2.0 equiv) in H2O (50 mL) portionwise at room temperature and then reacted at 80 °C for 6 h. After cooled to room temperature, the mixture was extracted with EtOH (3 × 50 mL) and the combined solution was evaporated, then dissolved in DMF (50 mL). Added 4-bromo-1-butene (6.3 g, 1.2 equiv) and reacted at 50 °C for 2 h. The reaction mixture was cooled to room temperature, diluted with EtOAc (50 mL), washed with brine (3 × 25 mL), dried with Na2SO4, concentrated under reduced pressure, and purified by silica gel flash chromatography with petroleum ether/ethyl acetate (8:1) to give S2 (3.9 g, 36%). 1H NMR (600 MHz, CDCl3): δ 7.80 - 7.78 (m, 2H), 7.74 - 7.72 (m, 2H), 5.73 (ddt, J = 16.8 Hz, 10.2 Hz, 6.6 Hz, 1H), 5.09 - 5.06 (m, 2H), 3.18 - 3.16 (m, 2H), 2.49 - 2.45 (m, 2H); 13C NMR (600 MHz, CDCl3): δ 138.00, 133.50, 132.68 (2C), 129.72 (2C), 129.17, 117.39, 55.41, 26.83; m/z (ESI): 296.99 and 298.99 ([M+Na]+, C10H11BrNaO2S+, requires 296.96 and 298.95). S2 (3.8 g, 13.8 mmol) was dissolved in CH2Cl2 (40 mL) and mCPBA (5.6 g, 85%) was added into the solution in portions at 0 °C. The mixture was stirred at room temperature until TLC analysis indicated complete consumption of S2. At the end of the reaction, the mixture was filtered using funnel, and the organic phase was first washed with aqueous Na2SO3 (1M, 2 × 30 mL), then washed with NaHCO3 (sat., aq., 2 × 30 mL) and finally washed with brine (2 × 20 mL), dried with Na2SO4, filtered, and concentrated under reduced pressure, purified by silica gel flash chromatography with petroleum ether/ethyl acetate (2:1) to give S3 (3.5 g, 87%). 1H NMR (600 MHz, CDCl3): δ 7.79 - 7.77 (m, 2H), 7.74 - 7.72 (m, 2H), 3.22 (m, 2H), 3.01 (dtd, J = 10.8 Hz, 3.0 Hz, 1.2 Hz, 1H), 2.78 (dd, J = 4.2 Hz, 4.2 Hz, 1H), 2.50 (dd, J = 4.5 Hz, 3.0 Hz, 1H), 2.21 -



2.15 (m, 1H), 1.83 - 1.77 (m, 1H); 13C NMR (600 MHz, CDCl3): δ 137.83, 132.76 (2C), 129.62 (2C), 129.32, 52.77, 49.97, 47.09, 25.83; m/z (ESI): 312.99 and 314.99 ([M+Na]+, C10H11BrNaO3S+, requires 312.95 and 314.95). Epoxide S3 (1.2 g, 4.0 mmol) was dissolved in THF (20 mL) and n-BuLi (2.5 M in hexane, 1.7 mL, 1.06 equiv) was added dropwise at 0 °C. After the addition was complete, the mixture was stirred a further 5 min before being quenched with sat. aq. NH4Cl. The organic layer was separated and the aqueous layer was extracted with EtOAc twice. The combined organic layers were dried with Na2SO4, concentrated under reduced pressure, and purified by silica gel flash chromatography with petroleum ether/ethyl acetate (2:1) to give S4 (0.9 g, 75%). 1H NMR (600 MHz, CDCl3): δ 7.77 - 7.76 (m, 2H), 7.71 - 7.69 (m, 2H), 3.74 (dd, J = 11.4 Hz, 4.8 Hz, 1H), 3.51 (dd, J = 11.4 Hz, 6.0 Hz, 1H), 2.46 (dt, J = 8.4 Hz, 4.8 Hz, 1H), 2.07 - 2.02 (m, 1H), 1.74 (s, 1H), 1.47 (dt, J = 9.6 Hz, 4.8 Hz, 1H), 1.12 - 1.09 (m, 1H); 13C NMR (600 MHz, CDCl3): δ 139.50, 132.60 (2C), 129.10 (2C), 128.72, 61.81, 36.80, 21.65, 10.07; m/z (ESI): 313.00 and 315.00 ([M+Na]+, C10H11BrNaO3S+, requires 312.95 and 314.95). S4 (5.1 g, 17.6 mmol) was dissolved in anhydrous CH2Cl2 (15 mL), anhydrous Et3N (2.8 mL, 1.1 equiv) and methanesulfonyl chloride (2.8 mL, 1.1 equiv) was added successively at room temperature. After stirring for 2 h, the reaction mixture was washed with brine twice, dried with Na2SO4, concentrated under reduced pressure, and purified by silica gel flash chromatography with petroleum ether/ethyl acetate (1:1) to give S5 (4.5 g, 69%). 1H NMR (600 MHz, CDCl3): δ 7.79 - 7.77 (m, 2H), 7.75 - 7.73 (m, 2H), 4.33 (dd, J = 11.4 Hz, 5.4 Hz, 1H), 3.99 (dd, J = 11.4 Hz, 7.2 Hz, 1H), 2.98 (s, 3H), 2.60 (dt, J = 8.4 Hz, 4.8 Hz, 1H), 2.21 - 2.15 (m, 1H), 1.72 (s, 1H), 1.66 (dt, J = 9.6 Hz, 5.4 Hz, 1H), 1.22 - 1.19 (m, 1H); 13C NMR (600 MHz, CDCl3): δ 138.92, 132.72 (2C), 129.28 (2C), 129.08, 68.72, 38.07, 37.82, 18.71, 10.90; m/z (ESI): 390.86 and 392.86 ([M+Na]+, C11H13BrNaO5S2+, requires 390.93 and 392.93). S5 (1.0 g, 2.7 mmol) was dissolved in THF (20 mL) and nBuLi (2.5 M in hexane, 1.0 mL, 0.95 equiv) was added dropwise at 0 °C. After the addition was complete, the mixture was stirred a further 5 min before being quenched with sat. aq. NH4Cl. The organic layer was separated and the aqueous layer was extracted with EtOAc twice. The combined organic layers were dried with Na2SO4, concentrated under reduced pressure, and purified by silica gel flash chromatography with petroleum ether/ethyl acetate (8:1) to give S6 (0.3 g, 40%). 1H NMR (600 MHz, CDCl3): δ 7.81 - 7.80 (m, 2H), 7.71 - 7.69 (m, 2H), 2.62 - 2.60 (m, 1H), 2.53 - 2.51 (m, 1H), 1.41 - 1.39 (m, 1H); 13 C NMR (600 MHz, CDCl3): δ 141.01, 132.45 (2C), 128.70 (2C), 128.12, 38.40, 22.87 (2C), 13.04; m/z (ESI): 294.95 and 296.95 ([M+Na]+, C10H9BrNaO2S+, requires 294.94 and 296.94). S6 (54.6 mg, 0.2 mmol) and B2(OH)4 (89.6 mg, 1.0 mmol) were dissolved in anhydrous CH3OH (2.0 mL), then the mixture was irradiated under UV light for 2 h in a water-cooled quartz reactor. The crude product was purified using HPLC to give S7 (10 mg, 21%). 1H NMR (600 MHz, CD3OD): δ 7.98 -

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7.82 (m, 4H), 2.65 - 2.60 (m, 1H), 2.46 - 2.43 (m, 2H), 1.43 1.40 (m, 2H); 13C NMR (600 MHz, CD3OD): δ 145.01, 136.55 (2C), 128.00 (2C), 39.46, 24.69 (2C), 14.24; m/z (ESI): 238.17 and 239.17 ([M+H]+, C10H12BO4S+, requires 238.06 and 239.05). Synthesis of stable reference compound (127I-S8). NaI (1.3 mg, 8.4 µmol) was added to a solution of S7 (1.0 mg, 4.2 µmol), Cu2O (0.6 mg, 4.2 µmol), 1,10-phenanthroline (1.7 mg, 8.4 µmol) in 50 µL acetonitrile, the mixture was stirred for 3 h, and then the crude product was purified using HPLC. m/z (ESI): 342.98 ([M+Na]+, C10H9INaO2S+, requires 342.93). Radioiodination of S7 to synthesize 131I-S8 or 125I-S8. Added precursor S7 (2 µmol) into one 1.5 mL reaction vial, 50 µL solution of Cu2O/1,10-phenanthroline in acetonitrile which contained Cu2O (0.4 µmol) and 1,10-phenanthroline (0.8 µmol) was then added into the vial, after that, Na131I or Na125I (about 37 MBq) in 5 - 10 µL water was added into the mixture and reacted for one hour at 25 °C. To separate the 131I-S8 or 125 I-S8 from the precursor S7, preparation silica gel plate was used with petroleum ether/ethyl acetate (4:1), 127I-S8 was used as standard substance. The radioiodination product 131I-S8 or 125 I-S8 was eluted from the silica gel using petroleum ether/ethyl acetate (1:1), and then was blown to dryness under a stream of argon. Radioiodination of peptide and amino acids. Peptide or amino acid (2 µmol) was dissolved in phosphate buffer (90 µL, 0.2 M, pH 8.0) which was degassed prior to use by bubbling argon through the solution to exclude oxygen, K2CO3 in water (20 µL, 0.2 M) was added into the peptide solution, then added the mixture into the 131I-S8 or 125I-S8 in DMF (40 µL) which was degassed prior to use by bubbling argon through the solution to exclude oxygen and reacted at 60 °C for 30 min, the radioiodination products were analyzed and purified by radio-HPLC. Biodistribution study. Biodistribution of radioiodinated c(RGDyC) were performed in normal ICR mice. The tracers were obtained by using this strain-release method and Iodogen method (see Supporting Information) respectively and purified by HPLC. About 0.185 MBq 131I-c(RGDyC) (in 100 µL saline solution) was injected through tail vein. At 30 min, 90 min, and 240 min after injection, mice (n=5 at each time point) were sacrificed, the tissues and organs of interest were collected, wet-weighted and counted in a γ-counter. The percentage injected dose (%ID/g and %ID) were calculated accordingly and expressed as mean ± standard deviation (n=5). SPECT imaging. 125I-c(RGDyC) was dissolved in phosphate buffer (0.05 M, pH 7.4) and passed through 0.22-µm Millipore filter. SPECT/CT imaging of male BALB/c nude mice bearing U87MG tumor were performed at 30 min, 1 h and 2 h after intravenously injected about 18.5 MBq 125I-c(RGDyC). The SPECT imaging parameter was 250 sec/frame. All images were performed in the same manner and reconstructed, processed and analyzed with Nucline software (Mediso Medical Imaging System).

ASSOCIATED CONTENT


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Bioconjugate Chemistry Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org. Synthesis route, optimization condition, cell culture and animal model, HPLC analysis method, NMR and mass spectra.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]

ORCID Xianzhong Zhang: 0000-0002-1001-1884 Xiaoyuan Chen: 0000-0002-9622-0870

ACKNOWLEDGMENT This research was supported by the National Key Basic Research Program of China (2014CB744503), the National Natural Science Foundation of China (81471707, 21271030), and the Scientific Research Foundation of State Key Laboratory of Molecular Vaccinology and Molecular Diagnostics (2016ZY002).

NOTES The authors declare no competing financial interest.

ABBREVIATION SIB, succinimidyl p-iodobenzoate; HPLC, high performance liquid chromatography; CAQK, cysteine-alanine-glutamine-lysine; RGDyC, arginine-glycine-(aspartic acid)-(D-tyrosine)-cysteine; DMF, N,N-dimethylformamide; SPECT, single photon emission computed tomography; RCY, radiochemical yield; FBS, fetal bovine serum; TLC, thin layer chromatography; NMR, nuclear magnetic resonance; ESI, electrospray ionization; mCPBA, mchloroperoxybenzoic acid; THF, tetrahydrofuran; %ID, percentage injected dose per organ; %ID/g, percentage injected dose per gram

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(2016) A peptide for targeted, systemic delivery of imaging and therapeutic compounds into acute brain injuries. Nat Commun 7, 11980.


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Bioconjugate Chemistry

Highly Efficient and Stable StrainRelease Radioiodination for Thiol Chemoselective Bioconjugation

A novel heterobifunctional agent for thiol selective radioiodination based on strain-release reaction was developed and demonstrated in thiol-contained amino acid and peptide radioiodine labelling successfully.


Highly efficient and stable strain-release radioiodination for thiol

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