Utilization of a hypoxia-responsive amino acid is indispensable in the preparation of hypoxic tumor-specific peptidyl prodrugs. Bioreduction of a nitro group is among the most attractive triggering reactions in the hypoxia-responsive prodrugs. In this paper, design and synthesis of a reduction-responsive amino acid that induces peptide bond cleavage after reduction of the nitro group are described. Application to hypoxia-responsive peptide bond cleavage system is also reported

Ebisuno, Koji

Hirakawa, Hiroko

Ishizawa, Keisuke

Miyamoto, Licht

Ogura, Keiji

Otaka, Akira

Shigenaga, Akira

Tsuchiya, Koichiro

Yamamoto, Jun

Tokushima University Institutional Repository

1 COMMUNICATIONSDOI: 10.1002/cbic.200((will be filled in by the editorial staff)) Hypoxia-responsive amino acid Development of Reduction-responsive Amino Acid that Induces Peptide Bond Cleavage in Hypoxic Cells Akira Shigenaga,*[a] Keiji Ogura,[a] Hiroko Hirakawa,[a] Jun Yamamoto,[a] Koji Ebisuno,[a] Licht Miyamoto,[b] Keisuke Ishizawa,[b] Koichiro Tsuchiya,[b] and Akira Otaka*[a] Bioreduction of a nitro group to an amine or a hydroxylamine is among the most attractive triggering reactions of antitumor prodrugs.[1,2] Prodrug 1, possessing a nitroarylmethyl group, can be enzymatically reduced to corresponding nitro anion radical 2 in living cells (Figure 1). However, anion radical 2 is usually oxidized by ROS (reactive oxygen species) to regenerate parent prodrug 1 in aerobic non-malignant cells. On the contrary, the oxidation of nitro radical 2 is suppressed in hyopxic solid tumor, because concentration of molecular oxygen in hypoxic cells is lower than that in non-malignant cells. Therefore, radical anion 2 can be converted to cytotoxic drug 3 via further reduction followed by removal of the arylmethyl group specifically in tumor cells.[2] On the other hand, antibody-directed enzyme prodrug therapy (ADEPT) and gene-directed enzyme prodrug therapy (GDEPT) evoke a great clinical interest for cancer therapy, and they also utilize prodrugs with the nitroarylmethyl protective group.[2,3] For ADEPT or GDEPT, a bacterial nitroreductase is assembled on a tumor by conjugating to a tumor-specific antibody or is expressed in tumor cells by use of a tumor-specific gene vector to achieve a tumor-specific release of cytotoxin 3, respectively. With these in mind, we decided to develop an NO2 reduction-responsive amino acid that induces peptide bond cleavage after reduction of the nitro group, because it would potentially facilitate preparation of hypoxia-responsive peptidyl prodrugs and bioprobes. Figure 1. Reduction-responsive antitumor prodrug possessing nitroarylmethyl group. (Ar: aromatic ring; R = H or OH; X: heteroatom) Scheme 1. Stimulus-responsive peptide bond cleavage. (PG: protective group removable by a corresponding stimulus.) Previously, we reported a stimulus-responsive amino acid[4,5] and its application to controlling peptidyl function in living cells.[4] When peptide 4, possessing the stimulus-responsive amino acid, was exposed to a corresponding stimulus, removal of PG (a protective group removable by the stimulus) followed by lactonization of trimethyl lock moiety[6] induced peptide bond cleavage (Scheme 1). In the present paper, development of the reduction-responsive amino acid and its application to a hypoxia-responsive peptide bond cleavage system are described. Because p-nitrobenzyl phenyl ether is known to be split under hypoxic conditions via reduction of the nitro group,[7] it was used as the PG group. Fmoc protected reduction-responsive amino acid 8, possessing the p-nitrobenzyl group, was synthesized as shown in Scheme 2. Phenol 5[8] was treated with p-nitrobenzyl bromide in the presence of K2CO3 to afford ether 6. The TBS group of 6 was then removed under acidic conditions. After two-step oxidation of generated alcohol 7, the Boc group was replaced with an Fmoc group to give reduction-responsive amino acid derivative 8. The amino acid derivative was then incorporated into model peptide 9 using Fmoc solid phase peptide synthesis (Fmoc SPPS) to demonstrate a capability to induce the reduction-responsive peptide bond cleavage. [a] Prof. Dr. A. Shigenaga, K. Ogura, H. Hirakawa, J. Yamamoto, K. Ebisuno, Prof. Dr. A. OtakaDepartment of Bioorganic Synthetic ChemistryInstitute of Health Biosciences andGraduate School of Pharmaceutical SciencesThe University of TokushimaShomachi, Tokushima 770-8505 (Japan)E-mail: ashige@ph.tokushima-u.ac.jp (A. Shigenaga)E-mail: aotaka@ph.tokushima-u.ac.jp (A. Otaka)[b] Dr. L. Miyamoto, Prof. Dr. K. Ishizawa, Prof. Dr. K. TsuchiyaDepartment of Medical PharmacologyInstitute of Health Biosciences andGraduate School of Pharmaceutical SciencesThe University of TokushimaShomachi, Tokushima 770-8505 (Japan)Supporting information for this article is available on the WWWunder http://www.chembiochem.org or from the author.This is the peer reviewed version of the following article: Shigenaga, A. , Ogura, K. , Hirakawa, H. , Yamamoto, J. , Ebisuno, K. , Miyamoto, L. , Ishizawa, K. , Tsuchiya, K. and Otaka, A. (2012), Development of a Reduction‐Responsive Amino Acid that Induces Peptide Bond Cleavage in Hypoxic Cells. ChemBioChem, 13: 968-971. doi:10.1002/cbic.201200141, which has been published in final form at https://doi.org/10.1002/cbic.201200141. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. 2  Scheme 2. Reagents and conditions. a) p-Nitrobenzyl bromide, K2CO3, DMF, 99%; b) AcOH aq., THF, quant.; c) PDC, DMF; d) NaClO2, 2-methyl-2-butene, NaH2PO4, acetone, H2O; e) HCl, AcOEt; f) FmocOSu, Na2CO3 aq., MeCN, 97% (4 steps); g) Fmoc SPPS. (A: alanine; D: aspartic acid; G: glycine; Q: glutamine; R: arginine; S: serine; Y: tyrosine). To examine whether a reduction of the nitro group triggers the peptide bond cleavage, chemical reduction of peptide 9 was performed as shown in Scheme 3. To peptide 9 in aqueous solution of NH4Cl was added a zinc powder under slightly acidic conditions, and the reaction mixture was incubated at 37 °C. During the reaction and HPLC purification under acidic conditions, removal of the p-aminobenzyl group of 10 was not observed. The obtained aniline 10 was incubated under physiological conditions (pH 7.4 of phosphate buffer, 37 °C). The reaction progress was monitored by HPLC, and the peptides were characterized by ESI-MS (Figure 2A). After 24 h of incubation, peptide 10 was completely converted to corresponding peptide fragments 12 and 13. Intermediate 11 was not observed during the reaction; therefore, we concluded that the rate determining step was the removal of the p-aminobenzyl group. The disappearance of peptide 10 showed first-order dependence on the concentration of starting material 10, and the half-life was determined as 3.1 h (Figure 2B). Because many tumor regions are known to be slightly acidic,[1] the half-life at pH 6.0 was also estimated, and was quite similar to that at pH 7.4 (t1/2 = 3.7 h at pH 6.0).  Scheme 3. Reagents and conditions. a) Zn, NH4Cl aq., 37 °C; b) Sodium phosphate buffer (pH 7.4 or 6.0, 20 mM), 37 °C.  ## Figure 2, see page 3 ## Next, we designed FRET (Fluorescence Resonance Energy Transfer)-based peptide 14 to demonstrate the capability of the reduction-responsive amino acid to induce peptide bond cleavage in living hypoxic cells (Scheme 4). Peptide 14 possesses a dabsyl group as a quencher, a fluoresceinyl group as a fluorophore,[9] and an octaarginine as a cell-penetrating peptide sequence.[10] Once added to a cell, peptide 14 should penetrate into the cell, but it does not show fluorescence because of quenching by FRET. Upon the peptide bond cleavage, strong fluorescence of generated peptide fragment 15 can be observed. Peptide 14 and reference peptide 16, possessing a tyrosine residue instead of the reduction-responsive amino acid, were synthesized using Fmoc SPPS (details are shown in Supporting Information). The peptide was incubated with Caco-2 cells under hypoxic (1% (v/v) O2 and 5% (v/v) CO2 in N2) or aerobic (5% (v/v) CO2 in air) conditions for 15 h, respectively. After exchanging medium followed by fluorescence microphotography, fluorescence intensity per cell area was calculated based on the fluorescence image. As shown in Figure 3, strong fluorescence was observed only when the cells were treated with peptide 14 under hypoxic conditions. Therefore, it was demonstrated that the reduction-responsive amino acid can induce the hypoxia-responsive peptide bond cleavage in living cells.  ## Scheme 4, see page 4 ##   Figure 3. Visualization of peptide bond cleavage in living cells. Caco-2 cells were incubated with 300 nM peptide 14 or 16 under hypoxic (1% (v/v) O2 and 5% (v/v) CO2 in N2) or aerobic (5% (v/v) CO2 in air) conditions for 15 h. A) Microscopic images after exchanging medium. FTC: Fluorescence images at λex = 460-490 nm and λem = 510 nm; B) Quantitative analysis of the fluorescence images. Fluorescence/cell area was average of 4 runs (10 cells/run), and statistical analyses were performed using Dunnett’s test (*p<0.001). Error bars are + s.d. In conclusion, we developed a reduction-responsive amino acid that induced peptide bond cleavage after reduction of a nitro group, and the cleavage products were obtained in high purity. Kinetic study of the peptide bond cleavage revealed that the half-life of peptide 10 at pH 6.0 was similar to that at pH 7.4. This suggests that the reduction-responsive amino acid could function even in acidic cancer cells. The capability of the reduction-responsive amino acid to induce hypoxia-responsive peptide  3 bond cleavage in living cells was then examined. Cell-based assay clarified that the peptide bond cleavage was efficiently induced only in the hypoxic cells. We believe that these results represent an indispensable step toward the development of hypoxia-responsive peptidyl prodrugs and bioprobes. Application of the reduction-responsive amino acid to a hypoxia-responsive antitumor prodrug is in progress. Acknowledgements This research was supported in part by a Grant-in-Aid for Young Scientist (B), Scientific Research (B), and Challenging Exploratory Research from the Japanese Society for the Promotion of Science (JSPS), and Scientific Research on Innovative Areas ‘Fusion Materials: Creative Development of Materials and Exploration of Their Function through Molecular Control’ (No. 2206) from the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT). The Takeda Science Foundation is also acknowledged. J.Y. is grateful for a SUNBOR Scholarship. Keywords: hypoxia-responsive · peptide bond cleavage · reduction-responsive · stimulus-responsive · trimethyl lock [1] Selected reviews: a) W. R. Wilson, M. P. Hay, Nat. Rev. Cancer 2011, 11, 393-410; b) H. Nagasawa, J. Pharmacol. Sci. 2011, 115, 446-452; c) S. Kizaka-Kondoh, S. Tanaka, H. Harada, M. Hiraoka, Adv. Drug Deliv. Rev. 2009, 61, 623-632; d) K. Tanabe, Z. Zhang, T. Ito, H. Hatta, S. Nishimoto, Org. Biomol. Chem. 2007, 5, 3745-3757; e) H. Nagasawa, Y. Uto, K. L. Kirk, H. Hori, Biol. Pharm. Bull. 2006, 29, 2335-2342. [2] Selected reviews: a) Y. Chen, L. Hu, Med. Res. Rev. 2009, 29, 29-64; b) H. Cerecetto, M. Gonzalez, M. L. Lavaggi, Med. Chem. 2006, 2, 315-327; c) W. A. Denny, Aust. J. Chem. 2004, 57, 821-828; d) W. A. Denny, Eur. J. Med. Chem. 2001, 36, 577-595. [3] Selected reviews: a) M. D. Roldan, E. Perez-Reinado, F. Castillo, C. Moreno-Vivian, FEMS Microbiol. Rev. 2008, 32, 474-500; b) D. Portsmouth, J. Hlavaty, M. Renner, Molec. Aspects Med. 2007, 28, 4-41. [4] A. Shigenaga, D. Tsuji, N. Nishioka, S. Tsuda, K. Itoh, A. Otaka, ChemBioChem 2007, 8, 1929-1231. [5] a) A. Shigenaga, H. Hirakawa, J. Yamamoto, K. Ogura, M. Denda, K. Yamaguchi, D. Tsuji, K. Itoh, A. Otaka, Tetrahedron 2011, 67, 3984-3990; b) A. Shigenaga, J. Yamamoto, Y. Sumikawa, T. Furuta, A. Otaka, Tetrahedron Lett. 2010, 51, 2868-2871; c) A. Shigenaga, J. Yamamoto, H. Hirakawa, K. Ogura, K. Morishita, N. Maeda, A. Otaka, Tetrahedron Lett. 2010, 51, 2525-2528. [6] a) K. L. Amsberry, R. T. Borchardt, J. Org. Chem. 1990, 55, 5867-5877; b) M. Caswell, G. L. Schmir, J. Am. Chem. Soc. 1980, 102, 4815-4821; c) S. Milstien, L. A. Cohen, J. Am. Chem. Soc. 1972, 94, 9158-9165; d) R. T. Borchardt, L. A. Cohen, J. Am. Chem. Soc. 1972, 94, 9166-9174; e) S. Milstien, L. A. Cohen, Proc. Natl. Acad. Sci. USA 1970, 67, 1143-1147; f) M. E. Jung, G. Piizzi, Chem. Rev. 2005, 105, 1735-1766 and references cited therein. [7] E. Nakata, Y. Yukimachi, H. Kariyazono, S. Im, C. Abe, Y. Uto, H. Maezawa, T. Hashimoto, Y. Okamoto, H. Hori, Bioorg. Med. Chem. 2009, 17, 6952-6958. [8] A. Shigenaga, J. Yamamoto, N. Nishioka, A. Otaka, Tetrahedron 2010, 66, 7367-7372. [9] A. Shigenaga, J. Yamamoto, H. Hirakawa, K. Yamaguchi, A. Otaka, Tetrahedron 2009, 65, 2212-2216. [10] I. Nakase, T. Takeuchi, G. Tanaka, S. Futaki, Adv. Drug Deliv. Rev. 2008, 60, 598-607 and references cited therein. Received: ((will be filled in by the editorial staff)) Published online: ((will be filled in by the editorial staff))    Figure 2. Reaction monitoring of the peptide bond cleavage of aniline 10 as shown in Scheme 3; A) HPLC profile of the peptide bond cleavage reaction. Analytical HPLC conditions: linear gradient of 0.1% (v/v) TFA/MeCN in 0.1% (v/v) TFA aq., 1-50% over 30 min; B) First order kinetic treatment of the data. Percentage of remaining substrate 10 was estimated based on HPLC peak area. Error bars are ± s.d.    4  Scheme 4. Design of FRET-based hypoxia-responsive peptide 14 and reference peptide 16. (G: glycine; K: lysine; R: arginine)  5 Entry for the Table of Contents (Please choose one layout)  COMMUNICATIONS  Utilization of a hypoxia-responsive amino acid is indispensable in the preparation of hypoxic tumor-specific peptidyl prodrugs. Bioreduction of a nitro group is among the most attractive triggering reactions in the hypoxia-responsive prodrugs. In this paper, design and synthesis of a reduction-responsive amino acid that induces peptide bond cleavage after reduction of the nitro group are described. Application to hypoxia-responsive peptide bond cleavage system is also reported.  A. Shigenaga,* K. Ogura, H. Hirakawa, J. Yamamoto, K. Ebisuno, L. Miyamoto, K. Ishizawa, K. Tsuchiya, A. Otaka* Page No. – Page No. Development of Reduction-responsive Amino Acid Which Induces Peptide Bond Cleavage in Hypoxic Cells      

Development of a Reduction‐Responsive Amino Acid that Induces Peptide Bond Cleavage in Hypoxic Cells

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Development of a Reduction‐Responsive Amino Acid that Induces Peptide Bond Cleavage in Hypoxic Cells

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