Suitable Stereostructures of vibrioferrin probes for iron uptake of Vibrio parahaemolyticus was revealed. Stereoisomers of dansyl labeled vibrioferrin at the 2′′-position were synthesized and their uptake activities were evaluated. Vibrio parahaemolyticus take in both isomers at the 2′′-position. In addition to Vibrio parahaemolyticus, several bacteria have also taken up the (R)-isomer

Inoue, Masaki

Karanjit, Sangita

Mera, Akane

Nakayama, Atsushi

Namba, Kosuke

Niwa, Kanji

Oda, Masataka

Okuda, Misa

Sato, Ryota

Sumida, Ryuichi

Tanaka, Naonobu

Tsutsumi, Tomohiro

English

Tokushima University Institutional Repository

1 Ryuichi Sumida,1 Misa Okuda,1 Akane Mera,1 Masaki Inoue,1 Karanjit Sangita,1 Kanji Niwa,1 Tomohiro Tsutsumi,1 Ryota Sato,1 Atsushi Nakayama,1 Naonobu Tanaka,1 Masataka Oda*2, Kosuke Namba*1 1Graduate School of Pharmaceutical Sciences, Tokushima University, 1-78-1 Shomachi, Tokushima 770-8505, Japan 2 Control of Innate Immunity, Collaborative Innovation Partnership, 761-0301, Kagawa, Japan. E-mail: namba@tokushima-u.ac.jp; masataka.oda@gmail.comSuitable Stereostructures of vibrioferrin probe for iron 1 uptake of Vibrio parahaemolyticus was revealed. 2 Stereoisomers of dansyl labeled vibrioferrin at the 2’’-3 position were synthesized and their uptake activities were 4 evaluated. Vibrio parahaemolyticus take in both isomers at 5 the 2’’-position. In addition to Vibrio parahaemolyticus, 6 several bacteria have also taken up the (R)-isomer. 7 Keywords: vibrioferrin, siderophore, fluorescent probe 8 Iron is an essential element for all creatures on the 9 earth. However, iron is mainly existing as water insoluble 10 trivalent iron salts under the current global environment of 11 high oxygen partial pressure, and many lives suffer from 12 iron deficiency.1 To acquire trivalent iron efficiently, 13 poaceous plants and many bacteria have developed unique 14 strategy characterized by the secretion of efficient iron 15 chelators called siderophore and take in iron as its complex 16 through the specific transporters.2 Mugineic acid (MA) was 17 the first identified phytosiderophore from the roots of barley 18 in 1972 by Takagi3 (Figure 1), and we succeeded in 19 development of the fluorescent probes of MA that could be 20 visualized the state being incorporated into the cells 21 overexpressing MA•Fe(III) complex transporter in 2010.4 22 On the other hand, vibrioferrin (VF) was initially isolated as 23 a siderophore of Vibrio parahaemolyticus, a type of 24 halophilic Gram-negative bacillus, by Yamamoto in 1992 25 (Figure 1).5 The same group later revealed the structure of 26 VF by chemical degradation analysis in 1994, 27 demonstrating that the C2’’’ position is epimeric mixture 28 and the configuration at the C2’’ position is S.6 In addition, 29 the chemical synthesis of VF by Takeuchi et al. revealed 30 that the configuration at the C2 position is R and its epimer 31 is inactive.7 However, the effect of the configuration at the 32 C2’’ position for iron uptake has been still unclear. In the 33 case of phytosiderophore MA, the epimer at the C2’ 34 position also showed similar iron transport activity to the 35 natural MA, and the fluorescent probe that connected the 36 fluorophore via the hydroxy group of the C2’ epimer 37 visualized cellular uptake.4 Recently, Wang’s group 38 reported an excellent fluorescent probe of VF for selective 39 targeting of vibrios, in which the fluorophore was linked 40 from the methyl group at the C2’’ position.8 Based on our 41 experience of fluorescent siderophores,4,9 we considered that 42 the epimer at the C2’’ position of VF would be also suitable 43 stereoisomer for introducing various labelling groups.     44 45 46 Figure 1. Structure of mugineic acid (MA) and vibrioferrin (VF) 47 First, we analyzed the conformation of iron complex of 48 VF by DFT calculation (Figure 2). The iron complexes of 49 possible epimers at C2’’’ and C2’’ positions were optimized. 50 The DFT analysis indicated that the epimer (R 51 configuration) at the C2’’ position, with R configuration at 52 the C2’’’ position, was the most stable structure (Complex 53 A) among four stereoisomers (Complexes A~D), suggesting 54 that the introduction of functional molecules such as 55 fluorophores to the methyl moiety of the epimer at the C2” 56 position likely provide a more stable iron complex than that 57 of natural form (Table 1). On the other hand, since bacteria 58 recognize their siderophore-iron complexes, the uptake 59 activity cannot be discussed solely by the stability of the 60 complexes. Therefore, to elucidate the suitable stereoisomer 61 of VF for probe development, we attempted to synthesize 62 the fluorescent probes of both stereoisomers of VF and 63 evaluate their incorporation into bacteria. 64  65 66 67 Figure 2. Optimized conformations of the iron complexes of VF and its epimer at the C2” position. 68 69 Vib.Fe(III)complex Complex A (R,R) Complex B (S,R) Complex C (R,S) Complex D (S,S) A suitable stereoisomer of vibrioferrin probes for iron uptake of Vibrio parahaemolyticus The final authenticated version is available online at: https://doi.org/10.1246/cl.2301662 E -1756.971694 -1756.956869 -1756.967251 -1756.962554ΔE (kcal/mol) 0 9.3 2.8 5.7 Table 1. Optimized conformations of the iron complexes of VF and their relative energies 1 2 Although optically pure citric acid fragments of VF 3 have been obtained by optical resolution in the previous 4 synthesis of VF and its analogs, we adopted the asymmetric 5 synthesis this time because the epimer at the C2 position 6 was known to be inactive. The chiral allyl dioxolanone 2 7 was obtained by the asymmetric allylation reaction of 1 8 derived from L-malic acid.10,11 Removal of acetal followed 9 by tert-butyl esters formation afforded the masked citric 10 acid 3. Finally, oxidative cleavage of the terminal olefin and 11 subsequent Pinnick oxidation12 of the resulting aldehyde 12 afforded the desired desymmetrized citric acid 4 (Scheme 1). 13  14 15 Scheme 1. Asymmetric synthesis of the citric acid fragment 4.  16 With reference to the Wang’s synthesis,8 we 17 synthesized a protected VF analog 7, in which the L-alanine 18 fragment of VF was replaced with L-lysine. Starting with 19 protected L-lysine L-5, sequential operation of a 20 condensation with aminoethanol, a protection with alloc 21 group, removal of Boc group, and a condensation with 22 ketoglutaric acid analog afforded L-6 in 52% yield from L-5. 23 Next, removal of alloc group followed by a condensation 24 with desymmetrized citric acid 4 gave the desired L-7. 25 While Wang’s group introduced a fluorescein by the 26 formation of propargyl carbamate with the primary amine in 27 the lysine moiety and subsequent click reaction13, we tried 28 to directly introduce organic fluorophores because each 29 fragment possessed different protecting groups. As the 30 organic fluorophore, a dansyl group was adopted due to its 31 good stability and fluorescence intensity. After 32 hydrogenation of L-7, the resulting free amine was 33 condensed with dansyl chloride followed by treatment with 34 TFA for removal of TMSE and tert-butyl groups to give 35 dansyl labeled VF L-8 as a 1:1 diastereomeric mixture at the 36 C2’’’ position (Scheme 2). To elucidate the effect of the 37 C2’’ position, we similarly synthesized the D-8 as the 38 epimer at the C2’’ position from D-lysine (Scheme 2). 39 Although diastereomers at the C2’’’ position could be 40 separated by PTLC before the deprotection leading to D-8, 41 the separated diastereomers were readily epimerized back to 42 diastereomeric mixtures.   43 44 Scheme 2. Synthesis of dansyl-VF analogs.  45 Having prepared fluorescent labeled VF analogs, the 46 uptake of these compounds into cells was evaluated (Figure 47 3). To 100 µL of a solution of Vibrio parahemolyticus strain 48 in culture medium was added 2 µL of a solution of iron 49 complex of L-8, which was prepared by treatment of L-8 50 with 1.0 equiv of FeCl3•6H2O in H2O (30 mM). The mixture 51 was incubated at room temperature for 5 min. Then, 10 µL 52 of the mixture was dropped on a glass slide and 53 fluorescently observed by confocal laser microscope. Vibrio 54 parahaemolyticus treated with dansyl labeled L-8 showed 55 fluorescence along the bacterial shape (Figures 3a-b), and it 56 means that L-8 was recognized as a siderophore and 57 incorporated into Vibrio parahaemolyticus. As reported by 58 Wang, the introduction of a fluorophore from the methyl 59 group at the C2’’ position was confirmed to be effective. 60 Next, we investigated the effect of the stereocenter at the 61 C2” position by using D-8. The bacteria treated with the 62 iron complex of D-8 also showed fluorescence similar to 63 that of L-8 (Figures 3c-d). Therefore, both stereoisomers at 64 the C2’’ position were found to be incorporated into bacteria 65 as the DFT analysis suggested. Furthermore, treatment of 66 only D-8 without iron source did not show fluorescence 67 from bacteria (Figures 3e-f), suggesting that the bacteria 68 recognized the iron complex of D-8, i.e., stereoisomer at the 69 3 C2” position was also recognized as their siderophore. 1 Moreover, treatment of only methanol and saline did not 2 show fluorescence, indicating that the fluorescence in 3 bacteria was not from cell death or autofluorescence 4 (Figures 3g and 3h). 5  6 7 Figure 3. Evaluation of labelled VF analogs for uptake of Vibrio 8 parahaemolyticus. Living bacteria solution (100 µL) in culture medium 9 was treated with 2 µL of various solutions such as 0.03 M solution of 10 Fe(III)•L-8 complex in H2O (a: bright field, b: fluorescence), 0.028 M 11 solution of Fe(III)•D-8 complex in MeOH (c: bright field, d: 12 fluorescence),  0.028 M solution of only D-8 in MeOH (e: bright field, 13 f: fluorescence), Saline (g: fluorescence) and MeOH (h: fluorescence). 14 The uptake of labelled VF analogs was monitored by confocal 15 microscopy (EVOS FL; Thermo) in a fluorescence image with 16 excitation and emission wavelengths of 360 and 447 nm, respectively 17 (b, d, f, g, h). Scale bar = 10 µm. 18 Next, we investigated whether the epimeric analog D-8 19 could be taken up by other bacteria as well as Vibrio 20 parahaemolyticus (Figure 4). Various bacteria were treated 21 with Fe(III)•D-8 complex under the similar condition and 22 observed by the confocal laser microscope. Interestingly, 23 while fluorescence was observed in Staphylococcus aureus 24 and Porphyromonas gingivalis (Figures 4a-d), E. coli and 25 Pseudomonas aeruginosa did not show fluorescence 26 (Figures 4e-h), demonstrating that there are bacteria that 27 take in Fe(III)•D-8 and bacteria that do not. In particular, 28 despite P. gingivalis is known not to produce siderophores,14 29 it strongly incorporated Fe(III)•D-8. The mechanistic 30 elucidation of these bacterial selectivity is the next subject. 31  32 33 Figure 4. Evaluation of labelled VF analogs for uptake of bacteria 34 other than Vibrio parahaemolyticus. 2 µL of 0.03 M solution of 35 Fe(III)•D-8 complex in H2O were applied to various living bacteria 36 solutions (100 µL) including Staphylococcus aureus (a: bright field, b: 37 fluorescence), Porphyromonas gingivalis (c: bright field, d: 38 fluorescence), E. coli (e: bright field, f: fluorescence), and 39 Pseudomonas aeruginosa (g: bright field, h: fluorescence). The uptake 40 of Fe(III)•D-8 was monitored by confocal microscopy (EVOS FL; 41 Thermo) in a fluorescence image with excitation and emission 42 wavelengths of 360 and 447 nm, respectively (b, d, f, h). Scale bar = 10 43 µm. 44 In conclusion, we demonstrated that the epimer at the 45 C2’’ position is also useful VF-based probe. We established 46 the asymmetric synthesis of citric acid fragment 4 and 47 applied it to the synthesis of VF analogs, in which L-alanine 48 fragment was replaced by L-lysine and D-lysine. The dansyl 49 group as the fluorophore was directly introduced to the 50 primary amine moiety in lysine fragment, and subsequent 51 global deprotection afforded the fluorescent probe of VF as 52 the diastereomeric mixture at the C2’’’ position. Both 53 analogs L-8 and D-8 derived from L-lysine and D-lysine 54 4 respectively were clearly showed fluorescence in bacteria. 1 Indeed, application of D-8 was able to fluorescently 2 visualize the movement of bacteria vibrio parahaemolyticus. 3 This study revealed that the D-lysine analog of VF that 4 corresponds to the epimer at the C2’’ position is also 5 suitable probe for the mechanistic investigation of VF in the 6 future.   7  8 This work was partially supported by JSPS KAKENHI 9 Grant Numbers JP21K19051 and JP22H00352, Adaptable 10 and Seamless Technology transfer Program through Target-11 driven R&D (A-STEP) from Japan Science and Technology 12 Agency (JST) Grant Number JPMJTR214D, and the 13 Research Clusters program of Tokushima University (No. 14 2201004). 15 16 Supporting Information is available on 17 http://dx.doi.org/10.1246/cl.******. 18 References and Notes 19 1 (a) S. Mori, Curr. Opin. Plant Biol. 1999, 2, 250. (b) J. F. Briat,20 C. Curie, F. Gaymard, Curr. Opin. Plant. Biol. 2007, 10, 276. 21 2 (a) R. C. Hider, X. Kong, Nat. Prod. Rep. 2010, 27, 637. (b) J. 22 Chen. Y. Guo, Y. Lu, B. Wang, J. Sun, H. Zhang, H. Wang, 23 Marine Drugs, 2019, 17, 562.24 3 (a) S. Takagi, Soil. Sci. Plant. Nutr. 1976, 22, 423. (b) T.25 Takemoto, K. Nomoto, S. Fushiya, R. Ouchi, H. Kusano, H.26 Hikino, S. Takagi, Y. Matsuura, M. Kakudo, Proc. Jpn. Acad.27 1978, 54, 469.28 4 K. Namba, K. Kobayashi, Y. Murata, H. Hirakawa, T. Yamagaki,29 T. Iwashita, M. Nishizawa, S. Kusumoto, K. Tanino, Angew.30 Chem. Int. Ed. 2010, 49, 9956.31 5 S. Yamamoto, Y. Fujita, N. Okujo, C. Minami, S. Matsuura, S.32 Shinoda, FEMS Micobiol. Lett.  1992, 94, 181.33 6 (a) S. Yamamoto, N. Okujo, T. Yoshida, S. Matsuura, S. Shinoda, 34 J. Biochem. 1994, 115, 868. (b) S. Yamamoto, N. Okujo, S.35 Matsuura, I. Fujiwara, Y. Fujita, S. Shinoda, Microbiol. Immunol.36 1994, 38, 687.37 7 (a) Y. Takeuchi, T. Akiyama, T. Harayama, Chem. Pharm. Bull. 38 1999, 47, 459. (b) Y. Takeuchi, Y. Nagano, K. Toma, Y.39 Yoshikawa, T. Akiyama, H. Nishioka, H. Abe, T. Harayama, S.40 Yamamoto, Chem. Pharm. Bull. 1999, 47, 1284.41 8 P.-H. C. Chen, S.-Y. Ho, P.-L. Chen, T.-C. Hung, A.-J. Liang,42 T.-F. Kuo, H.-C. Huang, T.-S. A. Wang, ACS Chem. Biol. 2017, 43 12, 2720. 44 9 T. Hayashi, A. Osawa, T. Watanabe, Y. Murata, A. Nakayama, K.45 Namba, Tetrahedron Lett. 2017, 58, 1961.46 10 D. Seebach, R. Naef, G. Calderari, Tetrahedron, 1984, 40, 1313. 47 11 Y. Huang, Y.-B., Zhang, Z.-C. Chen, P.-F., Xu. Tetrahedron 48 Asymmetry, 2006, 17, 3152. 49 12 (a) G. A. Kraus, B. Roth, J. Org. Chem. 1980, 45, 4825. (b) B. S.50 Bal, W. E. Childers Jr, H. W. Pinnick, Tetrahedron, 1981, 37, 51 2091. 52 13 (a) H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. Int.53 Ed. 2001, 40, 2004. (b) V. V. Rostovtsev, L. G. Green, V. V. 54 Fokin, K. B. Sharpless, Angew. Chem. Int. Ed. 2002, 41, 2596. 55 (c) C. W. Tornøe, C, Christnesen, M. J. Meldal, J. Org. Chem. 56 2002, 67, 3057.57 14 (a) T. E. Bramanti, S. C. Holt, Biochem Biophys Res Commun, 58 1990, 166, 1146. (b) T. E. Bramanti, S. C. Holt, J. Bacteriol, 59 1991, 173, 7330.  60 

A suitable stereoisomer of vibrioferrin probes for iron uptake of Vibrio parahaemolyticus

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A suitable stereoisomer of vibrioferrin probes for iron uptake of Vibrio parahaemolyticus

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