English

• 1.   Introduction

  Serving as one of the fundamental elements of genetic materials in organisms, phosphorus plays an irreplaceable role in the physiological activity of organisms. At molecular level, due to their unique chemistry, phosphorus-containing compounds participate in many physiological processes such as ATP-assisted enzyme reactions, posttranslational modifications of proteins and transmembrane transport. Penta-coordinated phosphorus as key important intermediate involved in many biological process, such as phosphoryl group transfer,^[1] ATP hydrolysis,^[2] RNA hydrolysis via internal transesterification,^[3] RNA self-splicing and twister ribozyme catalysis.^[4] Recently, the discovery of RNA fusion pairs could be formed with direct RNA-RNA interactions to regulate the specific gene transcriptions.^[5] Furthermore, it was found that RNA fusion was very closely to the development of human desease such as cancer. However, the molecular mechanisms of RNA hydrolysis and fusion with ligands exchange are unknown and should be understand firstly by using small chemical models, for example the penta-coordinated phosphorus compounds. Indeed, most of the phosphorus-containing bioactivity compounds which exist in a tetra-coordinated P(4) state would be activated firstly to produce penta- or hexa-coordinated phosphorus with or without enzyme catalysis in order to produce new structures with diverse functions. However, it is very difficult to synthesize and identify these very unstable intermediates under biological aqueous conditions. Decades of experimental and computational work has been made in order to determine the intrinsic chemistry of penta-coordinated phosphorus for the biological catalysis.^[6] For example, metal fluorides (MF_x), such as AlF₃^- and AlF₃, have been widely used as ligands with trigonal bipyramidal complexes to mimic the grometry of the phosphorus transition state for phosphoryl transfer process.^[7] Several computational models proposed with five-coordinate intermediates showed associative-type phosphoryl transfers mechanisms, such as the tyrosyl-DNA phosphodiesterase I (Tdp1),^[8] and phospholipase D superfamily with the five-coordinate phosphohistidine intermediate which could catalyze the cleavage of the headgroup of phosphatidylcholine to produce phosphatidic acid and choline.^[9] In order to systematically understand the intrinsic chemistry of penta-coordinated phosphorus, in this work we are trying to synthesize these model compounds with controllable stability and then investigate them by using ³¹P NMR.

  Oxyphosphoranes, especially penta-coordinated P(5) compounds, have been successfully prepared and identified by several groups.^{[10, 11]} The relatively stable trigonal bipyramidal structure endows oxyphosphoranes with the possibility of being the intermediates in the displacement reaction of biochemically important pyrophosphates (tetra-coordinated phosphorus compounds) such as AMP, ADP, ATP and phospholipid. In 1975, Ramirez's group^[12] set up an oxyphosphorane model to simulate the intermediate in the hydrolysis of ATP. From that time on, several groups have reported similar P(5)-centered chemical models for the study of biochemical reactions.^[13] In the past decades, Ramirez's group and other scientists have successfully prepared various penta-coordinated oxyphosphoranes stabilized by diketone ligands. Moreover, the alcoholysis of oxyphosphoranes has also been discovered.^[14] Herein, we identify an interesting intermolecular ligand-exchange reaction of one pentaoxyphosphorane for producing all three penta-coordinated phosphoranes with the combinatorial ligands under mild conditions with base catalysis, which might set up a chemical model for the possible intermediates of RNA hydrolysis and fusion (Scheme 1A).

  Scheme 1

  

  Scheme 1.  (A) Biological models of penta-coordinate phosphorus intermediate, and (B) chemical structure of penta-coordinated phosphorus with ¹H-coulpled ³¹P NMR spectra

  Molecular structure of compound c₃ with the aniso-tropic displacement parameters depicted at a 50% probability level. The hydrogen atoms are omitted for clarity. Selected bond lengths (Å and angles (°): P(1)—O(1) 1.767(1), P(1)—O(2) 1.573(1), P(1)—O(3) 1.656(1), P(1)—O(4) 1.609(1) P(1)—O(5) 1.573(1); O(1)—P(1)—O(3) 87.71(6), O(3)—P(1)—O(5) 123.85(6). The insert are ¹H-³¹P NMR spectra.

  下载: 全尺寸图片 幻灯片

  2.   Results and discussion

  Four penta-oxy phosphoranes (ab₂, a₃, b₃, and c₃) have been prepared with five-member cyclic structures as colorless crystal in high yields after recrystallization in toluene (Scheme 1B). The chemical structures were determined by using NMR for ab₂, a₃, b₃ and single crystal XRD analysis for c₃. For example, ab₂ with one methoxyl and two phenoxyl ligands was determined at ³¹P NMR δ -55 with four peaks (t), indicating that three hydrogen atoms are adjacent to phosphorus center. Compound ab₂ was stable at room temperature in anhydrous toluene solution without new peak formed (Figure 1A). In contrast, when 10% equivalent pyridine was added into the ab₂ solution, two new peaks at δ -51 and -60 were quickly formed with different ratios (Figure 1B). After 24 h incubation at rt, the reaction reached its equilibrium and the third novel peak was produced at δ -47 ppm with about 0.1% (a₃) as shown in Figure 1D. In order to confirm their structures,³¹P NMR tests with ¹H-coupling were performed and three new penta-oxy phosphoranes, namely a₃, a₂b and b₃ (s), could be simultaneously generated from the ab₂ with different ratio without side reaction under mild conditions. For example, ab₂ shows a quartet with J_H-P＝14.5 Hz in ¹H-coupled ³¹P NMR, indicating that there are only one methyl group linked to the phosphorus center of ab₂. Meanwhile, a₂b displays a heptet with J_H-P＝14.4 Hz in ¹H-coupled ³¹P NMR, indicating that there are two methyl groups linked to the phosphorus center of a₂b. The cleavage and formation of P—O ester bonds are atom economic with high efficiency. The diversities of chemical structures could be obtained for deserving more functions and information.

  Figure 1

  

  Figure 1.  Time-dependent, stacked ³¹P NMR spectra for the reaction of ligand exchange of ab₂ (250 mmol/L) with 10% pyridine in D₆-benzene solution at 25 ℃ for 24

  下载: 全尺寸图片 幻灯片

  In order to identify the ligand-exchange mechanism, a₃ with three methoxyl groups (³¹P NMR δ -45) was treated with b₃ with three phenol groups (³¹P NMR δ -60) in a ratio of 1:1 in C₆D₆ solution and monitored in situ by using ³¹P NMR. However, no new peaks could be formed even after 48 h incubation (Figure 2A). Addition of extra Lewis base will promote the transformation of penta-oxy phosphorus compounds.^[13] Thus, 10 mol% equivalent fresh distilled pyridine was added into the reaction mixture. The formation of a₂b and ab₂ could be obtained immediately in the ³¹P NMR spectrum (Figure 2B). After 12 h, two singlets at δ -51 and -55 were obtained, which could be identified as a₂b and ab₂, respectively. The NMR spectra of a₂b and ab₂ were also confirmed by comparing with authentic samples. The reaction proceeded smoothly with the addition of pyridine and more than 80% of a₃ and b₃ were transformed into a₂b and ab₂ after 24 h incubation. Other Lewis base such as 2, 6-lutidine showed similar behavior under the same reaction conditions. Similarly, c₃ with three ethoxyl groups could also smoothly exchange ligands with b₃ to form c₂b and cb₂ under base catalysis. The above results suggested that intermolecular ligand exchanges were the intrinsic reaction of penta-oxy phosphoranes with the economic cleavage and formation of the P—O ester bonds under base catalysis (Scheme 2).

  Figure 2

  

  Figure 2.  Time-dependent, stacked ³¹P NMR spectra for the reaction of ligand exchange between a₃ (250 mmol/L) and b₃ (250 mmol/L) without base (A) or with 10% pyridine (B) for 48 h

  下载: 全尺寸图片 幻灯片

  Scheme 2

  

  Scheme 2.  Ligand exchange of penta-coordinated phosphoranes with base catalysis

  下载: 全尺寸图片 幻灯片

  To determine the ligand exchange whether or not a unique phenomenon for penta-coordinated phosphorus, a₃ was treated with triphenyl phosphate 1b in a C₆D₆ solution for 5 d at 25 ℃, no exchange product was detected even with the catalysis of 2, 6-lutidine. Based on the experimental results, we assumed that the d_sp3 orbitals of penta-coordinated phosphorus were key to ligand exchange. Thus, the regular tetra-coordinated phosphate with tetrahedron sp³ orbitals could not exchange ligands with themselves or other d_sp3 orbitals of phosphoranes.

  To understand the mechanism, the reaction of a₃ (1 equiv.) with 2, 6-lutidine (1 equiv.) in anhydrous C₆D₆ solution was investigated. The reaction mixture was monitored by ³¹P NMR (Figure 3A). Two new peaks at δ 3 (2a) and -93 (1a) were obtained immediately with the gradient disappearing of a₃. 1a displays a heptet with J_H-P＝15.2 Hz in ¹H-coupled ³¹P NMR, indicating that there are two methyl groups linked to the phosphorus center. The singlet -93 could be postulated as an intermediate with a penta-coordinated phosphorous center as shown in Scheme 3. To determine the possible structures, 2D ¹H-³¹P HMBC NMR experiment was performed (Figure 3B). The signal at δ -93 was correlated to the protons at δ 3.76 with J_P—H＝15.2 Hz, which usually belongs to the penta-coordinated phosphorane. While the signal at δ 3 of the ³¹P NMR was correlated to the protons at δ 3.32 with J_P-H＝11.6 Hz, which usually belongs to the tetrahedron phosphates. Furthermore,¹H NMR showed that two sets of spectra with specific integrated areas were well corresponded to 1a with penta-coordinated phosphorus (Figure 3C) as compared with the ¹H NMR spectrum of the authentic 2, 6-lutidine (Figure S3, Supporting Information). In conclusion, the new signals at δ -93 could be a penta-coordinated phosphorane 1a, while the peak at δ 3 is a tetra-coordinated phosphate.

  Figure 3

  

  Figure 3.  (A) Time-dependent, stacked ³¹P NMR spectra for the reaction of a₃ with 1 equiv. of 2, 6-lutidine in D₆-benzen solution at 25 ℃ for 24 h, (B) ¹H-³¹P HMBC NMR spectrum of the mixture of a₃ with 1 equiv. of 2, 6-lutidine after 24 h, and (C) ¹H NMR spectrum of reaction mixture of a₃ with 2, 6-lutidine (1 equiv.) for 24 h

  The marked peaks with integration area are assigned to 1a with ³¹P NMR signal at δ-93

  下载: 全尺寸图片 幻灯片

  Scheme 3

  

  Scheme 3.  Possible mechanism of ligand exchange of penta-coordinated phosphoranes

  下载: 全尺寸图片 幻灯片

  Inspired by the experimental results, a possible mechanism was proposed as shown in Scheme 3. The starting material a₃ firstly reacted with pyridine or 2, 6-lutidine to produce a transition states or intermediate with hexa-coordinated phosphorus center.^[15] Then a slow demethylation of the unstable hexa-coordinated phosphorus afforded the ionic intermediate 1a as methyl group donor, which was in turn quickly nucleophilically attacked by another penta-coordinated ionic intermediate, leading to the intra-molecular ligand exchange. Meanwhile, phosphate 2a could be formed with the ring opening reaction of five-membered cycle when no other penta-coordinated phosphorus compounds were afforded.

  3.   Conclusions

  In this work, we provide a new chemical model of penta-coordinated phosphoranes with inter-molecular ligand exchange. In general, the experimental results demonstrated that the first generation compounds with ligands a₃ and b₃, could be hybridized and produced the second generation of compounds containing the mixed ligand units. The results show that penta-oxy phosphoranes a₃/b₃ or c₃/b₃ could inter-exchange their ligands of a/b or c/b to produce new chemical species with high efficiency. The self-ligand exchange within penta-oxy phosphorane ab₂ molecules could also produce three penta-oxy phosporanes with all of the ligands combination. Therefore, we wondered that if the uni-molecule ab₂, containing hetero-structure information could play the role of chemical information carrier. Furthermore, the reactivity of penta-oxy phosphorane could also be considered as a plausible chemical model for the study of the intermediates for phosphoryl group transfer, nucleoside hydrolysis, and RNA fusion at molecular level. It is possible that the gene transcription processes are closely related to RNA editing through the phosphate ligand exchange of the genetic bits. We then proposed that the ligand exchange reaction of the penta-coordinated phosphorus might be a general chemistry for translation of chemical information into biological signals as the phosphorus has done in living systems.

  4.   Experimental

  4.1   General

  All reactions were carried out in a glove box with Ar. Unless otherwise noted, all reagents were obtained from commercial suppliers and used without further purification.³¹P NMR (165 MHz),¹H NMR (400 MHz) and ¹³C NMR (100 MHz) spectra were measured on a Bruker AVIII 400M.

  4.2   General preparation for the oxy-phosphoranes

  Oxyphosphoranes were prepared from the corresponding trialkyl phosphite with 1 equiv. of tetrachloro-o-benzo-quinone in the toluene solution (Scheme 4). The oxyphosphoranes were obtained as colorless crystal in high yield after recrystallization from toluene. The molecular structure of c₃ was further characterized by single crystal XRD analysis.

  Scheme 4

  

  Scheme 4.  Preparation of oxyphosphoranes

  下载: 全尺寸图片 幻灯片

  Penta-oxy phosphorane (a₃): Colorless solid; ¹H NMR (C₆D₆, 400 MHz) δ: 3.33 (d, J_H-P＝13.6 Hz, 9 H); ¹³C NMR (C₆D₆, 100 MHz) δ: 140.8 (d, J_C-P＝6.7 Hz), 124.4, 114.4 (d, J_C-P＝17.35 Hz), 56.0, (d, J_C-P＝11.36 Hz); ³¹P NMR (C₆D₆, 164 MHz) δ: -45.

  Penta-oxy phosphorane (b₃): Colorless solid; ¹H NMR (C₆D₆, 400 MHz) δ: 7.06 (d, J＝8.1 Hz, 6H), 6.97 (t, J＝7.7 Hz, 6H), 6.83 (t, J＝7.2 Hz, 3H); ¹³C NMR (C₆D₆, 100 MHz) δ: 153.6 (d, J_C-P＝12.9 Hz), 140.3 (d, J_C-P＝6.4 Hz), 129.7, 125.4, 125.1 (d, J_C-P＝1.9 Hz), 121.1 (d, J_C-P＝5.2 Hz), 114.8 (d, J_C-P＝18.0 Hz); ³¹P NMR (C₆D₆, 164 MHz) δ: -60.

  Penta-oxy phosphorane (ab₂): Colorless solid; ¹H NMR (C₆D₆, 400 MHz) δ: 7.00~6.96 (m, 8 H), 6.84~6.81 (m, 2H), 3.43 (d, J＝14.3 Hz, 3H); ¹³C NMR (C₆D₆, 100 MHz) δ: 153.8 (d, J_C-P＝12.4 Hz), 140.4 (d, J_C-P＝6.0 Hz), 129.6 (d, J_C-P＝1.3 Hz), 125.1, 124.6 (d, J_C-P＝1.8 Hz), 121.3 (d, J_C-P＝5.3 Hz), 114.7 (d, J_C-P＝17.8 Hz); ³¹P NMR (C₆D₆, 164 MHz) δ: -55.

  Penta-oxy phosphorane (c₃): Colorless solid; ¹H NMR (C₆D₆, 400 MHz) δ: 3.88~3.82 (m, 6 H), 1.00 (t, J＝7.3 Hz, 9 H); ¹³C NMR (C₆D₆, 100 MHz) δ: 141.1 (d, J_C-P＝5.9 Hz), 124.2, 114.2 (d, J_C-P＝17.4 Hz), 65.0 (d, J_C-P＝11.5 Hz), 16.2 (d, J_C-P＝8.3 Hz); ³¹P NMR (C₆D₆, 164 MHz) δ: -48.

  SupportingInformation NMR spectral data for a₃, b₃, ab₂ and c₃, and crystal data for c3. The Supporting Information is available free of charge via the Internet at http://sioc-journal.cn/.

• 1. [1]

     Lassila, J. K.; Zalatan, J. G.; Herschlag, D. Ann. Rev. Biochem. 2011, 80, 669. doi: 10.1146/annurev-biochem-060409-092741

  2. [2]

     Petrovic, D.; Szeler, K.; Kamerlin, S. C. L. Chem. Commun. 2018, 54, 3077. doi: 10.1039/C7CC09504J

  3. [3]

     Guo, F. M.; Yue, Z. K.; Trajkovski, M.; Zhou, X. P.; Cao, D.; Li, Q.; Wang, B. F.; Wen, X.; Plavec, J.; Peng, Q.; Xi, Z.; Zhou, C. Z. J. Am. Chem. Soc. 2018, 140, 11893. doi: 10.1021/jacs.8b06313

  4. [4]

     (a) Messina, K. J.; Bevilacqua, P. C. J. Am. Chem. Soc. 2018, 140, 10578.
     (b) Wilson, T. J.; Liu, Y.; Domnick, C.; Kath-Schorr, S.; Lilley, D. M. J. Am. Chem. Soc. 2016, 138, 6151.

  5. [5]

     Nguyen, T. C.; Cao, X. Y.; Yu, P. F.; Xiao, S.; Lu, J.; Biase, F. H.; Sridhar, B.; Huang, N.; Zhang, K.; Zhong, S. Nat. Commun. 2016, 7, 12023. doi: 10.1038/ncomms12023

  6. [6]

     (a) Uraguchi, D.; Sasaki, H.; Kimura, Y.; Ito, T.; Ooi, T. J. Am. Chem. Soc. 2018, 140, 2765.
     (b) Yliniemela, A.; Uchimaru, T.; Tanabe, K.; Taira, K. J. Am. Chem. Soc. 1993, 115, 3032.
     (c) Westheimer, F. H. Acc. Chem. Res. 1968, 1, 70.

  7. [7]

     Jin, Y.; Richards, N. G.; Waltho, J. P.; Blackburn, G. M. Angew. Chem., Int. Ed. 2017, 56, 4110. doi: 10.1002/anie.201606474

  8. [8]

     DeYonker, N. J.; Webster, C. E. Biochemistry 2015, 54, 4236. doi: 10.1021/acs.biochem.5b00396

  9. [9]

     DeYonker, N. J.; Webster, C. E. J. Am. Chem. Soc. 2013, 135, 13764. doi: 10.1021/ja4042753

  10. [10]

      (a) Wittig, G.; Rieber, M. Justus Liebigs Ann. Chem. 1949, 562, 187.
      (b) Pajkert, R.; Röeschenthaler, G.-V. Organophosphorus Chem. 2017, 46, 323.
      (c) Swamy, K. C. K.; Kumar, N. S. Acc. Chem. Res. 2006, 39, 324.

  11. [11]

      (a) Hou, J. B.; Tang, G.; Guo, J. N.; Liu, Y.; Zhang, H.; Zhao, Y. F. Tetrahedron: Asymmetry 2009, 20, 1301.
      (b) Wang, T.; Zhang, P. B.; Hu, G. B.; Gao, Y. Z.; Wu, Y. L.; Xu, P. X.; Liu, Y.; Zhao, Y. F. ChemistrySelect 2018, 3, 7849.
      (c) Fu, H.; Li, Z. L.; Zhao, Y. F.; Tu, G. Z. J. Am. Chem. Soc.1999, 121, 291.
      (d) Ying, J. X.; Fu, S. S.; Li, X.; Feng, L. B.; Xu, P. X.; Liu, Y.; Gao, X.; Zhao, Y. F. Chem. Commun. 2018, 54, 8598.

  12. [12]

      (a) Ramirez, F. Acc. Chem. Res. 1968, 1, 168.
      (b) Ramirez, F.; Chaw, Y. F.; Marecek, J. F.; Ugi, I. J. Am. Chem. Soc. 1974, 96, 2429.

  13. [13]

      (a) Holmes, R. R. Acc. Chem. Res. 2004, 37, 746.
      (b) Timosheva, N. V.; Chandrasekaran, A.; Holmes, R. R. Inorg. Chem. 2006, 45, 3113.

  14. [14]

      (a) Ramirez, F.; Tasaka, K.; Desai, N. B.; Smith, C. P. J. Am. Chem. Soc. 1968, 90, 751.
      (b) Ramirez, F.; Loewengart, G. V.; Tsolis, E. A.; Tasaka, K. J. Am. Chem. Soc. 1972, 94, 3531.

  15. [15]

      Ramirez, F.; Marecek, J. F.; Okazaki, H. V. A. J. Am. Chem. Soc. 1976, 98, 5310. doi: 10.1021/ja00433a043

• 

  Scheme 1  (A) Biological models of penta-coordinate phosphorus intermediate, and (B) chemical structure of penta-coordinated phosphorus with ¹H-coulpled ³¹P NMR spectra

  Molecular structure of compound c₃ with the aniso-tropic displacement parameters depicted at a 50% probability level. The hydrogen atoms are omitted for clarity. Selected bond lengths (Å and angles (°): P(1)—O(1) 1.767(1), P(1)—O(2) 1.573(1), P(1)—O(3) 1.656(1), P(1)—O(4) 1.609(1) P(1)—O(5) 1.573(1); O(1)—P(1)—O(3) 87.71(6), O(3)—P(1)—O(5) 123.85(6). The insert are ¹H-³¹P NMR spectra.

  下载: 全尺寸图片 幻灯片
  

  Figure 1  Time-dependent, stacked ³¹P NMR spectra for the reaction of ligand exchange of ab₂ (250 mmol/L) with 10% pyridine in D₆-benzene solution at 25 ℃ for 24

  下载: 全尺寸图片 幻灯片
  

  Figure 2  Time-dependent, stacked ³¹P NMR spectra for the reaction of ligand exchange between a₃ (250 mmol/L) and b₃ (250 mmol/L) without base (A) or with 10% pyridine (B) for 48 h

  下载: 全尺寸图片 幻灯片
  

  Scheme 2  Ligand exchange of penta-coordinated phosphoranes with base catalysis

  下载: 全尺寸图片 幻灯片
  

  Figure 3  (A) Time-dependent, stacked ³¹P NMR spectra for the reaction of a₃ with 1 equiv. of 2, 6-lutidine in D₆-benzen solution at 25 ℃ for 24 h, (B) ¹H-³¹P HMBC NMR spectrum of the mixture of a₃ with 1 equiv. of 2, 6-lutidine after 24 h, and (C) ¹H NMR spectrum of reaction mixture of a₃ with 2, 6-lutidine (1 equiv.) for 24 h

  The marked peaks with integration area are assigned to 1a with ³¹P NMR signal at δ-93

  下载: 全尺寸图片 幻灯片
  

  Scheme 3  Possible mechanism of ligand exchange of penta-coordinated phosphoranes

  下载: 全尺寸图片 幻灯片
  

  Scheme 4  Preparation of oxyphosphoranes

  下载: 全尺寸图片 幻灯片
•