Chemical Synthesis of an Asymmetric Mimic of the Nitrogenase Active Site

  • Kazuki TanifujiEmail author
  • Yasuhiro OhkiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1876)


The synthetic inorganic chemistry of metal–sulfur (M-S, M = metals) clusters has played an important, complementary role to the biochemical analyses of nitrogenase toward a better understanding of the enzyme active site. The active site of nitrogenase (designated the M-cluster) can be extracted from the protein in a solvent-stabilized form, [(cit)MoFe7S9C] (cit = (R)-homocitrate). One important finding of the extracted M-cluster is its catalytic activity toward the reduction of C1-substrates (CN, CO, CO2) into C1–C5 hydrocarbons in solution. This catalytic property poses challenges for chemists to reproduce the function with synthetic mimics, not only because of the biochemical interests but also due to the potential significance in green chemistry and catalysis research. In this context, our successful synthesis of an asymmetric Mo-Fe-S cluster, [Cp*MoFe5S9(SH)]3−, is one of the recent important achievements in synthetic M-S chemistry, as this cluster catalyzes the reduction of C1-substrates in a similar manner to the extracted M-cluster. Even though the synthetic protocol for this cluster has been described in the literature, there are plenty of pitfalls for researchers unfamiliar with synthetic M-S chemistry. In this chapter, we provide general precautionary statements and detailed protocols for the synthesis of [Cp*MoFe5S9(SH)]3−, with a brief discussion of the experimental tips based on the authors’ experience in both biochemical and synthetic chemical fields.

Key words

Synthetic mimics Iron–molybdenum–sulfur clusters Nitrogenase Asymmetry Air-free techniques 



This work was financially supported by Takeda Science Foundation, the Hori Sciences and Arts Foundation, and Grant-in-Aids for Scientific Research (No. 16H04116) from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (to Y.O.). The authors thank Dr. Nathaniel S. Sickerman and Lee Rettberg (University of California, Irvine) for proofreading and fruitful discussions.


  1. 1.
    Bulen WA, LeComte JR (1966) The nitrogenase system from Azotobacter: two-enzyme requirement for N2 reduction, ATP-dependent H2 evolution, and hydrolysis. Proc Natl Acad Sci U S A 56:979–986CrossRefGoogle Scholar
  2. 2.
    Lancaster KM, Roemelt M, Ettenhuber P et al (2011) X-ray emission spectroscopy evidences a central carbon in the nitrogenase iron-molybdenum cofactor. Science 334:974–977CrossRefGoogle Scholar
  3. 3.
    Spatzal T, Aksoyoglu M, Zhang L et al (2011) Evidence for interstitial carbon in nitrogenase FeMo cofactor. Science 334:940–940CrossRefGoogle Scholar
  4. 4.
    Shah VK, Brill WJ (1977) Isolation of an iron-molybdenum cofactor from nitrogenase. Proc Natl Acad Sci U S A 74:3249–3253CrossRefGoogle Scholar
  5. 5.
    Lee SC, Holm RH (2004) The clusters of nitrogenase: synthetic methodology in the construction of weak-field clusters. Chem Rev 104:1135–1157CrossRefGoogle Scholar
  6. 6.
    Ohki Y, Tatsumi K (2013) New synthetic routes to metal-sulfur clusters relevant to the nitrogenase metallo-clusters. Z Anorg Allg Chem 639:1340–1349CrossRefGoogle Scholar
  7. 7.
    Lee SC, Lo W, Holm RH (2014) Developments in the biomimetic chemistry of cubane-type and higher nuclearity iron-sulfur clusters. Chem Rev 114:3579–3600CrossRefGoogle Scholar
  8. 8.
    Holm RH, Lo W (2016) Structural conversions of synthetic and protein-bound iron-sulfur clusters. Chem Rev 116:13685–13713CrossRefGoogle Scholar
  9. 9.
    Sickerman NS, Tanifuji K, Hu Y et al (2017) Synthetic analogues of nitrogenase metallocofactors: challenges and developments. Chem Eur J 23:12425–12432CrossRefGoogle Scholar
  10. 10.
    Herskovitz T, Averill BA, Holm RH et al (1972) Structure and properties of a synthetic analogue of bacterial iron-sulfur proteins. Proc Natl Acad Sci U S A 69:2437–2441CrossRefGoogle Scholar
  11. 11.
    Wolff TE, Berg JM, Warrick C et al (1978) The molybdenum-iron-sulfur complex [Mo2Fe6S9(SC2H5)8]3−. A synthetic approach to the molybdenum site in nitrogenase. J Am Chem Soc 100:4630–4632CrossRefGoogle Scholar
  12. 12.
    Demadis KD, Campana CF, Coucouvanis D (1995) Synthesis and structural characterization of the new Mo2Fe6S8(PR3)6(Cl4-cat)2 clusters. Double cubanes containing two edge-linked [MoFe3S4]2+ reduced cores. J Am Chem Soc 117:7832–7833CrossRefGoogle Scholar
  13. 13.
    Osterloh F, Achim C, Holm RH (2001) Molybdenum-iron-sulfur clusters of nuclearities eight and sixteen, including a topological analogue of the P-cluster of nitrogenase. Inorg Chem 40:224–232CrossRefGoogle Scholar
  14. 14.
    Ohki Y, Ikagawa Y, Tatsumi K (2007) Synthesis of new [8Fe-7S] clusters: a topological link between the core structures of P-cluster, FeMo-co, and FeFe-co of nitrogenases. J Am Chem Soc 129:10457–10465CrossRefGoogle Scholar
  15. 15.
    Hashimoto T, Ohki Y, Tatsumi K (2010) Synthesis of coordinatively unsaturated mesityliron thiolate complexes and their reactions with elemental sulfur. Inorg Chem 49:6102–6109CrossRefGoogle Scholar
  16. 16.
    Ohta S, Ohki Y, Hashimoto T et al (2012) A nitrogenase cluster model [Fe8S6O] with an oxygen unsymmetrically bridging two proto-Fe4S3 cubes: relevancy to the substrate binding mode of the FeMo cofactor. Inorg Chem 51:11217–11219CrossRefGoogle Scholar
  17. 17.
    Kawaguchi H, Yamada K, Lang J et al (1997) A new entry into molybdenum/tungsten sulfur chemistry: synthesis and reactions of mononuclear sulfido complexes of pentamethylcyclopentadienyl-molybdenum(VI) and -tungsten(VI). J Am Chem Soc 119:10346–10358CrossRefGoogle Scholar
  18. 18.
    Tanifuji K, Sickerman NS, Lee CC et al (2016) Structure and reactivity of an asymmetric synthetic mimic of nitrogenase cofactor. Angew Chem Int Ed 128:15862–15865CrossRefGoogle Scholar
  19. 19.
    Lee CC, Hu Y, Ribbe MW (2012) ATP-independent formation of hydrocarbons catalyzed by isolated nitrogenase cofactors. Angew Chem Int Ed 51:1947–1949CrossRefGoogle Scholar
  20. 20.
    Rebelein JG, Hu Y, Ribbe MW (2014) Differential reduction of CO2 by molybdenum and vanadium nitrogenases. Angew Chem Int Ed 53:11543–11546CrossRefGoogle Scholar
  21. 21.
    Coucouvanis D, Kanatzidis M, Simhon E et al (1982) Synthesis, molecular structure, and reactions of bis(tetraphenylphosphonium) hexakis(μ-thiophenolato)- tetrachlorotetraferrate(II), (Ph4P)2[Fe4(SPh)6Cl4]. Its reactions with dibenzyl trisulfide and the synthesis of the [Fe4S4Cl4]2− and [Fe4S4(Cl)2(SC6H5)2]2− “cubane”-type clusters. J Am Chem Soc 104:1874–1882CrossRefGoogle Scholar
  22. 22.
    Millar S (2013) Tips and tricks for the lab: Air-sensitive techniques. Accessed 1 Sep 2017
  23. 23.
    Pangborn AB, Giardello MA, Grubbs RH et al (1996) Safe and convenient procedure for solvent purification. Organometallics 15:1518–1520CrossRefGoogle Scholar
  24. 24.
    Armarego WLF, Chai CLL (2013) Common physical techniques used in purification. In: Purification of laboratory chemicals, 7th edn. Butterworth-Heinemann, BostonGoogle Scholar
  25. 25.
    Demadis KD, Coucouvanis D (1995) Synthesis, structural characterization, and properties of new single and double cubanes containing the MoFe3S4 structural unit and molybdenum-bound polycarboxylate ligands. Clusters with a molybdenum-coordination environment similar to that in the iron-molybdenum cofactor of nitrogenase. Inorg Chem 34:436–448CrossRefGoogle Scholar
  26. 26.
    Tatsumi K, Inoue Y, Kawaguchi H et al (1993) Structural diversity of sulfide complexes containing half-sandwich Cp*Ta and Cp*Nb fragments. Organometallics 12:352–364CrossRefGoogle Scholar
  27. 27.
    Gladysz JA, Wong VK, Jick BS (1979) New methodology for the introduction of sulfur into organic molecules. Tetrahedron 35:2329–2335CrossRefGoogle Scholar
  28. 28.
    Murray RC, Blum L, Liu AH et al (1985) Simple routes to mono(η5-pentamethylcyclopentadienyl) complexes of molybdenum(V) and tungsten(V). Organometallics 4:953–954CrossRefGoogle Scholar
  29. 29.
    Clark DE (2001) Peroxides and peroxide-forming compounds. Chem Health Saf 8:12–22CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Molecular Biology and BiochemistryUniversity of California, IrvineIrvineUSA
  2. 2.Department of Chemistry, Graduate School of ScienceNagoya UniversityNagoyaJapan

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