On the total synthesis of (S)-methanophenazine and the formal synthesis of (R)-methanophenazine from a common precursor

Article abstract of DOI:10.1016/S0040-4039(00)01669-5

Methanophenazine is a new cofactor from methanogenic archaea. (S)-Methanophenazine has been synthesized from three building blocks, i.e. 2-hydroxy-phenazine, ethyl (R)-3-methylglutarate and (E,E)-farnesyl acetone; a stereodivergent approach from ethyl (R)-3-methylglutarate warrants the formal synthesis of (R)-methanophenazine. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)01669-5

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 41 (2000) 9759–9763
On the total synthesis of (S)-methanophenazine and the
formal synthesis of (R)-methanophenazine from a common
precursor
Uwe Beifuss*,† and Mario Tietze
Institut fu¨r Organische Chemie der Georg-August-Universita¨t Go¨ttingen, Tammannstraße 2,
D-37077 Go¨ttingen, Germany
Received 29 August 2000; accepted 21 September 2000
Abstract
Methanophenazine is a new cofactor from methanogenic archaea. (S)-Methanophenazine has been
synthesized from three building blocks, i.e. 2-hydroxy-phenazine, ethyl (R)-3-methylglutarate and (E,E)-
farnesyl acetone; a stereodivergent approach from ethyl (R)-3-methylglutarate warrants the formal
synthesis of (R)-methanophenazine. © 2000 Elsevier Science Ltd. All rights reserved.
Keywords: electron transfer; natural products; nitrogen heterocycles; phenazines; terpenoids.
Methanogenesis is the terminal step in the degradation of complex organic compounds and is
essential for the mineralization of organic material. The formation of methane from simple
compounds such as hydrogen and carbon dioxide, formic acid, methanol, methylamines or
acetic acid, is the characteristic feature of the strictly anaerobic methanogenic archaea. The
metabolic pathways leading to the generation of methane are unique and involve a number of
enzymes and cofactors that only occur in methanogens. Methyl-S-CoM, the central intermediate
of methanogenesis, is reductively demethylated to yield methane by methyl CoM reductase.1
Both electrons for this process come from CoBꢀSH to yield the heterodisulfide CoBꢀSꢀSꢀCoM.2
The energy-conserving step in the metabolism of methylotrophic methanogens is the reduction
of CoBꢀSꢀSꢀCoM with H2 or F420H2.3 The H2:hetero-disulfide oxidoreductase- and the
F420H2:hetero-disulfide oxidoreductase systems are responsible for the reductive regeneration of
HSꢀCoM and HSꢀCoB from CoBꢀSꢀSꢀCoM. Both the membrane-bound hydrogenase and the
F420H2 dehydrogenase are key enzymes of these electron transport systems. The electrons are
transferred to the heterodisulfide reductase and finally to CoBꢀSꢀSꢀCoM. The most interesting
question relates to the nature of the electron carriers mediating the electron transfer.4
* Corresponding author. E-mail: ubeifus@gwdg.de
†
Dedicated to Professor Dr. Horst Kunz on the occasion of his 60th birthday.
0040-4039/00/$ - see front matter © 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)01669-5

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Recently methanophenazine (1) has been isolated from the cytoplasmic membranes of
Methanosarcina mazei Go¨1,5 and racemic methanophenazine (rac-1) was shown to function as
an electron carrier in the enzyme catalyzed heterodisulfide reduction with either H2 or F420H2.6
Most importantly, these results indicate that the role of the new cofactor in the energy
metabolism of methanogenic archaea corresponds to that of ubiquinone in bacteria and
mitochondria. The absolute configuration of the new natural product could not be elucidated by
analytical or spectroscopic methods. An alternative relies on chemical degradation. As only
small amounts of methanophenazine are available from the natural source it will be necessary
to carry out the degradation first with synthetic material. This is why we designed a route that
can easily be tailored to (S)- and (R)-methanophenazine and is flexible enough to synthesize a
number of methanophenazine derivatives for degradation and biological studies. Here we report
on both the first total synthesis of (S)-methanophenazine [(S)-1] and the formal synthesis of
(R)-methanophenazine [(R)-1] from a common enantiomerically pure precursor.
The retrosynthetic analysis results in 2-hydroxy-phenazine (2) and the terpenoid unit 3 to be
linked by etherification. 3 was divided into the alkyl iodide 4 and the (E)-vinyl iodide 5 to be
coupled by a Pd(0) catalyzed cross coupling reaction in order to ensure the correct geometry of
the C-6%,C-7% double bond of the side chain (Scheme 1).
Scheme 1.
Particular attention has been paid to develop a stereodivergent approach for (R)-4 and (S)-4
from a common precursor. Ethyl (R)-3-methylglutarate [(R)-6] was found to be suitable for the
synthesis of both enantiomerically pure bisfunctionalized C6-building blocks. For the synthesis
of enantiomerically pure (S)-methanophenazine [(S)-1] we had to prepare the monoprotected
diol (S)-11. In accordance with earlier reports7 the direct conversion of (R)-6 into (R)-7b via
chemoselective reduction of the carboxyl group with BH3·Me2S and silylation of the resulting
hydroxy group could not be achieved. In all experiments mixtures of varying compositions of
(R)-7b and the lactone (R)-8 were isolated. This is why we had to develop a new protocol for
the transformation of (R)-6 into (S)-11. First the lactone (R)-8 was prepared in 92% yield by
chemoselective reduction of (R)-6 with BH3·Me2S, followed by cyclization of the resulting
5-hydroxy ester (R)-7a under basic conditions (Scheme 2).8 We found that the lactone could be
opened reliably with piperidine to afford the acyclic 5-hydroxy-amide (R)-9 with 97% yield.
After silylation of the hydroxyl group with TBDMSCl/NEt3 the amide function of (R)-10 was
reduced selectively with lithium triethylborohydride9 to the corresponding protected alcohol
(S)-11 in enantiomerically pure form.10 Finally (S)-11 was transformed into the iodide (R)-4 via

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mesylate (R)-12. To summarize, a reliable and practical synthesis of (R)-4 in seven steps with a
total yield of 75% was developed providing the material in gram quantities.
Scheme 2. (i) BH3·SMe2 (1.2 equiv.), THF, 36 h, rt; (ii) 2 M aq. KOH, 10% MeOH, 8 h, 6N HCl, rt, 92% (2 steps);
(iii) piperidine (8.0 equiv.), sealed tube, 12 h, 70°C, 97%; (iv) TBDMSCl (1.1 equiv.), NEt3 (1.3 equiv.), CH2Cl2, 4 h,
0°C, 98%; (v) LiBHEt3 (2.5 equiv.), THF, 2 h, 0°C to rt, 92%; (vi) MsCl (1.1 equiv.), NEt3 (1.3 equiv.), THF, 15 min,
0°C; (vii) LiI (1.4 equiv.), THF, 2 h, 80°C, 93% (2 steps)
Preparation of 5 started with the transformation of (E,E)-farnesyl acetone (13) into the
terminal alkyne 14 (Scheme 3).11 Carboalumination of 14 with Me3Al and Cp2ZrCl2 followed by
12
a stereospecific quench of the resulting aluminium species with I2 afforded the (E)-vinyl iodide
5 in 74% yield.13 Due to the low reactivity the cross-coupling between 4 and 5 this was difficult
to achieve. However, the reaction of 4 with anhydrous ZnCl2 and tert-BuLi provided the
Scheme 3. (i) 13 (1.0 equiv.), LiTMP (1.05 equiv.), THF, 1 h, −78°C, then ClPꢁO(EtO)2 (1.05 equiv.), 3 h, −78°C to
rt, transfer to LiTMP (2.25 equiv.), 3 h, −78°C to rt, then H2O, 75%; (ii) 14 (1.0 equiv.), Cp2ZrCl2 (0.25 equiv.),
Me3Al (3.0 equiv.), CH2ClCH2Cl, 15 h, 0°C to rt, I2 (1.2 equiv.), THF, 1 h, −40 to 0°C, 74%; (iii) (R)-4 (1.5 equiv.),
ZnCl2 (1.5 equiv.), Et2O, 1 h, rt, then tert-BuLi (4.5 equiv.), 2 h, −90°C to rt, transfer to 5 (1.0 equiv.), Pd[P(Ph)3]4
(cat.), 8 h, rt, 65%; (iv) TBAF (3.0 equiv.), THF, 30 min, 70°C, 98%; (v) (S)-16 (1.0 equiv.), MsCl (1.1 equiv.), NEt3
(1.3 equiv.), THF, 20 min, 0°C; (vi) (S)-3 (1.0 equiv.), 2 (1.2 equiv.), KOH (1.2 equiv.), Aliquat 336 (cat.), toluene,
2 h, 110°C, 87% (2 steps)

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corresponding organozinc derivative. This species underwent a clean Pd(0) catalyzed sp3–sp2
coupling with the (E)-vinyl iodide 5 to afford (S)-15 in stereoisomeric pure form14 with 65%
yield. Using PdCl2(dppf) and PdCl2[P(Ph)3]2 as catalysts instead of Pd[P(Ph)3]4 lower yields of
(S)-15 were isolated. With enantio- and diastereomerically pure (S)-15 in hand the last three
steps of the total synthesis of (S)-methanophenazine [(S)-1] were performed with 85% yield.
Cleavage of the TBDMS ether with TBAF provided the alcohol (S)-16,15 which itself delivered
the mesylate (S)-3 upon treatment with MsCl/Et3N. Etherification of 2-hydroxy-phenazine (2)
and (S)-3 with Aliquat as a phase transfer catalyst16 finally yielded (S)-methanophenazine
[(S)-1]. The spectral data for the synthetic methanophenazine are identical to those of the
natural product.17
As the lactone (S)-8 can also be prepared from (R)-6 by reduction of its ester group with
LiBH4 and subsequent lactonization of the resulting 5-hydroxy carboxylic acid (S)-7a with 84%
yield18 (Scheme 4), our approach will also allow the synthesis of (R)-methanophenazine [(R)-1].
Scheme 4. (i) LiBH4 (1.5 equiv.), MeOH (1.5 equiv.), DME, 2 h, 84°C; (ii) 2 M aq. NaOH, 12 h, rt, 6N HCl, 84%
(2 steps)
In conclusion, we have developed a highly convergent route to enantiomerically pure
(S)-methanophenazine [(S)-1]. By the modular building block strategy numerous
methanophenazine derivatives will be accessible. With (S)-1 in hand further studies will be
directed to its chemical degradation and finally the determination of the absolute configuration
of natural methanophenazine as well as to studies related to the electron transport and the
energy conservation in methanogenic archaea.
Acknowledgements
This work was supported by the Deutsche Forschungsgemeinschaft (SFB 416) and the Fonds
der Chemischen Industrie. We are grateful to BASF AG, Ludwigshafen and Hoffmann La
Roche Ltd., Basel for the generous donations of chemicals.
References
1. Thauer, R. K. Microbiology 1998, 144, 2377–2406.
2. Thauer, R. K.; Hedderich, R.; Fischer, R. In Methanogenesis; Ferry, J. G., Ed.; Chapman & Hall: New York,
1993; pp. 209–252.
3. Ellermann, J.; Hedderich, R.; Bo¨cher, R.; Thauer, R. K. Eur. J. Biochem. 1988, 172, 669–677.
4. Deppenmeier, U.; Lienard, T.; Gottschalk, G. FEBS Lett. 1999, 457, 291–297.
5. Abken, H.-J.; Tietze, M.; Brodersen, J.; Ba¨umer, S.; Beifuss, U.; Deppenmeier, U. J. Bact. 1998, 180, 2027–2032.
6. Beifuss, U.; Tietze, M.; Ba¨umer, S.; Deppenmeier, U. Angew. Chem., Int. Ed. 2000, 39, 2470–2472.
7. Herold, P.; Mohr, P.; Tamm, C. Helv. Chim. Acta 1983, 66, 744–754.

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8. So far only the acidic cyclization of (R)-7a to yield (R)-8 has been reported; see for example: Theisen, P. D.;
Heathcock, C. H. J. Org. Chem. 1993, 58, 142–146.
9. Brown, H. C.; Kim, S. C.; Krishnamurthy, S. J. Org. Chem. 1980, 45, 1–12.
1
10. Selected data for (S)-11: [h]D=+2.3° (c=1, CHCl3); H NMR (C6D6, 300 MHz) 0.17 [s, 6H, Si(CH3)2], 0.93 (s
br, 1H, OH), partially superimposed by 0.94 (d, J=6.5 Hz, 3H, CH3), 1.09 [s, 9H, SiC(CH3)3], 1.26–1.47 (m, 2H,
3-H, 2-HA or 4-HA), 1.55 (dq, J=14.0 Hz, J=7.0 Hz, 1H, 2-HB or 4-HB), 1.65 (dtd, J=14.0 Hz, J=7.0 Hz,
J=6.0 Hz, 1H, 4-HB or 2-HB), 1.84 (mc, 1H, 4-HA or 2-HA), 3.45-3.60 (m br, 2H, 1-H2 or 5-H2), 3.68 (mc, 2H,
5-H2 or 1-H2). The 1H NMR spectrum (500 MHz) of the Mosher ester, prepared from (S)-(−)-a-methoxy-tri-
fluoromethyl-phenylacetic acid and alcohol (S)-11, exhibited only the signals of one diastereoisomer.
11. Negishi, E.; King, A. O.; Klima, W. L. J. Org. Chem. 1980, 45, 2526–2528.
12. For a review see: Negishi, E.; Takahashi, T. Synthesis 1988, 1–19.
13. Selected data for 5: 1H NMR (C6D6, 300 MHz) 1.49 (s, 3H; 6-CH3 or 10-CH3), 1.56 (s, 3H, 14-CH3), 1.60 (s, 3H,
10-CH3 or 6-CH3), 1.66 (s, 3H, 2-CH3), 1.67 (s, 3H, 15-H3), 1.93 (mc, 4H, 3-H2, 4-H2), 2.00–2.22 (m, 8H, 7-H2,
8-H2, 11-H2, 12-H2), 5.04 (mc, 1H, 13-H), 5.22–5.27 (m, 2H, 5-H, 9-H), 5.71 (d, J=0.7 Hz, 1H, 1-H).
14. For a review see: Negishi, E.; Liu, F. In Metal-catalyzed Cross-coupling Reactions; Diederich, F.; Stang, P. J.,
Eds.; Wiley-VCH: Weinheim, 1998, pp. 1–47.
1
15. Selected data for (S)-16: [h]D=−2.6° (c=1, CHCl3); H NMR (C6D6, 200 MHz) 0.90 (d, J=6.0 Hz, 3H, 3-CH3),
1.10–1.51 (m, 5H, 2-H2, 4-H2, OH), 1.55 (mc, 1H, 3-H), 1.66 (s, 3H, 19-CH3), 1.71 (s, 9H, 7-CH3, 11-CH3,
15-CH3), 1.78 (s, 3H, 20-H3), 2.05–2.43 (m, 14H, 5-H2, 8-H2, 9-H2, 12-H2, 13-H2, 16-H2, 17-H2), 3.44–3.70 (m,
2H, 1-H2), 5.21–5.50 (m, 4H, 6-H, 10-H, 14-H, 18-H). The 13C NMR spectrum (125 MHz) of the Mosher ester,
prepared from (S)-(−)-a-methoxy-trifluoromethyl-phenylacetic acid and alcohol (S)-16, exhibited only the signals
of one diastereoisomer.
16. Loupy, A.; Sansoulet, J.; Vaziri-Zand, F. Bull. Soc. Chim. Fr. 1987, 1027–1035.
1
17. Selected data for (S)-1: [h]D=−3.7° (c=1, C6H6); H NMR (C6D6, 200 MHz) 0.89 (d, J=6.5 Hz, 3H, 3%-CH3),
1.12–1.58 (m, 4H, 4%-H2, 2%-H2), 1.61 (s, 3H, 19%-CH3), 1.70–1.80 (m, 1H, 3%-H) superimposed by 1.64–1.71 (3s,
9H, 7%-CH3, 11%-CH3, 15%-CH3) and 1.73 (s, 3H, 20%-H3), 2.02–2.29 (m, 14H, 5%-H2, 8%-H2, 9%-H2, 12%-H2, 13%-H2,
16%-H2, 17%-H2), 3.83 (t, J=6.5 Hz, 2H, 1%-H2), 5.21–5.48 (m, 4H, 6%-H, 10%-H, 14%-H, 18%-H), 7.27–7.47 (m, 3H,
3-H, 7-H, 8-H), 7.56 (d, J=3.0 Hz, 1H, 1-H), 8.15 (d, J=9.6 Hz, 1H, 4-H), 8.34 (dd, J=7.0 Hz, J=2.5 Hz, 2H,
6-H, 9-H).
18. Francis, C. J.; Jones, J. B. J. Chem. Soc., Chem. Commun. 1984, 579–580.
.
.