Selectivity of biohydroxylation with Beauveria bassiana of trans-2-fluorocycloalkyl N-phenylcarbamates

Article abstract of DOI:10.1021/jo016332j

Biohydroxylation with Beauveria bassiana of racemates and the pure enantiomers of trans-2- fluorocyclohexyl- 3 and trans-2-fluorocycloheptyl N-phenylcarbamates 6 were investigated and compared with results found for the corresponding nonfluorinated parent compounds. In all cases, mixtures of diastereomeric products hydroxylated in the 4-position were isolated, besides products of p-hydroxylation of the aromatic ring and succeeding compounds derived from these primary reaction products. The regioselectivity of hydroxylation by this fungus is not changed by a single fluorine substituent attached closely to the electron-rich anchoring group in the trans-2-position. There is a different influence on the diastereoselectivity of hydroxylation depending on the absolute configuration of the fluorinated substrates. While the transformation of the (S,S)-2-fluorocycloalkyl N-phenylcarbamates is not diastereoselective giving almost 1:1 mixtures of cis- and trans-4-hydroxyl compounds, the corresponding reactions of the (R,R)-isomers led preferentially to the products transhydroxylated in the 4-position. The transformation of the racemic fluorinated six-membered N-phenylcarbamate 3 led to products having a very small enantiomeric excess. The fluorine substituent slightly increased the enantioselectivity of transformation of the racemic seven-membered substrate 6 compared to the C₈-symmetric nonfluorinated carbamate. Thus, the fluorine substituent in the trans-2-position in these examples did not change the regioselectivity but rather influenced the stereochemistry of biotransformation, depending on the absolute configuration of the substrate and ring size.

Full text of DOI:10.1021/jo016332j

3022
J . Org. Chem. 2002, 67, 3022-3028
Selectivity of Bioh yd r oxyla tion w ith Bea u ver ia ba ssia n a of
tr a n s-2-F lu or ocycloa lk yl N-P h en ylca r ba m a tes
Gu¨nter Haufe,* Do¨rthe Wo¨lker, and Roland Fro¨hlich†
Organisch-Chemisches Institut, Westfa¨lische Wilhelms-Universita¨t Mu¨nster,
Corrensstr. 40, D-48149 Mu¨nster, Germany
haufe@uni-muenster.de
Received November 29, 2001
Biohydroxylation with Beauveria bassiana of racemates and the pure enantiomers of trans-2-
fluorocyclohexyl- 3 and trans-2-fluorocycloheptyl N-phenylcarbamates 6 were investigated and
compared with results found for the corresponding nonfluorinated parent compounds. In all cases,
mixtures of diastereomeric products hydroxylated in the 4-position were isolated, besides products
of p-hydroxylation of the aromatic ring and succeeding compounds derived from these primary
reaction products. The regioselectivity of hydroxylation by this fungus is not changed by a single
fluorine substituent attached closely to the electron-rich anchoring group in the trans-2-position.
There is a different influence on the diastereoselectivity of hydroxylation depending on the absolute
configuration of the fluorinated substrates. While the transformation of the (S,S)-2-fluorocycloalkyl
N-phenylcarbamates is not diastereoselective giving almost 1:1 mixtures of cis- and trans-4-hydroxyl
compounds, the corresponding reactions of the (R,R)-isomers led preferentially to the products trans-
hydroxylated in the 4-position. The transformation of the racemic fluorinated six-membered
N-phenylcarbamate 3 led to products having a very small enantiomeric excess. The fluorine
substituent slightly increased the enantioselectivity of transformation of the racemic seven-
membered substrate 6 compared to the Cs-symmetric nonfluorinated carbamate. Thus, the fluorine
substituent in the trans-2-position in these examples did not change the regioselectivity but rather
influenced the stereochemistry of biotransformation, depending on the absolute configuration of
the substrate and ring size.
In tr od u ction
having any electron-rich functional binding group may
be hydroxylated.5b Moreover, it is well-known that this
fungus can also p-hydroxylate aromatic rings of N-
benzoylamines, N-phenylcarbamates,11 or substituted
heteroaromatics.12 Thioethers have been shown to be
The selective hydroxylation of nonactivated hydrocar-
bon positions is continuing to attract the attention of
organic chemists.1 However, there are almost no chemical
methods available2 that can compete in selectivity with
microbiological oxygenations involving mono-oxygenases3
in whole-cell systems.4 The fungus Beauveria bassiana
is one of the most frequently used microorganisms for
this purpose, and its biocatalytic reactions have been
recently reviewed.5 This fungus is known to hydroxylate,
in addition to amides4-6 and carbamates,7-9 saturated
N-containing heterocycles.4a,10 Even saturated hydrocar-
bon positions of cycloalkanes, saturated steroids or
terpenes, and side chains of aromatic hydrocarbons not
(4) (a) Fonken, G. S.; J ohnson, R. A. Chemical Oxidations with
Microorganisms; Marcel Dekker: New York, 1972; Chapter 1, pp 1-83.
(b) J ohnson, R. A. Oxidation with Microorganisms. In Oxidation in
Organic Chemistry; Trahanowsky, W. S., Ed.; Academic Press: New
York, 1978; Vol. 5C, pp 131-210. (c) Holland, H. L. Organic Synthesis
with Oxidative Enzymes, VCH: New York, 1992. (d) Enzyme Catalysis
in Organic Synthesis; Drauz, K., Waldmann, H., Eds.; VCH: Wein-
heim, 1995; pp 667-807. (e) Holland, H. L. In Biotechnology, 2nd ed.;
Rehm, H.-J ., Reed, G., Eds.; Kelly, D. R., Ed. Vol. 8a; Wiley-VCH:
Weinheim, 1998; pp 475-533. (f) Faber, K. Biotransformations in
Organic Chemistry, 4th ed.; Springer: Berlin, 2000; pp 225-236.
(5) (a) Holland, H. L.; Morris, T. A.; Nava, P. J .; Zabic, M.
Tetrahedron 1999, 55, 7441-7460. (b) Grogan, G. J .; Holland, H. L. J .
Mol. Catal. B, Enzym. 2000, 9, 1-32 and references cited therein.
(6) (a) Fonken, G. S.; Herr, M. E.; Murray, H. C.; Reineke, L. M. J .
Am. Chem. Soc. 1967, 89, 672-675. (b) Fonken, G. S.; Herr, M. E.;
Murray, H. C.; Reinecke, L. M. J . Org. Chem. 1968, 33, 3182-3187.
(c) Fourneron, J . D.; Archelas, A.; Furstoss, R. J . Org. Chem. 1989,
54, 2478-2483. (d) J ohnson, R. A.; Herr, M. E.; Murray, H. C.;
Chidester, C. G.; Han, F. J . Org. Chem. 1992, 57, 7209-7212. (e)
J ohnson, R. A.; Herr, M. E.; Murray, H. C.; Krueger, W. C.; Pschigoda,
L. M.; Duchamp, D. J . J . Org. Chem. 1992, 54, 7212-7216.
(7) Pietz, S.; Fro¨hlich, R.; Haufe, G. Tetrahedron 1997, 53, 17055-
17066.
* To whom correspondence should be addressed. Fax: +49-251-
83-39772.
† X-ray analyses.
(1) (a) Shelve, A. V. Activation of Saturated Hydrocarbons by
Transition Metal Complexes; Reedier: Boston, 1984. (b) Hill, C. L.
Activation and Funktionalisation of Alkanes; Wiley: New York, 1989.
(c) Davis, J . A.; Watson, P. L.; Liebman, J . F.; Greenberg, A. Selective
Hydrocarbon Activation; VCH Publishers: New York, 1990. (d) Barton,
D. H. R.; Be´vie`re, S. D.; Chavasiri, W.; Csuhai, E.; Doller, D.; Liu, W.-
G. J . Am. Chem. Soc. 1992, 114, 2147-2156. (e) Meunier, B. Chem.
Rev. 1992, 92, 1411-1456. (f) Reiser, O. Angew. Chem. 1994, 106, 73-
76; Angew. Chem., Int. Ed. Engl. 1994, 33, 69-72. (g) Asensio, G.;
Castellano, G.; Mello, R.; Nu´n˜ez, M. E. G. J . Org. Chem. 1996, 61,
5564-5566. (h) Holland, H. L., Adv. Appl. Microbiol. 1997, 44, 125-
165. (i) Holland, H. L. Curr. Opin. Chem. Biol. 1999, 3, 22-27. (j)
Lehmann, L. R.; Stewart, J . D. Curr. Org. Chem. 2001, 5, 439-470.
(2) Ostovic, D.; Bruice, T. C. Acc. Chem. Res., 1992, 25, 314-320.
(3) (a) Ortiz de Montellano, P. R. Cytochrome P-450, Mechanism and
Biochemistry, 2nd ed.; Plenum Press: New York, 1995. (b) Sono, M.;
Roach, M. P.; Coulter, E. D.; Dawson, J . H. Chem. Rev. 1996, 96, 2841-
2887. (c) Woggon, W. D. Top. Curr. Chem. 1996, 184, 39-96.
(8) Pietz, S.; Wo¨lker, D.; Haufe, G. Tetrahedron 1997, 53, 17067-
17078.
(9) (a) Furstoss, R.; Archelas, A.; Fourneron, J . D.; Vigne, B. In
Organic Synthesis an Interdisciplinary Challange; Streith, J ., Prinz-
bach, H., Schill, G., Eds.; Blackwell: Oxford, 1985; pp 215-226. (b)
Vigne, B.; Archelas, A.; Fourneron, J . D.; Furstoss, R. Nouv. J . Chim.
1987, 11, 297-298. (c) Vigne, B.; Archelas, A.; Furstoss, R. Tetrahedron
1991, 47, 1447-1458. (d) Davis, C. R.; J ohnson, R. A.; Cialdella, J . I.;
Liggett, W. F.; Mizsak, S. A.; Marsshall, V. P. J . Org. Chem. 1997, 62,
2244-2251.
10.1021/jo016332j CCC: $22.00 © 2002 American Chemical Society
Published on Web 04/06/2002

Biohydroxylation with Beauveria bassiana
J . Org. Chem., Vol. 67, No. 9, 2002 3023
oxygenated to sulfoxides,13 and epoxides were formed
from olefins.6a,10a Even a Baeyer-Villiger oxidation of a
ketone has been facilitated by this microorganism.14
Recently, a new concept for anchoring/protecting groups
has been introduced for hydroxylation reactions with B.
bassiana.15
Sch em e 1a
In 1997, we investigated the regio-, diastereo-, and
enantioselectivity of hydroxylation by B. bassiana of
cycloheptyl N-phenylcarbamate7 and extended8 the origi-
nal distance models by Fonken et al.6a and Furstoss et
al.16 By using the racemic trans-2-fluorocycloheptyl N-
phenylcarbamate, the first information regarding the
influence of a single fluorine substituent attached neigh-
bored to the anchoring group was obtained.7 This is most
interesting in connection with the enzyme-inhibiting
abilities of several fluorinated compounds,17 the well-
known ability of the fluorine substituent to modify the
electronic properties of molecules,18 and the compound’s
ability to act as a hydrogen-bond acceptor.19
We now present our results on the biohydroxylation
of the pure enantiomers of six- and seven-membered
trans-2-fluorocycloalkyl N-phenylcarbamates.
a
Reagents and conditions: (a) neat Et3N‚3HF, 115 °C, 4 h for
1 and 155 °C, 4 h for 4; (b) phenyl isocyanate, petroleum ether
(110-140 °C), reflux, 4 h, recrystallization; (c) PCL, vinyl acetate,
(i-Pr)2O, rt, 50% cv, (d), KOH, MeOH, rt.
(10) Recent papers: (a) Palmer, C. F.; Webb, B.; Broad, S.; Casson,
S.; McCague, R.; Willetts, A. J .; Roberts, S. M. Bioorg. Med. Chem.
Lett. 1997, 7, 1299-1302. (b) Aitken, S. J .; Grogan, G.; Chow, C. S.-
Y.; Turner, N. J .; Flitsch, S. L. J . Chem. Soc., Perkin Trans. 1 1998,
3365-3370. (c) Olivo, H. F.; Hemenway, M. S. J . Org. Chem. 1999,
64, 6312-6318. (d) De Raadt, A.; Fetz, B.; Griengl, H.; Klinger, M. F.;
Krenn, B.; Mereiter, K.; Mu¨nzer, D. F.; Plachota, P.; Weber, H.; Saf,
R. Tetrahedron 2001, 57, 8151-8157.
Resu lts a n d Discu ssion
The racemic fluorinated N-phenylcarbamates 3 and 6
were prepared in good yields from the corresponding
epoxides 1 and 4 by ring opening with triethylamine
trihydrofluoride (Et3N‚3HF)20 and subsequent treatment
with the phenyl isocyanate of the fluorohydrins 2 and 5.
The corresponding enantiopure carbamates were avail-
able from the optically active fluorohydrins obtained by
enzymatic deracemization using Pseudomonas cepacia
lipase (PCL) and vinyl acetate in diisopropyl ether. The
enantiomerically enriched fluorohydrins 2 and 5 were
separated from the also-formed acetates 7 and 8, which
were hydrolyzed afterward.21 Following treatment with
phenyl isocyanate of both enantiomeric fluorohydrins, the
enantiopure carbamates 3 and 6 were isolated after
recrystallization from petroleum ether (Scheme 1). The
optical purity of the carbamates was determined after
reduction with LiAlH4 in ether to the respective fluoro-
hydrins 2 and 5 and subsequent gas-chromatographic
analysis using a chiral â-cyclodextrin stationary phase.
All biotransformations of compounds 3 and 6 were done
following a standard procedure using a 2 L fermenter
with 200 mg of substrate per liter of a growing culture
of B. bassiana. In a standard medium,7 the culture with
the substrate was aerated with 1.5 L air per minute at
30 °C for 72 h.
(11) (a) Vigne, B.; Archelas, A.; Fourneron, J . D.; Furstoss, R.
Tetrahedron 1986, 42, 2451-2456. (b) Griffith, D. A.; Brown, D. E.;
J ezequel, S. G. Xenobiotica 1993, 23, 1085-1100.
(12) Gotor, V.; Quiro´s, M.; Liz, R.; Frigola, J .; Ferna´ndez, R.
Tetrahedron 1997, 53, 6421-6432.
(13) Holland, H. L.; Brown, F. M. Tetrahedron: Asymmetry 1998, 9,
535-538.
(14) Donzelli, F.; Fuganti, C.; Mendozza, M.; Pedrocchi-Fantoni, G.;
Servi, S.; Zucchi, G. Tetrahedron: Asymmetry 1996, 7, 3129-3134.
(15) (a) Braunegg, G.; de Raadt, A.; Feichenhofer, S.; Griengl, H.;
Kopper, I.; Lehmann, A.; Weber, H. Angew. Chem. 1999, 111, 2946-
2949; Angew. Chem., Int. Ed. 1999, 38, 2763-2766. (b) de Raadt, A.;
Fetz, B.; Griengl, H.; Klingler, M. F.; Kopper, I.; Krenn, B.; Mu¨nzer,
D. F.; Ott, R. G.; Plachota, P.; Weber, H. J .; Braunegg, G.; Mosler, W.;
Saf, R. Eur. J . Org. Chem. 2000, 3835-3847. (d) De Raadt, A.; Griengl,
H.; Weber, H. Chem. Eur. J . 2001, 7, 27-31.
(16) (a) Archelas, A.; Furstoss, R.; Waegell, B.; Le Petit, J .; Deveza,
L. Tetrahedron 1984, 40, 355-367. (b) Furstoss, R.; Archelas, A.;
Fourneron, J . D.; Vigne, B. In Enzymes as Catalysts in Organic
Synthesis; Schneider, M. P., Ed.; NATO ASI Ser.; D. Reidel: Norwell,
MA, 1986; Vol. 178, pp 361-370.
(17) (a) O’Hagan, D.; Rzepa, H. S. J . Chem. Soc., Chem. Commun.
1997, 645-652. (b) Yamazaki, T.; Kitazume, T. Coordination Ability
of Fluorine to Proton or Metals Based on Experimental and Theoretical
Evidence. In Enantiocontrolled Synthesis of Fluoro-Organic Compound.
Stereochemical Challanges and Biomedical Targets; Soloshonok, V. A.,
Ed.; J ohn Wiley & Sons: Chichester, 1999; pp 575-600.
(18) (a) Liebman, J F.; Greenberg, A.; Dolbier, W. R., J r. Fluorine-
containing Molecules; VCH Publishers: New York, 1988. (b) Welch, J .
T. Selective Fluorination in Organic and Bioorganic Chemistry; ACS
Symposium Series 456; American Chemical Society: Washington DC,
1991. (c) Ojima, I.; McCarthy, J . R.; Welch, J . T. Biomedical Frontiers
of Fluorine Chemistry; ACS Symposium Series 639; American Chemical
Society: Washington DC, 1996. (d) Plenio, H. Chem. Rev. 1997, 97,
3363-3384. (e) Alderfer, J . L.; Eliseev, A. V. J . Org. Chem. 1997, 62,
8225-8556.
(19) (a) Huang, J .; Hedberg, K. J . Am. Chem. Soc. 1989, 111, 6909-
6913. (b) J effrey, G. A.; Saenger, W. Hydrogen Bonding in Biological
Structures; Springer-Verlag: Berlin, 1991; pp 29-33. (c) Welch, J . T.;
Eswarakrishnan, S. Fluorine in Bioorganic Chemsitry; J ohn Wiley &
Sons: New York, 1991. (d) Banks, R. E., Smart, B. E., Tatlow, J . C.,
Eds. Organofluorine Chemistry: Principles and Commercial Applica-
tions; Plenum Press: New York, 1994. (e) Schlosser, M.; Michel, D.
Tetrahedron 1996, 52, 99-108. Dunitz, J . D.; Taylor, R. Chem. Eur.
J . 1997, 3 89-98. (f) Bramonte, M. A.; Vasella, A. Helv. Chim. Acta
1998, 81, 695-717. (g) Schlosser, M. Angew. Chem. 1998, 110, 1538-
1556; Angew. Chem., Int. Ed. 1998, 37, 1496-1513. (h) Desiraju, G.
R.; Steiner, T. The Weak Hydrogen Bond in Structural Chemistry and
Biology; IUCr Monographs on Crystallography, Vol. 9; Oxford Uni-
versity Press: Oxford, 1999; pp 202-215.
The biotransformation of the racemic trans-2-fluoro-
cyclohexyl N-phenylcarbamate (()-3 using a fresh culture
of B. bassiana gave six products. Some amount of starting
material was always recovered (Scheme 2).
The two products (-)-9 and (+)-10, hydroxylated
exclusively in the 4-position, were formed in a 1:1 ratio
as determined by 19F NMR spectroscopy of the crude
reaction product. The two compounds were isolated in
6.9% (6% ee) and 7.1% (33% ee) yield, respectively. The
(20) (a) Sattler, A.; Haufe, G. J . Fluorine Chem. 1994, 69, 185-190.
(b) Goj, O.; Haufe, G. Liebigs Ann. 1996, 1289-1294. (c) Haufe, G. J .
Prakt. Chem. 1996, 338, 99-113 and references cited therein.
(21) Wo¨lker, D.; Haufe, G. J . Org. Chem. 2002, 67, 3015-3021.

3024 J . Org. Chem., Vol. 67, No. 9, 2002
Haufe et al.
Sch em e 2
Ta ble 1. Com p a r ison of th e Ra tio (19F NMR) of P r od u cts Hyd r oxyla ted in th e Cycloh exa n e Rin g a n d Isola ted Yield s of
All P r od u cts Obta in ed by Biotr a n sfor m a tion of tr a n s-2-F lu or ocycloh exyl N-P h en ylca r ba m a te (()-3 a n d th e P u r e
En a n tiom er s (1S,2S)-(+)-3 or (1R,2R)-(-)-3
product ratio (%)a
Σ 9 + 11 Σ 10 + 12
49 51
absolute configuration, yield [%], and enantiomeric excess (%)b
carbamate 3
(recovered (%))
9
10
11
12
13
(S,S)-(+)-3
[20]
(R,R)-(-)-3
[13]
(()-3
[15]
(S,S,S)-(+)-
[7.8]
(R,R,R)-(-)-
(S,S,R)-(+)-
[7.3]
(R,R,S)-(-)-
[3.4]
(S,S,R)-(+)-
[7.1] (33% ee)
n.d.
[trace]
n.d.
[trace]
n.d.
trace
n.d.
[trace]
n.d.
[trace]
n.d.
trace
(S,S)-(+)-
[2.3]
(R,R)-(-)-
[5.8]
(R,R)-(-)-
[3.1] (36% ee)
69
50
31
50
[12.3]
(R,R,R)-(-)-
[6.9] (6% ee)
a
b
Determined from the crude product mixture by 19F NMR. The enantiomeric excesses (ee) of the transformation products of racemic
3 were determined by comparison of the measured optical rotations with those of the tranformation products of the pure enantiomers (cf.
the Experimental Section).
1
structures of these products follow from their H, 13C, and
19F NMR spectra and their mass spectra. These struc-
tures were further confirmed by X-ray analyses22 of the
enantiopure compounds obtained by biohydroxylation
(vide infra) of (1S,2S)-(+)-2-fluorocyclohexyl N-phenyl-
carbamate (1S,2S)-(+)-3 (cf. Figures 1 and 2 in the
Supporting Information).
and the already existing carbamate function. Interest-
ingly enough, studies on biotransformations of fluori-
nated steroids23 and terpenes24 using microorganisms
other than Beauveria bassiana, when compared to the
nonfluorinated parents, have demonstrated that a fluo-
rine substituent frequently alters the regiochemistry of
hydroxylation and occasionally also the enantioselectivity
of hydroxylation.25
The enantiomeric excesses of all products were deter-
mined by comparison of the measured rotations with
those of the products formed from the enantiopure
carbamates (1S,2S)-(+)-3 or (1R,2R)-(-)-3, respectively.
Two succeeding products, 11 and 12, which were derived
most probably from the primary formed compounds (-)-9
and (+)-10 by para hydroxylation, were detected only in
traces by 19F NMR spectroscopy of the crude product
mixture and by GC/MS coupling experiments of the
silylated crude product mixture. The other two products,
13 (3.1% yield, 36% ee) and 14 (14.9% yield), were not
hydroxylated in any alicyclic position at all, but were
formed by para hydroxylation of the phenyl ring. The OH
group was partially glycosylated afterward by the mi-
croorganism. However, this glycosylation was observed
only in cases when a culture not older than 10 days was
used. When we used a 5-month-old culture, the same
products 9-13 were formed in very similar amounts as
with the fresh culture, except that the glycoside 14 was
absent. All of the isolated products that were hydroxy-
lated in an alicyclic position bore the OH group at carbon
4. Furthermore, the isolated main hydroxylation products
were optically active. The enantiomeric excesses were
rather small in all cases, showing that there was a quite
weak influence of the fluorine substituent. This fact
would be expected if there is only a small geometric
deviation from the Cs symmetry of the nonfluorinated
parent carbamate. The diastereoselectivity of 9:1 in the
nonfluorinated case9b changed to a 1:1 ratio of the
respective cis/trans isomers 9 and 10 with regard to the
relative configuration of the introduced hydroxyl group
To check the influence of the fluorine in more detail,
we investigated the biotransformation of both pure
enantiomers. Treatment of (1S,2S)-(+)-3 or (1R,2R)-
(-)-3 with a 5-month-old culture of B. bassiana gave the
corresponding enantiopure products 9-13. The ratio of
diastereomeric products hydroxylated in the cyclohexane
ring was determined by 19F NMR spectroscopy of the
crude product mixtures. The results are shown in Table
1, together with the isolated yields of products 9-13.
From comparison of the results of the enantiopure
compounds with the racemic 3, one can conclude that the
differences in transition-state energies and hence in the
rates of hydroxylation of the enantiomers are not sig-
nificantly different. Consequently, the enantiodifferen-
tiation of the racemate is quite inefficient as indicated
by the low enantiomeric excess in the products derived
from the six-membered ring compound (()-3.
(22) Details of the X-ray crystal structure analyses are available in
the Supporting Information.
(23) (a) Kieslich, K.; Petzold, K.; Kosmol, H.; Koch, W. Liebigs Ann.
Chem. 1969, 726, 168-176. (b) Bird, T. G. C.; Fredericks, P. M.; J ones,
E. R. H.; Meakins, G. D. J . Chem. Soc., Perkin Trans. 1 1980, 750-
755.
(24) (a) Cross, B. E.; Erasmuson, A. J . Chem. Soc., Chem. Commun.
1978, 1013-1015. (b) Eble, K. S.; Dawson, J . H. J . Biol. Chem. 1984,
259, 14389-14393. (c) Kadkhodayan, S.; Coulter, E. D.; Maryniak, D.
M.; Bryson, T. A.; Dawson, J . H. J . Biol. Chem. 1995, 270, 28042-
28048.
(25) (a) Holland, H. L.; Bergen, E. J .; Chenchaiah, P. C.; Khan, S.
H.; Munoz, B.; Ninniss, R. W.; Richards, D. Can. J . Chem. 1987, 65,
502-507. (b) Holland, H. L.; Allen, L. J .; Chernishenko, M. J .; Diez,
M.; Kohl, A.; Ozog, J .; Gu, J .-X. J . Mol. Catal. B: Enzym. 1997, 3,
311-324.

Biohydroxylation with Beauveria bassiana
J . Org. Chem., Vol. 67, No. 9, 2002 3025
Sch em e 3
Ta ble 2. Com p a r ison of th e Ra tio (19F NMR) of P r od u cts Hyd r oxyla ted in th e Cycloh ep ta n e Rin g a n d Absolu te
Con figu r a tion of P r od u cts of Biotr a n sfor m a tion of tr a n s-2-F lu or ocycloh ep tyl N-P h en ylca r ba m a te (()-67 a n d th e P u r e
En a n tiom er s (1R,2R)-(-)-6 or (1S,2S)-(+)-6
absolute configuration, ratio, yield [%], and enantiomeric excess of products (%)a
carbamates
15
16
17
18
(R,R)-(-)-6
(R,R,R)-(-)-
78
(R,R,S)-(-)-
15
(R,R,R)-(-)-
1
(R,R,S)-(-)-
6
(S,S)-(+)-6
(()-6
(S,S,S)-(+)-
(S,S,R)-(+)-
(S,S,S)-(+)-
(S,S,R)-(+)-
23
34
27
16
(R,R,R)-(-)-
54, [24%] (82% ee)
n.d.b
24, [8%] (16% ee)
(S,S,S)-(+)-
14, [5%] (79% ee)
(S,S,R)-(+)-
8, [4.5%] (52% ee)
a
The enantiomeric excesses (ee) of the transformation products of racemic 6 were determined by comparison of the measured optical
b
rotations with those of the tranformation products of the pure enantiomers (cf. the Experimental Section). Should be the (S,S,R)-
enantiomer by comparison of the amounts of the respective 16 obtained by transformation of the pure enantiomers of 6.
With the seven-membered compounds 6, more pro-
nounced effects have been observed. The products, iso-
lated yields, and enantiomeric excesses of biotransfor-
mation of racemic trans-2-fluorocycloheptyl N-phenylcar-
bamate (()-6 have already been reported (Scheme 3).7
Two trans-4-hydroxylated compounds (with regard to the
haptophoric carbamate function), (-)-15 (24% yield, 82%
ee) and (+)-17 (4.5% yield, 79% ee), were isolated. One
of those was a 1,4-fluorohydrin, while the other was a
1,3-fluorohydrin, but both of them were hydroxylated in
a formal 4-position in relation to the anchoring group.
Moreover, two cis-configurated products, 16 (8% yield,
16% ee), which should be the (S,S,R)-enantiomer, and
(+)-18 (5% yield, 52% ee), were isolated having the same
regiochemistry as the trans compounds. Furthermore, the
4-keto derivative (+)-19 (1%, absolute configuration has
not been determined) and the phenol 20 (2%), showing
no rotation, were isolated. All compounds that were
hydroxylated in the alicyclic ring were optically active
and posessed higher enantiomeric excesses than the
corresponding six-membered analogues.
We next determined the product ratio in the crude
reaction mixture of treatment of both enantiomers of 6.
After transformation of (1R,2R)-(-)-6 and (1S,2S)-(+)-6
under standard conditions, all products isolated in our
former study7 were identified in the crude product
mixture by 19F NMR. Relative amounts and absolute
configurations of the products are given in Table 2
together with the ratios, yields, and enantiomeric ex-
cesses of the products isolated in our earlier study.7 In
contrast to the transformation of the six-membered
compounds 3, no products hydroxylated both in an
alicyclic and aromatic position were found in the product
mixtures of the transformation of 6 under the same
conditions.
compounds were formally hydroxylated nondiastereose-
lectively. From (S,S)-(+)-3 a 49:51 ratio of trans (Σ 9 +
11) and cis (Σ 10 + 12) and from (S,S)-(+)-6 a 50:50 ratio
of trans (Σ 15 + 17) and cis compounds (Σ 16 + 18) was
observed. In contrast, (R,R)-(-)-3 gave a 69:31 ratio of
trans and cis compounds, and for the seven-membered
ring (R,R)-(-)-6 this ratio is even 79:21. However, for the
products of compounds 6 this is a formal ratio, because
two different positions of the seven-membered ring have
been hydroxylated in the two trans and the two cis
compounds. Another comparison of transformation prod-
ucts of the seven-membered ring enantiomers is interest-
ing, namely the regioselectivity of hydroxylation (posi-
tions 4 or 5, i.e., formation of 1,4-fluorohydrins 15 and
16 compared to 1,3-fluorohydrins 17 and 18, respec-
tively). While from (R,R)-(-)-6, 93% of the isomeric
2-fluoro-5-hydroxycycloheptyl N-phenylcarbamates 15
and 16 were formed, the transformation of (S,S)-(+)-8
gave only 57% of the corresponding carbamates 15 and
16. Furthermore, for the products of transformation of
(()-3 only 6% ee and 33% ee were found for the trans or
the cis products 9 and 10, respectively. The enantiomeric
excesses for the corresponding trans products 15 (82%
ee) and 17 (79% ee) or the cis products 16 (16% ee) and
18 (52% ee) were larger, showing a stronger influence of
the fluorine substituent in the transformation of the
seven-membered ring compounds.
Con clu sion
This study has shown that the fungus B. bassiana
selectively monohydroxylates fluorinated cycloalkyl N-
phenylcarbamates. Comparing these results with those
of the nonfluorinated cyclohexyl-9b or cycloheptyl N-
phenylcarbamates,7 one can conclude that in case of the
six-membered and the seven-membered substrates the
regiochemistry of hydroxylation with respect to the
anchoring group was not changed by fluorine substitution
in the trans 2-position. The trans diastereoselectivity of
Comparing the results of biotransformation of the
enantiomers of the six- and the seven-membered ring
trans-2-fluorocycloalkyl N-phenylcarbamates 3 and 6,
respectively, one can see that the two (S,S)-configurated

3026 J . Org. Chem., Vol. 67, No. 9, 2002
Haufe et al.
isotherm, 91 or 107 °C, respectively, for the six- or the seven-
membered ring fluorohydrins, N2 as carrier gas).
hydroxylation, which was 88:12 for the nonfluorinated
compound,9b decreased to 69:31 for (R,R)-(-)-3 and
decreased to 49:51 for (S,S)-(+)-3. For the products of
transformation of the nonfluorinated seven-membered
ring carbamate a 92:8 ratio of trans to cis 4-hydroxylated
compounds was found, considering that the ketone
isolated in 12% yield was formed exclusively from trans-
4-hydroxycycloheptyl N-phenylcarbamate.7 From (R,R)-
(-)-6, the trans-5-hydroxy derivative 15 was formed with
a 84:16 selectivity over the cis isomer 16 and the trans-
4-hydroxy derivative 17 was formed with a 16:84 selec-
tivity compared to the cis isomer 18. The reverse dia-
stereoselectivity was observed for (S,S)-(+)-6. While the
5-hydroxy compounds 15 and 16 were formed in a 40:60
ratio, the 4-hydroxy isomers 17 and 18 were formed in a
63:37 ratio (cf. Table 2). Thus, the fluorine substituent
in the trans 2-position to the electron-rich anchoring
group in these examples did not change the regioselec-
tivities, but influenced the stereochemistry of biotrans-
formation differently, depending on the absolute config-
uration of the substrates.
(()-tr a n s-2-F lu or ocycloh exyl N-P h en ylca r ba m a te 3.
1
Yield: 0.80 g (84%). Mp: 118 °C (petroleum ether). H NMR:
2
δ 1.21-1.85 (m, 6H), 2.04-2.23 (m, 2H), 4.44 (dddd, J H,F
)
3
3
3
50.3 Hz, J Ha,Ha ) 10.3 Hz, J Ha,Ha ) 8.4 Hz, J Ha,He ) 4.8 Hz,
3
1H), 4.79-4.90 (m, 1H), 6.77 (br s, 1H), 7.05 (t, J H,H ) 7.3
3
3
Hz, 1H), 7.27 (t, J H,H ) 7.9 Hz, 2H), 7.38 (br d, J H,H ) 7.6
Hz, 2H). 13C NMR: δ 22.8 (dt, J C,F ) 10.2 Hz), 23.0 (t), 29.7
3
(dt, 3J C,F ) 7.6 Hz), 30.4 (dt, 2J C,F ) 17.8 Hz), 75.1 (dd, 2J C,F
)
20.4 Hz), 92.1 (dd, 1J C,F ) 178.0 Hz), 118.7 (2d), 123.4 (d), 129.0
(2d), 137.8 (s), 152.8 (s). 19F NMR: δ -181.7 (d, J F,H ) 47.7
2
Hz). GC-MS m/z: 237 (46), 150 (5), 138 (4), 137 (23), 132 (18),
119 (58), 118 (7), 94 (8), 93 (100), 91 (22), 81 (56), 77 (17), 72
(9), 64 (14), 59 (20), 57 (53), 55 (21), 51 (10), 41 (35).
(S,S)-(+)-3. Yield: 0.58 g (61%). Mp: 119 °C (petroleum
ether). [R]22D: +58.9 (c 1.0, CHCl3), >98% ee. Anal. Calcd for
C
13H16NO2F (237.3): C, 65.81; H, 6.80; N, 5.90. Found: C,
65.78; H, 6.77; N, 6.09.
(R,R)-(-)-3. Yield: 0.63 g (66%). Mp: 118-119 °C (petro-
leum ether). [R]22D: -57.8 (c 1.0, CHCl3), >98% ee. Anal. Calcd
for C13H16NO2F (237.3): C, 65.81; H, 6.80; N, 5.90. Found: C,
66.00; H, 6.60; N, 6.09.
(()-tr a n s-2-F lu or ocycloh ep tyl N-P h en ylca r ba m a te 6.
Yield: 0.89 g (89%). Mp: 94 °C (petroleum ether). 1H NMR:
Exp er im en ta l Section
2
δ 1.38-2.05 (m, 10H), 4.62 (dm, J H,F ) 48.4 Hz, 1H), 4.90-
3
4
5.06 (m, 1H), 6.56 (br s, 1H), 7.04 (tt, J H,H ) 6.9 Hz, J H,H
)
1
Gen er a l Meth od s. H NMR (300.1 MHz), 13C NMR (75.5
3
3
1.3 Hz, 1H), 7.28 (t, J H,H ) 8.0 Hz, 2H), 7.36 (dd, J H,H ) 8.6
MHz), and 19F NMR spectra (282.3 MHz) were recorded from
ca. 20% solutions in CDCl3. Chemical shifts are reported as δ
values (ppm) relative to TMS (1H), CDCl3 (13C) or CFCl3 (19F),
respectively, as internal standards. The multiplicity of 13C
signals was determined by the DEPT operation. Mass spectra
(electron impact ionization, 70 eV) were recorded by GC/MS
coupling. The enantiomeric excesses of the fluorohydrins 2 and
5 and the corresponding acetates 7 and 8 or the carbamates 3
and 6, respectively, after hydrolysis were determined by chiral
GC with FID using a â-cyclodextrin column, 30 m × 0.25 mm,
0.25 µm, , isotherm 96 °C for 2 and 111 °C for 5 , N2 as carrier
gas. The product ratio of microbial transformations were
determined by 19F NMR spectroscopy of the crude product
mixtures. The products were separated by column chroma-
tography (silica gel, 70-230 mesh, diethyl ether/pentane 1:1).
Optical rotations were determined at NaD line, λ ) 589 nm.
Microanalyses were carried out by the Mikroanalytisches
Laboratorium, Organische Chemie, University of Mu¨nster.
The (()-trans-2-fluorocycloalkanols have been prepared from
the corresponding epoxides by ring opening with triethylamine
trishydrogen fluoride (Et3N‚3HF)20 and cleaved by lipase-
catalyzed deracemization as described previously.21
Hz, J H,H ) 1.0 Hz, 2H). 13C NMR: δ 21.8 (dt, J C,F ) 7.6 Hz),
4
3
23.1 (t), 28.3 (t), 29.8 (dt, 3J C,F ) 7.6 Hz), 30.9 (dt, 2J C,F ) 20.3
2
1
Hz), 78.7 (dd, J C,F ) 22.9 Hz), 95.8 (dd, J C,F ) 172.9 Hz),
118.8 (2d, C-10), 123.5 (d), 129.1 (2d, C-11), 137.9 (s), 152.9
(s). 19F NMR: δ -171.7 (m). GC-MS m/z: 252 (6), 251 (37),
231 (4), 138 (6), 137 (45), 124 (13), 119 (26), 95 (53), 93 (100),
91 (11), 77 (15), 73 (19), 67 (13), 57 (18), 55 (28), 43 (7), 41
(23).
(S,S)-(+)-6. Yield: 0.62 g (62%). Mp: 93-94 °C (petroleum
ether). [R]22 +44.1 (c 1.0, CHCl3), >98% ee. Anal. Calcd for
D
C
14H18NO2F (251.3): C, 66.91; H, 7.22; N, 5.57. Found: C,
66.84; H, 7.20; N, 5.91.
(R,R)-(-)-6. Yield: 0.59 g (59%). Mp: 94-95 °C (petroleum
ether). [R]22D: -44.2 (c 0.5, CHCl3), >98% ee. Anal. Calcd for
C
14H18NO2F (251.3): C, 66.91; H, 7.22; N, 5.57. Found: C,
66.72; H, 7.16; N, 5.86.
Biotr a n sfor m a tion w ith B. ba ssia n a .7 All media and
equipment were sterilized in an autoclave at 121 °C and 2.3
bar for 20 min prior to use. The fungus B. bassiana ATCC
7159 was mobilized on potato dextrose agar at 30 °C in Petri
dishes for 70 h. Subsequently, the vegetative cells of the light
yellow culture were incubated in 75 mL of a culture medium
made from 20 g/L corn steep liquor, pH 5.0 and 10 g/L glucose.
This mixture was shaken (180 rpm) under nitrogen for 48 h.
This culture broth was used to inoculate the main culture
(1.425 L). The mixture was stirred (200 rpm) in a 2 L autoclave
while being aerated with 1.5 L of air per min at 30 °C for 24
h. Then 300 mg of the respective carbamate 3 or 6 in DMF (3
mL) was added and fermented under the mentioned conditions
for 72 h. Subsequently, the mycelium was separated by
centrifugation. The mycelium was stirred with CH2Cl2 (300
mL) for 48 h and separated by filtration. The aqueous layer
was continuously extracted with CH2Cl2 (200 mL) for 7 days.
The combined organic layer was dried over MgSO4, and the
solvent was removed in a vacuum. The crude product mixture
was analyzed by 19F NMR spectroscopy and gas chroma-
tography prior to separation by column chromatography (silica
gel, cyclohexane/ethyl acetate, gradient). The partially resolved
products were purified by HPLC, Nucleosil 50/7, CHCl3/
methanol, 50:1.
Syn th esis of N-P h en ylca r ba m a tes 3 a n d 6 fr om F lu o-
r oh yd r in s. The racemic or enantiopure fluorohydrins 2 or 5
(4 mmol) were dissolved in petroleum ether (110-140 °C, 40
mL), and phenyl isocyanate (571 mg, 4.8 mmol) was added
(syringe). The mixture was refluxed for 4 h and subsequently
concentrated to ∼20 mL. This solution was stored in
a
refrigerator for 12-24 h, and the precipitate was separated
and recrystallized from petroleum ether. To get the optically
active carbamates with >98% ee, the carbamates 3 and 6 had
to be recrystallized three to five times, depending on the ee of
the fluorohydrin that was used in the preparation.
Deter m in a tion of th e En a n tiop u r ity of th e Ca r ba m -
a tes 3 or 6. The enantiomeric excess has been determined
using the fluorohydrins obtained by reduction of the carbam-
ates 3 or 6, respectively. Analogously to a known protocol,26
the respective carbamate (0.15 mmol) was refluxed with
LiAlH4 (23 mg, 0.6 mmol) in diethyl ether (5 mL) for 5 h. After
careful addition of water (5 mL), the phases were separated
and the aqueous phase was extracted with diethyl ether (5
mL). The combined organic phase was dried (MgSO4), the
solvent was evaporated, and the residue was analyzed by
chiral GC (â-cyclodextrin column, 30 m × 0.25 mm, 0.25 µm,
Biotr a n sfor m a tion of (()-tr a n s-2-F lu or ocycloh exyl N-
P h en ylca r ba m a te (()-3. From the transformation of car-
bamate (()-3 (450 mg, 1.90 mmol) in addition to the trans-
formation products 9-14 was recovered 69 mg (15%) of (+)-3.
[R]20D: +3.8 (c, 1.2, CHCl3); 9% ee (calculated from the value
of optical rotation of the pure enantiomer). The relative
(26) Organikum, 18th ed.; Deutscher Verlag der Wissenschaften:
Berlin, 1990; p 430.

Biohydroxylation with Beauveria bassiana
J . Org. Chem., Vol. 67, No. 9, 2002 3027
amounts of compounds 9-13 found in the crude product
mixture are given in Table 1.
(1R,2R,4R)-(-)-2-F lu or o-4-h yd r oxycycloh exyl N-P h en -
ylca r ba m a te (-)-9. Yield: 33 mg (6.9%). Mp: 162-164 °C
(cyclohexane/ethyl acetate). [R]22D: -2.8 (c 0.5, CHCl3), 6% ee.
1H NMR: δ 1.31-2.48 (m, 6H), 3.67-3.83 (m, 1H), 4.49 (dddd,
eluent. Yield: 27 mg (97%). [R]20D: -22.9 (c 1.2, CHCl3). 1H
NMR: δ 1.15-2.18 (m, 8H), 2.03 (s, 3H), 2.06 (s, 3H), 2.07 (s,
3
3H), 3.42 (t, J Ha,Ha ) 7.1 Hz, 1H), 3.42 (s, 3H), 3.56-3.69 (m,
1H), 4.24 (dd, J H,H ) 11.9 Hz, J H,H ) 5.5 Hz, 1H), 4.39 (dd,
2
J H,H ) 11.9 Hz, J H,H ) 2.4 Hz, 1H), 4.41 (dddd, J H,F ) 52.6
3
3
3
Hz, J Ha,Ha ) 10.3 Hz, J Ha,Ha ) 8.3 Hz, J Ha,He ) 4.8 Hz, 1H),
3
3
3
3
2J H,F ) 49.8 Hz, J Ha,Ha ) 10.7 Hz, J Ha,Ha ) 8.4 Hz, J Ha,He
)
4.72-4.86 (m, 1H), 4.94 (d, J Ha,Ha ) 7.6 Hz, 1H), 5.10 (dd,
3
3
4.8 Hz, 1H), 4.79-4.93 (m, 1H, 1-CH), 6.57 (br. s, 1H), 7.05
3J Ha,Ha ) 9.5 Hz, J Ha,Ha ) 7.6 Hz, 1H), 5.21 (t, J Ha,Ha ) 9.2
3
4
(tt, 3J H,H ) 7.2 Hz, 4J H,H ) 1.4 Hz, 1H), 7.29 (t, 3J H,H ) 8.0 Hz,
Hz, 1H), 6.59 (br s, 1H), 6.91 (dt, J H,H ) 9.1 Hz, J H,H ) 2.7
3
4
3
2H), 7.36 (dd, J H,H ) 7.9 Hz, J H,H ) 1.2 Hz, 2H). 13C NMR:
Hz, 2H), 7.26 (br d, J H,H ) 9.1 Hz, 2H). 13C NMR: δ 20.7,
δ 25.0 (dt, J C,F ) 5.1 Hz), 32.0 (t), 39.0 (dt, J C,F ) 15.3 Hz),
20.8, 20.9 (3q), 22.9 (dt, 3J C,F ) 10.1 Hz), 23.1 (t), 29.8 (dt, 3J C,F
) 5.8 Hz), 30.5 (dt, 2J C,F ) 18.4 Hz), 60.3 (q), 62.8 (t), 71.8 (d),
73.1 (d), 74.9 (d), 77.7 (d), 75.3 (d), 92.2 (dd, 1J C,F ) 178.1 Hz),
99.6 (d), 117.9 (2d), 118.9 (2d), 132.3 (s), 153.0 (s) 153.2 (s),
3
2
3
2
67.3 (dt, J C,F ) 10.1 Hz), 74.4 (dd, J C,F ) 20.3 Hz), 89.9 (dd,
1J C,F ) 180.6 Hz), 118.8 (2d), 123.7 (d), 129.1 (2d), 137.6 (s),
152.7 (s). 19F NMR: δ -183.2 (m). GC-MS m/z: 254 (4), 253
(29), 138 (4), 137 (16), 120 (6), 119 (22), 99 (56), 93 (100), 79
(17), 77 (17), 73 (18), 65 (15), 55 (10), 43 (9), 41 (30). Anal.
Calcd for C13H16NO3F (253.3): C, 61.65; H, 6.37; N, 5.53.
Found: C, 61.28; H, 6.38; N, 5.72.
169.7, 170.0, 170.6, (3s). 19F NMR: δ -181.8 (d, J F,H ) 53.4
2
Hz). ESI-MS, nanospray, m/z: 554 (12), 437 (25), 436 (100),
394 (8), 352 (5), 301 (13), 232 (13).
cis- a n d tr a n s-2-F lu or o-4-h yd r oxycycloh exyl N-(p-Hy-
d r oxyp h en yl)ca r ba m a tes 11 a n d 12. In the proton-de-
coupled 19F NMR spectra of the crude product mixtures of
biotransformations of 3 two more signals, δ ) -182.7 and δ )
-188.7 ppm, were detected (both <1% of the total integral of
all fluorinated compounds) very close to the signals of products
9 (-183.2) or 10 (-189.0), respectively. These signals were
assigned to compounds 11 and 12, respectively. The structures
of these compounds are supported by the fact that in the mass
spectra of a silylated sample of the crude product mixture
(after treatment with N-methyl-N-trimethylsilyltrifluoroacet-
amide) the molecular ions of the disilylated compounds 11 and
12 were identified. Compound 11. GC-MS m/z: 415 (8), 414
(10), 413 (27), 282 (5), 225 (20), 207 (100), 192 (21), 181 (13),
180 (6), 134 (4), 111 (4), 99 (10), 97 (6), 79 (11), 75 (16), 73
(43), 69 (7), 45 (9). Compound 12. GC-MS m/z: 413 (27), 304
(2), 303 (8), 253 (21), 225 (18), 207 (34), 192 (75), 189 (27), 169
(39), 129 (8), 107 (10), 99 (57), 97 (72), 79 (83), 73 (100), 69
(36), 67 (16), 55 (14), 41 (19).
Tr a n sfor m a tion of (1S,2S)-(+)-2-F lu or ocycloh exyl N-
P h en ylca r ba m a te (+)-3. From the transformation of car-
bamate (+)-1 (450 mg, 1.90 mmol) in addition to the trans-
formation products 9-13 was recovered 91 mg (20%) of (+)-3.
The spectroscopic data of the isolated products agree with
those determined for the products obtained by biotransforma-
tion of racemic 3.
(1S,2S,4S)-(+)-2-F lu or o-4-h yd r oxycycloh exyl N-P h en -
ylca r ba m a te (+)-9. Yield: 37 mg, (7.8%). Mp: 163-164 °C
(cyclohexane/ethyl acetate). [R]20D: +51.0 (c 1.0, CHCl3). High-
resolution MS: calcd for C13H16NO3F + NH4+ 271.1458, found
271.1461.
(1S,2S,4R)-(+)-2-F lu or o-4-h yd r oxycycloh exyl N-P h en -
ylca r ba m a te (+)-10. Yield: 34 mg (7.1%). Mp: 123 °C
(CHCl3). [R]22D: +11.3 (c, 2.8, CHCl3), 33% ee. 1H NMR: δ
1.50-2.21 (m, 6H), 4.09-4.20 (m, 1H), 4.82 (dddd, 2J H,F ) 33.6
3
3
3
Hz , J Ha,Ha ) 8.6 Hz, J Ha,Ha ) 7.4 Hz, J Ha,He ) 4.3 Hz, 1H),
3
4.81-4.98 (m, 1H, 1-CH), 6.69 (br s, 1H), 7.05 (tt, J H,H ) 7.3
4
3
4
Hz, J H,H ) 1.4 Hz, 1H), 7.29 (tt, J H,H ) 7.9 Hz, J H,H ) 1.9
Hz, 2H), 7.37 (dd, J H,H ) 8.5 Hz, J H,H ) 1.2 Hz, 2H). 13C
3
4
3
2
NMR: δ 24.2 (dt, J C,F ) 3.9 Hz), 30.0 (t), 36.9 (dt, J C,F
)
3
2
18.5 Hz), 66.1 (dt, J C,F ) 8.7 Hz), 73.1 (dd, J C,F ) 21.6 Hz),
1
89.5 (dd, J C,F ) 174.9 Hz), 118.7 (2d), 123.6 (d), 129.1 (2d),
137.7 (s), 152.7 (s). 19F NMR: δ -189.0 (m). GC-MS m/z: 254
(6), 253 (38), 138 (4), 137 (20), 120 (7), 119 (46), 117 (2), 99
(26), 93 (100), 91 (5), 77 (16), 69 (14), 65 (8), 57 (7), 43 (6), 41
(24). High-resolution MS: calcd for C13H16NO3F 253.1114,
found 253.1102.
(1R,2R)-(-)-2-F lu or ocycloh exyl N-(p-Hyd r oxyp h en yl)-
ca r ba m a te (-)-13. Yield: 15 mg (3.1%). [R]23D: -16.7 (c, 1.7,
CHCl3). 1H NMR: δ 1.18-1.82 (m, 6H), 2.02-2.20 (m, 2H),
2
3
3
4.42 (dddd, J H,F ) 50.5 Hz, J Ha,Ha ) 10.2 Hz, J Ha,Ha ) 8.4
3
Hz, J Ha,He ) 4.8 Hz, 1H), 4.72-4.88 (m, 1H), 6.50 (br s, 1H),
3
4
3
6.73 (dt, J H,H ) 9.5 Hz, J H,H ) 2.8 Hz, 2H), 7.17 (br d, J H,H
) 8.8 Hz, 2H). 13C NMR: δ 22.9 (dt, J C,F ) 7.5 Hz), 23.1 (t),
3
3
2
29.8 (dt, J C,F ) 5.1 Hz), 30.5 (dt, J C,F ) 17.8 Hz), 75.2 (dd,
2J C,F ) 17.8 Hz), 92.2 (dd, J C,F ) 178.0 Hz), 115.8 (2d), 121.6
1
(2d), 130.5 (s), 152.5 (s) 153.7 (s). 19F NMR: δ -181.7 (d, 2J F,H
) 50.5 Hz). GC-MS m/z: 255 (0.5), 254 (10), 253 (77), 154
(4), 153 (63), 140 (12), 135 (22), 110 (8), 109 (100), 108 (24),
107 (6), 82 (7), 81 (71), 79 (10), 65 (4), 59 (16), 55 (21), 53 (15),
52 (9), 51 (4), 41 (38). High-resolution MS: calcd for C13H16
NO3F + NH4 271.1458, found 271.1440.
(-)-tr a n s-2-F lu or ocycloh exyl N-[p-(4′-Meth yl-â-D-glu -
cop yr a n osyl)]p h en ylca r ba m a te (-)-14. Yield: 121 mg
-
+
(1S,2S,4R)-(+)-2-F lu or o-4-h yd r oxycycloh exyl N-P h en -
ylca r ba m a te (+)-10. Yield: 35 mg (7.3%). Mp: 122 °C
(CHCl3). [R]22
: +34.4 (c 1.2, CHCl3). High-resolution MS:
D
(14.9%). Mp: 186-188 °C (cyclohexane/ethyl acetate). [R]22
:
D
+
calcd for C13H16NO3F + NH4 271.1458, found 271.1441.
(1S,2S)-(+)-2-F lu or ocycloh exyl N-(p-Hyd r oxyp h en yl)-
ca r ba m a te (+)-13. Yield: 11 mg (2.3%). [R]22D: +46.2 (c 0.6,
CHCl3). High-resolution MS: calcd for C13H16NO3F + NH4:
271.1458, found 271.1409.
1
-49.3 (c 0.5, methanol). H NMR: δ 1.26-2.25 (m, 8H), 3.23
3
3
(dd, J Ha,Ha ) 9.8 Hz, J Ha,Ha ) 9.5 Hz), 3.38-3.94 (m, 6H),
2
3
3.63 (s, 3H), 4.50 (dddd, J H,F ) 50.3 Hz, J Ha,Ha ) 10.0 Hz,
3J Ha,Ha ) 8.6 Hz, 3J Ha,He ) 4.8 Hz, 1H), 4.69-4.89 (m, 1H), 7.08
3
4
3
(dt, J H,H ) 9.1 Hz, J H,H ) 2.7 Hz, 2H), 7.37 (br d, J H,H ) 8.8
Tr a n sfor m a tion of (1R,2R)-(-)-2-F lu or ocycloh exyl N-
P h en ylca r ba m a te (-)-3. From the transformation of car-
bamate (-)-3 (450 mg, 1.90 mmol) in addition to the trans-
formation products 2-6 was recovered 59 mg (13%) of (-)-3.
The spectroscopic data of the isolated products agree with
those determined for the products obtained by biotransforma-
tion of racemic 3.
Hz, 2H). 13C NMR: δ 24.1 (dt, J C,F ) 10.2 Hz), 24.4 (t, C-5),
3
3
2
31.0 (dt, J C,F ) 5.1 Hz), 31.8 (dt, J C,F ) 17.8 Hz), 61.1 (q),
2
62.4 (t), 75.3, 77.4, 78.3, 80.9 (4d), 72.2 (dd, J C,F ) 20.3 Hz),
1
93.6 (dd, J C,F ) 175.5 Hz), 103.0 (d), 118.6 (2d), 121.8 (2d),
135.0 (s), 155.3 (s) 155.9 (s). 19F NMR: δ -180.9 (d, J F,H
)
2
53.4 Hz). ESI-MS, nanospray, m/z: 474 (40), 428 (100), 310
(18), 133 (13).
(1R,2R,4R)-(-)-2-F lu or o-4-h yd r oxycycloh exyl N-P h en -
ylca r ba m a te (-)-9. Yield: 59 mg (12.3%). Mp: 159-161 °C
(cyclohexane/ethyl acetate). [R]20D: -49.6 (c 1.0, CHCl3). High-
resolution MS: calcd for C13H16NO3F + H+ 254.1192, found
254.1177.
(1R,2R,4S)-(-)-2-F lu or o-4-h yd r oxycycloh exyl N-P h en -
ylca r ba m a te (-)-10. Yield: 16 mg (3.4%). Mp: 122 °C
(CHCl3). [R]22D: -33.2° (c 0.5, CHCl3). High-resolution MS:
calcd for C13H16NO3F 253.1142, found 253.1144.
To determine the structure of (-)-14, this compound was
acetylated and the structure of the product was determined.
(-)-tr a n s-2-F lu or ocycloh exyl N-[p-(4′-Meth yl-â-D-glu -
cop yr a n oa cetyl)]p h en ylca r ba m a te. Compound (-)-14 (22
mg, 0.05 mmol) was dissolved in acetic anhydride (1 mL, 10
mmol) and pyridine (2 mL, 25 mmol) and stored at rt for 12 h.
Pyridine and acetic anhydride were removed in vacuo, and the
residue was dissolved in CH2Cl2. The solution was washed with
2 N HCl (5 × 2 mL) and dried (MgSO4) overnight. The solvent
was removed, and the residue was filtered through a short
silica gel column using cyclohexane/ethyl acetate 4:1 as an
(1R,2R)-(-)-2-F lu or ocycloh exyl N-(p-Hyd r oxyp h en yl)-
ca r ba m a te (-)-13. Yield: 28 mg (5.8%). [R]22D: -49.9° (c 0.9,

3028 J . Org. Chem., Vol. 67, No. 9, 2002
Haufe et al.
+
CHCl3). High-resolution MS: calcd for C13H16NO3F + NH4
(0.781 e T e 0.881), Z ) 8, monoclinic, space group P21/n (No.
14), λ ) 1.541 78 Å, T ) 223 K, ω/2θ scans, 5310 reflections
collected ((h, +k, +l), [(sin θ)/λ] ) 0.62 Å-1, 5182 independent
(Rint ) 0.151) and 3941 observed reflections [I g 2σ(I)], 354
refined parameters, R ) 0.047, wR2 ) 0.131, max. residual
electron density 0.22 (-0.23) e Å-3, fluorine atoms disordered
between the C2/C6 (C22/C26) positions with the ratios of 0.85-
(1):0.15 (0.53(1):0.47), two almost identical independent mol-
ecules in the asymmetric unit, connected by hydrogen bonds,
hydrogen atoms at nitrogen atoms from difference Fourier
calculations, others calculated, all refined as riding atoms.
271.1458, found 271.1424.
Biotr a n sfor m a tion of (()-tr a n s-2-F lu or ocycloh ep tyl
N-P h en ylca r ba m a te (()-6. From the transformation of
carbamates (()-6, (+)-6, and (-)-6 (450 mg, 1.79 mmol) with
B. bassiana under standard conditions, in addition to some
amount of starting material, the transformation products 15-
18 were detected in the 19F NMR spectra of the crude product
mixtures. The relative amounts of compounds 15-18 deter-
mined by integration of the signals in the 19F NMR spectra
are given in Table 2. The structures of the products have
already been determined.7
X-r a y cr ysta l str u ctu r e a n a lysis of (S,S,S)-(+)-9: for-
mula C13H16FNO3, M ) 253.27, colorless crystal 0.20 × 0.20
× 0.20 mm, a ) 9.162(1) Å, b ) 11.137(1) Å, c ) 12.587(1) Å,
â ) 99.20(1)°, V ) 1267.8(2) Å3, Fcalcd ) 1.327 g cm-3, µ ) 8.71
cm-1, empirical absorption correction via ψ scan data (0.845
e T e 0.845), Z ) 4, monoclinic, space group P21(No. 4), λ )
1.541 78 Å, T ) 223 K, ω/2θ scans, 2880 reflections collected
Ack n ow led gm en t. Financial support by the Fonds
der Chemischen Industrie is gratefully acknowledged.
We are grateful to Dr. H. Luftmann and Dr. K. Ber-
gander for their help concerning the mass spectra and
the NMR spectra. We wish to thank the Bayer AG,
Leverkusen, Germany, and Novo Nordisk A/S, Copen-
hagen, Denmark, for kind donation of chemicals and
enzymes, respectively.
(+h, +k, (l), [(sin θ)/λ] ) 0.62 Å-1, 2712 independent (Rint
)
0.032) and 2519 observed reflections [I g 2σ(I)], 334 refined
parameters, R ) 0.034, wR2 ) 0.093, maximum residual
electron density 0.18 (-0.17) e Å-3, Flack parameter 0.0(2),
two almost identical independent molecules in the asymmetric
unit, connected by hydrogen bonds, hydrogen atoms at nitro-
gen atoms from difference Fourier calculations, others calcu-
lated, all refined as riding atoms.
X-r a y cr ysta l str u ctu r e a n a lysis of (S,S,R)-(+)-10: for-
mula C13H16FNO3, M ) 253.27, colorless crystal 0.30 × 0.25
× 0.15 mm, a ) 8.783(2) Å, b ) 12.746(3) Å, c ) 22.848(6) Å,
â ) 93.54(2)°, V ) 2552.9(11) Å3, Fcalcd ) 1.318 g cm-3, µ )
8.66 cm-1, empirical absorption correction via ψ scan data
Su p p or tin g In for m a tion Ava ila ble: 1H, 13C, and 19F
NMR spectral data including assignments of the signals and
the EI mass spectral data of the carbamates 3 and 6 and their
transformation products, and details of the X-ray crystal
structure analyses of (S,S,S)-(+)-9 and (S,S,R)-(+)-10. This
material is available free of charge via the Internet at
http://pubs.acs.org.
J O016332J