Syntheses of new phosphorus-containing azabicycloalkanes and their microbial hydroxylation using Beauveria bassiana

Article abstract of DOI:10.1021/jo9904664

Representative novel phosphorus-containing azabicyclic substrates have been synthesized and subsequently microbially hydroxylated in fair to good yields using the microorganism Beauveria bassiana. (7-Azabicyclo[2.2.1]hept- 7-yl)phosphonic acid diethyl ester was hydroxylated at the unactivated methylene carbon to give (2-endo-hydroxy-7-azabicyclo[2.2.1]hept-7- yl)phosphonic acid diethyl ester in 43% yield and 64% ee, while N- (diphenylphosphinoyl)-7-azabicyclo[2.2.1]heptane was similarly hydroxylated to give 2-endo-hydroxy-7-(diphenylphosphinoyl)-7-azabicyclo[2.2.1]heptane in 35% yield and 20% ee. (7-Azabicyclo[2.2.1]hept-7-yl)phosphonic acid diphenyl ester yielded two distinct hydroxylated products: monohydroxylated (2-endo- hydroxy-7-azabicyclo[2.2.1]hept-7-yl)phosphonic acid diphenyl ester in 7% yield and 7% ee and dihydroxylated (2-endo-hydroxy-7-azabicyclo[2.2.1]hept-7- yl)phosphonic acid phenyl, p-hydroxyphenyl ester in 37% yield and 77% ee. HPLC studies indicated that the monohydroxylated metabolite is formed first during fermentation, and becomes a substrate for a second enzymatic hydroxylation at one of the aromatic rings with induced enantioselection, to give the dihydroxylated metabolite. All microbially hydroxylated metabolites were easily N-deprotected using TFA-CH₂Cl₂ (1:1). Thus, N-phosphinyl groups are good facilitators of hydroxylation reactions with B. bassiana and offer a new choice for an N-substituent when substrates are hydroxylated with this microorganism. By offering a new N-substituent, this work extends the general utility of B. bassiana as a preparatively useful unactivated methylene hydroxylator.

Full text of DOI:10.1021/jo9904664

6312
J . Org. Chem. 1999, 64, 6312-6318
Syn th eses of New P h osp h or u s-Con ta in in g Aza bicycloa lk a n es a n d
Th eir Micr obia l Hyd r oxyla tion Usin g Bea u ver ia ba ssia n a
Michael S. Hemenway and Horacio F. Olivo*
Division of Medicinal & Natural Products Chemistry, College of Pharmacy and The Center for
Biocatalysis and Bioprocessing, The University of Iowa, Iowa City, Iowa 52242
Received March 15, 1999
Representative novel phosphorus-containing azabicyclic substrates have been synthesized and
subsequently microbially hydroxylated in fair to good yields using the microorganism Beauveria
bassiana. (7-Azabicyclo[2.2.1]hept-7-yl)phosphonic acid diethyl ester was hydroxylated at the
unactivated methylene carbon to give (2-endo-hydroxy-7-azabicyclo[2.2.1]hept-7-yl)phosphonic acid
diethyl ester in 43% yield and 64% ee, while N-(diphenylphosphinoyl)-7-azabicyclo[2.2.1]heptane
was similarly hydroxylated to give 2-endo-hydroxy-7-(diphenylphosphinoyl)-7-azabicyclo[2.2.1]-
heptane in 35% yield and 20% ee. (7-Azabicyclo[2.2.1]hept-7-yl)phosphonic acid diphenyl ester
yielded two distinct hydroxylated products: monohydroxylated (2-endo-hydroxy-7-azabicyclo[2.2.1]-
hept-7-yl)phosphonic acid diphenyl ester in 7% yield and 7% ee and dihydroxylated (2-endo-hydroxy-
7-azabicyclo[2.2.1]hept-7-yl)phosphonic acid phenyl, p-hydroxyphenyl ester in 37% yield and 77%
ee. HPLC studies indicated that the monohydroxylated metabolite is formed first during fermenta-
tion, and becomes a substrate for a second enzymatic hydroxylation at one of the aromatic rings
with induced enantioselection, to give the dihydroxylated metabolite. All microbially hydroxylated
metabolites were easily N-deprotected using TFA-CH2Cl2 (1:1). Thus, N-phosphinyl groups are
good facilitators of hydroxylation reactions with B. bassiana and offer a new choice for an
N-substituent when substrates are hydroxylated with this microorganism. By offering a new
N-substituent, this work extends the general utility of B. bassiana as a preparatively useful
unactivated methylene hydroxylator.
In tr od u ction
Microbial hydroxylation of unactivated methylene car-
bons1 still remains one of the most impressive examples
of transformations not easily emulated with conventional
organic chemistry.2 Our laboratory has become increas-
ingly interested in microbial hydroxylations of unacti-
vated methylenes in certain azabicyclic substrates of the
type illustrated in Figure 1. These 7-azabicyclo[2.2.1]-
heptanes are of interest to us because they are easy to
prepare and constitute intermediates in a synthesis of
the natural product epibatidine.3 The field of microbial
hydroxylations is well-documented, with pioneering work
F igu r e 1. 7-Azabicyclo[2.2.1]heptanes for microbial hydroxy-
lation.
done by Fonken and J ohnson et al.,1c,4 and later work
1h,6
done by the research groups of Holland,1d,5 Furstoss
and Roberts.1b,7 More recent contributions from Griengl,8
(3) For references on epibatidine see: (a) Spande, T. F.; Garraffo,
H. M.; Edwards, M. W.; Yeh, H. J . C.; Pannell, L.; Daly, J . W. J . Am.
Chem. Soc. 1992, 114, 3475. (b) Fletcher, S. R.; Baker, R.; Chambers,
M. S.; Herbert, R. H.; Hobbs, S. C.; Thomas, S. R.; Verrier, H. M.; Watt,
A. P.; Ball, R. G. J . Org. Chem. 1994, 59, 1771. (c) Sza´ntay, C.; Kardos-
Balogh, Z.; Sza´ntay, C., J r. In The Alkaloids; Cordell, G. A., Ed.;
Academic Press: San Diego, 1995; Vol. 46, p 95.
* To whom correspondence should be addressed. Fax: (319) 335-
8766. E-mail: Horacio-Olivo@uiowa.edu.
(1) For reviews see: (a) J ohnson, C. R.; Wells, G. W. Curr. Opin.
Chem. Biol. 1998, 2, 70. (b) Roberts, S. M.; Turner, N. J .; Willetts, A.
J .; Turner, M. K. Introduction to Biocatalysis Using Enzymes and
Micro-Organisms; Cambridge University Press: Cambridge, 1995. (c)
J ohnson, R. A. Oxidation with Microorganisms. In Oxidation in
Organic Chemistry; Trahanowsky, W. S., Ed.; Academic Press: New
York, 1978; Vol. 50. (d) Holland, H. L. Organic Synthesis with Oxidative
Enzymes; VCH: New York, 1992. (e) Faber, K. Biotransformations in
Organic Chemistry; Springer-Verlag: Berlin, 1995. (f) J ones, B. J .; Sih,
G. J .; Perlman, D. Applications of Biochemical Systems in Organic
Chemistry; Wiley and Sons: New York, 1976; Vol. 10. (g) Kieslich, K.
Biotransformations in Biotechnology: A Comprehensive Treatise; Rehm,
H. J ., Reed, G., Eds.; VCH: Weinheim, 1984; Vol. 6a. (h) Furstoss, R.;
Archelas, A.; Fourneron, J . D.; Vigne, B. In Enzymes as Catalysts in
Organic Synthesis; Schneider, M. P., Ed.; NATO ASI Series; Reidel
Dortrecht: Norwell, MA, 1986; Vol. 178, p 361.
(2) For reviews see: (a) Davis, J . A.; Watson, P. L.; Liebmann, J .
F.; Greenberg, A. Selective Hydrocarbon Activation; VCH: New York,
1990. (b) Hill, C. L. Activation and Functionalization of Alkanes; Wiley
and Sons: New York, 1989. (c) Shilov, A. V. Activation of Saturated
Hydrocarbons by Transition Metal Complexes; Reisel: Boston, 1984.
(d) Reiser, O. Angew. Chem., Int. Ed. Engl. 1994, 33, 69. (e) Ostovic,
D.; Bruice, T. C. Acc. Chem. Res. 1992, 25, 314. (f) Barton, D. H. R.;
Doller, D. Acc. Chem. Res. 1992, 25, 504.
(4) (a) Fonken, G. S.; Herr, M. E.; Murray, H. C.; Reineke, L. M. J .
Am. Chem. Soc. 1967, 89, 672. (b) Fonken, G. S.; Herr, M. E.; Murray,
H. C.; Reineke, L. M. J . Org. Chem. 1968, 33, 3182. (c) J ohnson, R. A.;
Herr, M. E.; Murray, H. C.; Fonken, G. S. J . Org. Chem. 1968, 33,
3187. (d) J ohnson, R. A.; Herr, M. E.; Murray, H. C.; Reineke, L. M.;
Fonken, G. S. J . Org. Chem. 1968, 33, 3195. (e) Herr, M. E.; J ohnson,
R. A.; Murray, H. C.; Reineke, L. M.; Fonken, G. S. J . Org. Chem. 1968,
33, 3201. (f) J ohnson, R. A.; Murray, H. C.; Reineke, L. M.; Fonken,
G. S. J . Org. Chem. 1968, 33, 3207. (g) J ohnson, R. A.; Herr, M. E.;
Murray, H. C.; Fonken, G. S. J . Org. Chem. 1968, 33, 3217. (h) J ohnson,
R. A.; Herr, M. E.; Murray, H. C.; Fonken, G. S. J . Org. Chem. 1970,
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C.; Pschigoda, L. M. J . Org. Chem. 1970, 35, 3607. (j) J ohnson, R. A.;
Herr, M. E.; Murray, H. C.; Reineke, L. J . Am. Chem. Soc. 1971, 22,
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Han, F. J . Org. Chem. 1992, 57, 7209. (l) J ohnson, R. A.; Herr, M. E.;
Murray, H. C.; Krueger, W. C.; Pschigoda. L. M.; Duchamp, D. J . J .
Org. Chem. 1992, 57, 7212. (m) Davis, C. R.; J ohnson, R. A.; Cialdella,
J . I.; Liggett, W. F.; Mizsak, S. A.; Marshall, V. P. J . Org. Chem. 1997,
62, 2244. (n) Davis, C. R.; J ohnson, R. A.; Cialdella, J . I.; Liggett, W.
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10.1021/jo9904664 CCC: $18.00 © 1999 American Chemical Society
Published on Web 07/29/1999

Syntheses of Azabicycloalkanes
J . Org. Chem., Vol. 64, No. 17, 1999 6313
Haufe,9 and Turner10 have helped to solidify the field of
microbial hydroxylations and establish this biotransfor-
mation as a routine endeavor.
Our first report in this field explored the microbial
hydroxylation of N-benzoyl substrate 1 and N-benzene-
sulfonyl substrate 211 using the popular and well-studied
fungus Beauveria bassiana (ATCC 7159).12 Other N-
substituents and related functionalities have included
various amides,4,6 lactams,6f,i N-carbamoyl,6e-g,9 and
N-acetyl4e,7c groups. Recently, J ohnson’s research group
reported on the microbial hydroxylation of a variety of
N-substituted azabicycloalkanes, utilizing N-BOC, N-
Cbz, N-tosyl, and N-phenyloxycarbonyl functional
groups.4m All of the above-mentioned functional groups
are electron-rich centers which, according to the prevail-
ing theory,1c,4a,g-j act as anchoring/directing groups while
the substrate is at the active site of the enzyme system-
(s) performing the oxidation. Recent modifications to this
theory include Haufe’s refined distance model9b to allow
F igu r e 2. Metabolic activation step for cyclophosphamide.
greater prediction of the hydroxylation position, and the
suggestion by Turner that the active site of the hydroxy-
lating enzyme contains a defined aromatic binding
pocket.10 Given these literature precedents, we hypoth-
esized that N-phosphinyl substituents, which are also
electron-rich moieties, might also constitute acceptable
anchoring groups for these microbial hydroxylations.
Shown in Figure 1 are the specific phosphinyl substitu-
ents that we examined (3-5).
There are several reasons why we envisaged these
azabicyclic phosphoramidates and phosphinamides to be
good substrates. First, the PdO bond of these substrates
would mimic the carbonyl and sulfonyl functionalities of
known anchoring groups and thus might be expected to
function in the same manner while at the enzyme’s active
site. Roberts et al. postulated that the carbonyl/sulfonyl
might be participating in noncovalent bonding with an
amino acid residue at the active site of the oxidating
enzyme.7d Second, these molecules are structurally simi-
lar to phosphates, which are ubiquitous in nature and
living systems. Thus, we anticipated that these sub-
strates would be biologically compatible with the fungal
cells of B. bassiana without being toxic to them.
One final piece of suggestive evidence comes from
cyclophosphamide,13 an anti-cancer drug that has been
in clinical use for decades (Figure 2). Cyclophosphamide
becomes metabolically activated to the actual drug
substance via methylene hydroxylation at C-4 by a
cytochrome P450 enzyme system in the liver.14 Once it
enters tumor cells, the six-membered ring opens and the
drug then alkylates the cell’s DNA. Given the structural
similarity between cyclophosphamide and our proposed
phosphorus-containing substrates, it was reasonable to
expect that these molecules would be taken up by the
cultures and be compatible with their cellular physiology.
For these reasons, we have pursued the synthesis of a
few representative phosphorus-containing azabicyclic
substrates and their subsequent microbial hydroxylation
using B. bassiana. Herein, we describe the results of
these experiments.
(5) (a) Holland, H. L.; Auret, B. J . Can. J . Chem. 1975, 53, 845. (b)
Holland, H. L.; Auret, B. J . Can. J . Chem. 1975, 53, 2041. (c) Holland,
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Chem. 1981, 59, 1651. (h) Holland, H. L.; Taylor, G. J . Can. J . Chem.
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Chenchaiah, P. C.; Brown, F. M. Tetrahedron Lett. 1988, 29, 6393.
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L. Tetrahedron Lett. 1980, 21, 451. (b) Furstoss, R.; Archelas, A.;
Waegell, B.; Le Petit, J .; Deveze, L. Tetrahedron Lett. 1981, 22, 445.
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Archelas, A.; Furstoss, R.; Waegell, B.; Le Petit, J .; Deveze, L.
Tetrahedron 1984, 40, 355. (e) Vigne, B.; Archelas, A.; Fourneron, J .
D.; Furstoss, R. Tetrahedron 1986, 42, 2451. (f) Archelas, A.; Furstoss,
R.; Srairi, D.; Maury, G. Bull. Chem. Soc. Fr. 1986, 2, 234. (g)
Fourneron, J . D.; Archelas, A.; Vigne, B.; Furstoss, R. Tetrahedron
1987, 43, 2273. (h) Vigne, B.; Archelas, A.; Fourneron, J . D.; Furstoss,
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(j) Fourneron, J . D.; Archelas, A.; Furstoss, R. J . Org. Chem. 1989,
54, 2478. (k) Fourneron, J . D.; Archelas, A.; Furstoss, R. J . Org. Chem.
1989, 54, 4686. (l) Zhang, X. M.; Archelas, A.; Furstoss, R. J . Org.
Chem. 1991, 56, 3814. (m) Vigne, B.; Archelas, A.; Furstoss, R.
Tetrahedron 1991, 47, 1447. (n) Pedragosa-Moreau, S.; Archelas, A.;
Furstoss, R. J . Org. Chem. 1993, 58, 5533.
(7) (a) Davies, H. G.; Dawson, M. J .; Lawrence, G. C.; Mayall, J .;
Noble, D.; Roberts, S. M.; Turner, M. K.; Wall, W. F. Tetrahedron Lett.
1986, 27, 1089. (b) Carruthers, W.; Prail, J . D.; Roberts, S. M.; Willetts,
A. J . J . Chem. Soc., Perkin Trans. 1 1990, 2854. (c) Floyd, N.;
Munyemana, F.; Roberts, S. M.; Willetts, A. J . J . Chem. Soc., Perkin
Trans. 1 1993, 881. (d) Ridyard, C. H.; Whittaker, R. A.; Higgins, S.
D.; Roberts, S. M.; Willetts, A. J .; Bailey, P. D.; Rosair, G. M. J . Chem.
Soc., Perkin Trans. 2 1996, 1811. (e) Bailey, P. D.; Higgins, S. D.;
Ridyard, C. H.; Roberts, S. M.; Rosair, G. M.; Whittaker, R. A.; Willetts,
A. J . Chem. Commun. 1996, 1833. (f) Palmer, C. F.; Webb, B.; Broad,
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N.; Weber, H.; Braunegg, G.; Kopper, I.; Kreiner, M.; Zeiser, A.;
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Griengl, H.; Petsch. M.; Plachota, P.; Schoo, N.; Weber, H.; Braunegg,
G.; Kopper, I.; Kreiner, M.; Zeiser, A. Tetrahedron: Asymmetry 1996,
7, 473 (c) de Raadt, A.; Griengl, H.; Petsch. M.; Plachota, P.; Schoo,
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Tetrahedron: Asymmetry 1996, 7, 491. (d) Morwaski, B.; Griengl, H.;
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Resu lts
Hassner has reported a synthesis of certain N-alkyl-
7-azabicyclo[2.2.1]heptanes in four steps starting from
monoprotected 1,4-cyclohexanedione.15 However, we chose
(11) Olivo, H. F.; Hemenway, M. S.; Gezginci, M. H. Tetrahedron
Lett. 1998, 39, 1309.
(12) Previously known as Sporotrichum sulfurescens and later
reclassified as Beauveria sulfurescens; see: Taylor, J . J . Mycol. 1970,
62, 797. This microorganism is currently listed as Beauveria bassiana
(ATCC 7159) in the catalog of the American Type Culture Collection.
(13) Hill, D. L. A Review of Cyclophosphamide; Thomas: Springfield,
IL, 1975.
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1709.

6314 J . Org. Chem., Vol. 64, No. 17, 1999
Hemenway and Olivo
Ta ble 1. Biotr a n sfor m a tion Da ta for Su bstr a tes 3-5
medium Aa
medium Ba
%
%
%
%
%
abs
substrate metab convb yieldc convb yieldc eed [R]25 conf
D
3
4
4
5
13
14
16
15
97
100
100
99
36
11
34
3
92
100
100
88
43
7
37
35
64 +2.7 (1S)
7
+3.5 (1S)
77 +4.7
20 -8.1 (1R)
a
The compositions of medium A and medium B are provided
b
in the Experimental Section. Based on the amount of substrate
recovered by column chromatography. c After isolation and puri-
fication by column chromatography. Based on 1H and 19F NMR
d
analyses of Mosher ester derivatives and confirmed by HPLC
resolution of the same derivatives. These % ee values apply to
metabolites isolated from both growth media.
phorylating agents22 or N-blocking and coupling reagents
in peptide synthesis.23 Although it might appear that we
are also using these phosphinyl moieties as N-protecting
groups, our primary interest in them lies in their function
as facilitators of unactivated methylene hydroxylation
using B. bassiana.24
For all three substrates examined, we indeed observed
the desired hydroxylation, and in moderate to good yields
(Table 1). Originally, only medium A was utilized in these
biotransformations, but at the end of the fermentation
the pH of the cultures was found to be very acidic (1.3-
3.7) when using this medium. Although this acidity did
not appear to be detrimental to the yields of metabolites
13, 14, and 16, we suspected that it might have contrib-
uted to the very low yield (3%) of metabolite 15. For this
reason, medium B was added to our experimental pro-
tocol and all substrates were reexamined. Table 1 indi-
cates that medium B (ending pH 5.1-7.5) is the preferred
growth medium, with the isolated yields of metabolites
13 and 16 slightly higher, and the yield of metabolite 15
much higher (35%). Hydroxylation of the aromatic ring
of substrate 4 was an unforeseen discovery, since our
F igu r e 3. Synthesis and microbial hydroxylation of substrates
3-5. Reagents and conditions: (a) R2P(O)Cl, Et3N, DMF, 0
°C; (b) MsCl, Et3N, CH2Cl2, 0 °C; (c) KOtBu, 1:1 benzene-DMF;
(d) stage II B. bassiana cultures, initial pH 5.0, 29 °C, 7 days.
to use our shorter route starting from commercially
available trans-4-aminocyclohexanol hydrochloride.11 Af-
ter the hydrochloride salt was converted to its free base
6, it was treated with the appropriate phosphorus
chloride (diethylchlorophosphate, diphenylchlorophos-
phate, or diphenylphosphinic chloride) and triethylamine
in DMF (Figure 3). The resulting alcohols 7-9 were
converted to mesylates 10-12 using methanesulfonyl
chloride and triethylamine in CH2Cl2. Cyclization oc-
curred in good yields in the presence of potassium tert-
butoxide to give the azabicyclic substrates 3-5. Thus,
these substrates are synthesized in only three steps and
in good overall yields.
Substrates 3-5 were subjected to stage II liquid
cultures of B. bassiana at 28-29 °C, 250 rpm, and 50%
relative humidity for 168 h (7 days) of continuous bio-
transformation (Figure 3). Diethylphosphoramidate 3
was hydroxylated at the unactivated methylene to give
metabolite 13 in 43% yield and 64% enantiomeric excess,
with 92% conversion. Diphenylphosphinamide 5 was
hydroxylated in 35% yield, 20% enantiomeric excess, and
88% conversion to provide metabolite 15. Hydroxylation
of diphenyl phosphoramidate 4 resulted in metabolite 14
in 7% yield and 7% enantiomeric excess and dihydroxy-
lated metabolite 16 in 37% yield and 77% enantiomeric
excess. Table 1 compares biotransformation data using
two different growth media.
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(23) (a) Wolfrom, M. L.; Conigliaro, P. J .; Soltes, E. J . J . Org. Chem.
1967, 32, 653. (b) Kenner, G. W.; Moore, G. A.; Ramage, R. Tetrahedron
Lett. 1976, 40, 3623. (c) Zhao, Y.-F.; Xi, S.-K.; Song, A.-T.; J i, G.-J . J .
Org. Chem. 1984, 49, 4549. (d) Zwierzak, A. Osowska, K. Synthesis
1984, 223. (e) Dudash, J ., J r.; J iang, J .; Mayer, S. C.; J oullie´, M. M.
Synth. Commun. 1993, 23, 349.
(24) We were unable to isolate hydroxylated metabolites when the
free amine 7-azabicyclo[2.2.1]heptane was subjected to cultures of B.
bassiana following our biotransformation protocol.
Discu ssion
Research concerning phosphoramidates and phos-
phinamides continues to accumulate in the literature,
although papers on the former are more abundant.16-21
Diethylchlorophosphate, diphenylchlorophosphate, and
diphenylphosphinic chloride have all been used as phos-

Syntheses of Azabicycloalkanes
J . Org. Chem., Vol. 64, No. 17, 1999 6315
Ta ble 2. Dep r otection Da ta for Meta bolites 13-16
metab
% yielda
time (h)
metab
% yielda
time (h)
13
14
91
95
6
10
16
15
97
99
10
3
a
Determined indirectly using gas chromatography.
substrate. While the yield and % ee for monohydroxylated
metabolite 14 are much lower than those for J ohnson’s
N-phenyloxycarbonyl substrate, the yield for the dihy-
droxylated metabolite 16 is quite comparable (37% vs
46%), but the ee is somewhat higher (77% vs 51%).
Moreover, J ohnson et al. did not observe any aromatic
ring para-hydroxylation. Diphenylphosphinamide 5 could
be compared with our previously studied N-benzoyl-7-
azabicyclo[2.2.1]heptane substrate 1.11 Compared to the
metabolite derived from 1, the yield of metabolite 15 is
noticeably lower (35% vs 57%), but the enantioselectivi-
ties are quite close (20% vs 22%).
We have found that all four microbial metabolites 13-
16 are readily N-deprotected to give the free secondary
amine using TFA-CH2Cl2 (1:1) at room temperature
(Table 2). These reaction conditions have been previously
studied by Ramage et al.27 Deprotection occurs in rela-
tively high yield and in reasonable reaction times. The
short reaction time for deprotection of diphenylphosphi-
namide metabolite 15 highlights the extreme acid-lability
of the N-P bond of diphenylphosphinamides found by
Ramage et al.27 The ability to easily deprotect these
N-phosphinyl metabolites after the biotransformation
step further extends their potential synthetic utility as
valuable intermediates.
In conclusion, we have demonstrated that N-phosphi-
nyl substituents are good facilitators of these hydroxy-
lations of unactivated carbons with B. bassiana. The
extent of bioconversion of substrates, the fair to good
isolated yields of hydroxylated metabolites, and the
enantiomeric excess (in two cases) are all encouraging
results, even though the yields and ee are not yet high
enough to be synthetically useful. These N-phosphinyl
substituents are easily cleaved under acidic conditions,
a fact which extends their potential as intermediates.
Finally, results with these N-phosphinyl substituents
suggest that there may be other anchoring/activating
groups yet to be discovered in the search for ways to effect
hydroxylation of unactivated carbons with B. bassiana.
F igu r e 4. Time course of the biotransformation of substrate
4 (closed circles) to metabolite 14 (open circles) and metabolite
16 (closed triangles) by B. bassiana.
previous work with N-benzoyl and N-benzenesulfonyl
substrates11 did not display any aromatic ring hydroxy-
lation.6e,h,7f,9a The observation that substrate 4 gave rise
to metabolites 14 and 16 caused us to speculate that
perhaps monohydroxylated product 14 is formed first
during the fermentation, and dihydroxylated product 16
is formed second and accumulates at the expense of
product 14. To test this hypothesis, a biotransformation
of substrate 4 was performed, during which 1 mL aliquots
of culture were taken at predetermined time points.
Normal-phase HPLC analysis of organic extracts of the
aliquots, summarized as a time course plot in Figure 4,
clearly indicates that metabolite 14 was indeed formed
first, with significant amounts of product formed within
the first 3 h of fermentation. Metabolite 16 formed soon
afterward, its accumulation occurring at the expense of
metabolite 14. The amount of substrate 4 decreases
exponentially over the course of the experiment. This
observation in which an enzymatic hydroxylation product
itself becomes a substrate for a second enzymatic hy-
droxylation has previously been reported for B. bassiana.
Furstoss et al. reported double hydroxylation of a cyclo-
hexyl N-phenyl carbamate6h over a decade ago, and more
examples of this are found in a review.25 Furthermore,
the hydroxyl group of metabolite 14 (7% ee by 19F NMR
analysis of the corresponding Mosher26 esters) apparently
has induced enantioselection in the second hydroxylation
step to give metabolite 16 (77% ee, as determined using
the same method). Although Furstoss et al. have reported
glucosylation of phenol metabolites,6e,h we did not isolate
any glucoside conjugate of the phenol of metabolite 16.
In comparing our results with those of J ohnson et al.
in hydroxylations of 7-azabicyclo[2.2.1]heptane systems
using B. bassiana, we find a similar range of percent
yields and enantioselectivities overall, but also some
important differences.4m Our diethyl phosphoramidate
substrate 3 is structurally most similar to J ohnson’s
t-BOC substrate, yet both our isolated yield (43% vs 10%)
and ee (64% vs 26%) are higher. Diphenyl phosphorami-
date 4 is similar to J ohnson’s N-phenyloxycarbonyl
Exp er im en ta l Section
Gen er a l Meth od s. The microorganism used was B. bassi-
ana, ATCC 7159 (Manassas, VA).12 Melting points were
determined in open capillary tubes and are uncorrected. IR
spectra were recorded using NaCl film or KBr disk techniques.
Optical rotations were determined using a J ASCO P-1020
polarimeter. 1H NMR spectra were recorded at 360 MHz, with
chemical shifts referenced to CDCl3 (δ 7.27), (CD3)2SO (δ 2.50),
or TMS (δ 0.00) as an internal standard. 13C NMR spectra were
recorded at 90 MHz, with chemical shifts referenced to either
CDCl3 (δ 77.23) or (CD3)2SO (δ 39.51). 19F NMR spectra were
recorded at 282 MHz, with chemical shifts referenced to CFCl3
(δ -98.07) as an external standard. 31P NMR spectra were
recorded at 360 MHz, with chemical shifts referenced to H3-
PO4 (δ 0.00) as an external standard. All mass spectra were
obtained at an ionizing voltage of 70 eV, in EI or FAB
(25) Furstoss, R.; Archelas, A.; Fourneron, J . D.; Vigne, B. Biohy-
droxylation of nonactivated carbon atoms by fungus Beauveria sulfer-
escens. In Organic SynthesissAn Interdisciplinary Challenge; Streith,
J ., Prinzbach, H., Schill, G., Eds.; Blackwell: Oxford, 1985; p 215.
(26) Dale, J . A.; Dull, D. L.; Mosher, H. S. J . Org. Chem. 1969, 34,
2543.
(27) (a) Ramage, R.; Hopton, D.; Parrott, M. J .; Kenner, G. W.;
Moore, G. A. J . Chem. Soc., Perkin Trans. 1 1984, 1357. (b) Ramage,
R.; Atrash, B.; Hopton, D.; Parrott, M. J . J . Chem. Soc., Perkin Trans.
1 1985, 1217.

6316 J . Org. Chem., Vol. 64, No. 17, 1999
Hemenway and Olivo
ionization modes, at The University of Iowa Mass Spectrom-
etry Facility. Analytical HPLC was performed on a normal-
phase column with UV detection at 254 nm. Elemental
analyses were performed at Galbraith Laboratories, Inc.,
Knoxville, TN. Methylene chloride and triethylamine were
each distilled from calcium hydride before use. Column chro-
matography was carried out using silica gel 60 (230-400
mesh). All reactions were monitored by TLC on Merck 60 F254
precoated silica gel plates (250 µm thickness), and spots were
visualized with UV light or with a series of two dip solutions:
ninhydrin, followed by p-anisaldehyde (heating after each dip).
Commercially available (Aldrich) trans-4-aminocyclohexanol
hydrochloride was converted to the free base form by exhaus-
tively extracting a solution of the HCl salt in 4 N aqueous
NaOH with ethyl acetate and subsequently removing the
solvent. All other solvents and materials were of reagent grade
and used without further purification.
HP LC An a lysis of F er m en ta tion Extr a cts. Aliquots (1
mL) of culture medium were taken at predetermined time
points. Each aliquot was shaken with 1 mL of CH2Cl2 and then
centrifuged. The organic layer was transferred to a clean vial,
concentrated to the absence of solvent, and redissolved in 100
µL of mobile phase. A 20 µL sample of the resulting solution
was injected for analysis. Chromatographic parameters: normal-
phase silica analytical column (250 mm × 4 mm, 5 µm
packing); 7:3 n-heptane-2-propanol mobile phase; UV detec-
tion at 254 nm; 1.0 mL/min flow rate.
J ) 8.6 Hz), 7.18 (2 H, d, J ) 7.3 Hz), 5.72 (1 H, dd, J ) 9.4,
13.2 Hz), 4.49 (1H, d, J ) 4.1 Hz), 3.30 (1 H, br s), 2.97 (1 H,
br s), 1.72 (2 H, d, J ) 8.8 Hz), 1.65 (2 H, d, J ) 8.8 Hz), 1.15
(4 H, m); 13C NMR ((CD3)2SO) δ 150.8 (d, J CP ) 6.7 Hz), 129.7,
124.7, 120.2 (d, J CP ) 5.3 Hz), 68.0, 50.4, 34.0, 32.7 (d, J CP
)
4.4 Hz); 31P NMR (CDCl3) δ -1.29; IR (KBr) νmax 3476, 3235,
1193, 947 cm-1; EIMS m/z (relative intensity) 347 (M+, 13),
329 (21), 289 (100), 250 (67), 77 (78); HRMS calcd for C18H22
NO4P 347.1286, found 347.1279.
-
N-(Dip h en ylp h osp h in oyl)-tr a n s-1,4-a m in ocycloh ex-
a n ol (9). A solution containing trans-4-aminocyclohexanol free
base 6 (1.16 g, 10.09 mmol) at 0 °C in DMF (30 mL) was
treated with triethylamine (1.23 g, 12.12 mmol) under argon.
The reaction mixture was treated 10 min later with diphen-
ylphosphinic chloride (2.27 g, 9.60 mmol). The reaction mixture
was stirred for 4 h, diluted with water, and extracted with
ethyl acetate. The organic layer was washed with brine and
dried over Na2SO4. The solvent was concentrated under
diminshed pressure to afford a residue which was purified by
flash chromatography on a silica gel column. Elution with 9:1
chloroform-methanol afforded 9 as a white solid: yield 1.29
g (41%); Rf 0.29 (9:1 chloroform-methanol); mp 215-216 °C;
1H NMR ((CD3)2SO) δ 7.79 (2 H, dd, J ) 1.5, 11.5 Hz), 7.77 (2
H, dd, J ) 1.8, 11.7 Hz), 7.47 (6 H, m), 5.15 (1 H, t, J ) 9.5
Hz), 4.44 (1 H, d, J ) 4.5 Hz), 3.30 (1 H, m), 2.69 (1 H, m),
1.85 (2 H, d, J ) 11.0 Hz), 1.74 (2 H, d, J ) 11.0 Hz), 1.33 (2
H, qd, J ) 2.9, 13.2 Hz), 1.02 (2 H, qd, J ) 2.9, 13.2 Hz); 13C
GC An a lysis for Deter m in a tion of Yield of N-Dep r o-
tection . A linear standard curve was generated using pure
2-endo-hydroxy-7-azanorbornane product. After TLC analysis
had indicated a completed deprotection reaction, an aliquot
of the reaction mixture was injected for analysis. The area
under the product peak was translated into a percent yield
using the standard curve and the corresponding equation for
a straight line. Chromatographic parameters: Alltech Heliflex
AT-1 capillary column (30 m × 0.53 mm i.d.); helium carrier
gas; flame ionization detector at 220 °C; injector temperature
200 °C; initial column temperature at 100 °C, increasing 30
°C/min to 250 °C, and then hold; 1.0 µL injection volume.
(tr a n s-1,4-Cycloh exa n olyl)p h osp h or a m id ic Acid Di-
eth yl Ester (7). A solution containing trans-4-aminocyclo-
hexanol free base 6 (1.84 g, 15.97 mmol) at 0 °C in DMF (50
mL) was treated with triethylamine (1.93 g, 19.10 mmol) under
argon. The reaction mixture was treated 10 min later with
diethylchlorophosphate (2.62 g, 15.21 mmol) under argon. The
reaction mixture was stirred for 3.5 h and then filtered through
filter paper to remove white solids which had developed. The
DMF was removed by distillation, leaving a residue which was
purified by flash chromatography on a silica gel column.
Elution with 9:1 chloroform-methanol afforded 7 as a colorless
oil: yield 3.06 g (76%); Rf 0.30 (9:1 chloroform-methanol); 1H
NMR (CDCl3) δ 4.06 (4 H, qd, J ) 1.4, 7.1 Hz), 3.60 (1 H, m),
2.98 (1 H, br s), 2.45 (1 H, br s), 1.99 (4 H, t, J ) 15.0 Hz),
1.87 (1 H, br s), 1.33 (6 H, td, J ) 7.1, 0.7 Hz), 1.43-1.15 (4
H, m); 13C NMR (CDCl3) δ 69.3, 62.3 (d, J CP ) 5.0 Hz), 50.1,
34.1, 33.5 (d, J CP ) 5.1 Hz), 16.3 (d, J CP ) 7.2 Hz); 31P NMR
(CDCl3) δ 8.84; IR (thin film) νmax 3253 (br), 1453, 1235, 1040,
977 cm-1; EIMS m/z (relative intensity) 251 (M+, 2.5), 192
(100), 136 (71); HRMS calcd for C10H22NO4P 251.1286, found
251.1292.
(tr a n s-1,4-Cycloh exa n olyl)p h osp h or a m id ic Acid Di-
p h en yl Ester (8). A solution containing trans-4-aminocyclo-
hexanol free base 6 (1.26 g, 10.97 mmol) at 0 °C in DMF (30
mL) was treated with triethylamine (1.33 g, 13.18 mmol) under
argon. The reaction mixture was treated 5 min later with
diphenylchlorophosphate (2.80 g, 10.43 mmol) under argon.
The reaction mixture was stirred for 4 h, diluted with water,
and extracted with ethyl acetate. The organic layer was
washed with brine and dried over Na2SO4. The solvent was
concentrated under diminished pressure to afford a residue,
which was purified by flash chromatography on a silica gel
column. Elution with 4:1 ethyl acetate-petroleum ether and
then ethyl acetate afforded 8 as a white solid: yield 2.82 g
(74%); Rf 0.36 (95:5 chloroform-methanol); mp 136-137 °C;
1H NMR ((CD3)2SO) δ 7.39 (4 H, t, J ) 7.8 Hz), 7.23 (4 H, d,
NMR ((CD3)2SO) δ 134.5 (d, J CP ) 126.0 Hz), 131.7 (d, J CP )
9.4 Hz), 131.3 (d, J CP ) 2.2 Hz), 128.4 (d, J CP ) 11.5 Hz), 68.1,
49.6, 34.4, 33.4 (d, J CP ) 5.1 Hz); 31P NMR (CDCl3) δ 25.62;
IR (KBr) νmax 3350 (br), 3256, 1438, 1179, 1104 cm-1; EIMS
m/z (relative intensity) 315 (M+, 15), 257 (60), 201 (100); HRMS
calcd for C18H22NO2P 315.1388, found 315.1386.
Gen er a l P r oced u r e for P r ep a r a tion of Mesyla tes 10-
12. A solution containing alcohol (7-9) in CH2Cl2 at 0 °C was
treated under argon with triethylamine (3.0 equiv). The
reaction mixture was treated 10 min later with methanesulfo-
nyl chloride (2.0 equiv) under argon. The reaction mixture was
stirred at 0 °C for 3 h and then added to 2 volumes of water.
The organic layer was washed with brine and dried over
Na2SO4. The solvent was concentrated under diminished
pressure to afford a residue which was purified by flash
chromatography on a silica gel column.
(tr a n s-1,4-Cycloh exa n ol m eth a n esu lfon yl)p h osp h or -
a m id ic Acid Dieth yl Ester (10). Elution with 95:5 methylene
chloride-methanol afforded 10 as white, needlelike crystals:
yield 2.47 g (62%); Rf 0.42 (9:1 chloroform-methanol); mp 70-
71 °C; 1H NMR (CDCl3) δ 4.62 (1 H, m), 4.05 (4 H, qd, J ) 1.5,
7.1 Hz), 3.04 (1 H, br s), 3.01 (3 H, s), 2.71 (1 H, br s), 2.10 (4
H, m), 1.66 (2 H, qd, J ) 2.7, 12.7 Hz), 1.33 (8 H, t, J ) 7.1
Hz); 13C NMR (CDCl3) δ 79.9, 62.0 (d, J CP ) 5.0 Hz), 48.7, 38.5,
32.1 (d, J CP ) 5.0 Hz), 30.8, 16.1 (d, J CP ) 7.3 Hz); 31P NMR
(CDCl3) δ 8.60; IR (KBr) νmax 3219, 1455, 1350, 1223, 1032
cm-1; EIMS m/z (relative intensity) 329 (M+, 1.1), 233 (36),
192 (100). Anal. Calcd for C11H24NO6PS: C, 40.12; H, 7.34; N,
4.25. Found: C, 40.06; H, 7.49; N, 4.21.
(tr a n s-1,4-Cycloh exa n ol m eth a n esu lfon yl)p h osp h or -
a m id ic Acid Dip h en yl Ester (11). Gradient elution with 1:1
ethyl acetate-petroleum ether, ending with ethyl acetate,
afforded 11 as a white, crystalline solid: yield 3.40 g (98%);
Rf 0.56 (9:1 chloroform-methanol); mp 116-117 °C; 1H NMR
((CD3)2SO) δ 7.40 (4 H, t, J ) 7.8 Hz), 7.25 (4 H, d, J ) 8.5
Hz), 7.19 (2 H, t, J ) 7.3 Hz), 5.85 (1 H, dd, J ) 9.4, 12.8 Hz),
4.50 (1 H, m), 3.15 (3 H, s), 3.13 (1 H, br s), 1.97 (2 H, d, J )
9.3 Hz), 1.74 (2 H, d, J ) 10.1 Hz), 1.53 (2 H, qd, J ) 3.1, 12.3
Hz), 1.31 (2 H, qd, J ) 2.4, 12.3 Hz); 13C NMR ((CD3)2SO) δ
150.7 (d, J CP ) 6.5 Hz), 129.8, 124.8, 120.2 (d, J CP ) 4.3 Hz),
79.9, 48.9, 37.7, 31.6 (d, J CP ) 5.7 Hz), 30.5; 31P NMR (CDCl3)
δ -1.42; IR (KBr) νmax 3226, 1347, 1180 cm-1; EIMS m/z
(relative intensity) 425 (M+, 0.5), 329 (59), 275 (79), 250 (100).
Anal. Calcd for C19H24NO6PS: C, 53.64; H, 5.67; N, 3.29.
Found: C, 53.09; H, 5.79; N, 3.09.
N-(Dip h en ylp h osp h in oyl)-tr a n s-1,4-a m in ocycloh ex-
a n ol Meth a n esu lfon a te (12). Elution with 95:5 chloroform-
methanol afforded 12 as a white solid: yield 1.33 g (94%); Rf

Syntheses of Azabicycloalkanes
J . Org. Chem., Vol. 64, No. 17, 1999 6317
0.47 (10% MeOH/CHCl3); mp 175-176 °C; 1H NMR (CDCl3) δ
7.89 (2 H, dd, J ) 1.3, 11.9 Hz), 7.87 (2 H, dd, J ) 1.5, 12.0
Hz), 7.48 (6 H, m), 4.59 (1 H, m), 3.05 (1 H, m), 2.97 (3 H, s),
2.74 (1 H, dd, J ) 6.0, 10.0 Hz), 2.20 (2 H, d, J ) 12.7 Hz),
2.11 (2 H, d, J ) 12.7 Hz), 1.57 (2 H, qd, J ) 3.3, 12.8 Hz),
1.39 (2 H, qd, J ) 3.1, 12.7 Hz); 13C NMR (CDCl3) δ 132.9 (d,
J ) 139.5 Hz), 132.2 (d, J CP ) 9.5 Hz), 128.8 (d, J CP ) 12.3
Hz), 79.9, 49.1, 38.9, 33.8 (d, J CP ) 5.1 Hz), 31.5; 31P NMR
(CDCl3) δ 24.25; IR (KBr) νmax 3137, 1438, 1351, 1198, 1112
cm-1; EIMS m/z (relative intensity) 394 (M + H, 100)+, 298
(79), 201 (52); HRMS (M + H)+ calcd for C19H25NO4PS
394.1242, found 394.1246.
Gen er a l P r oced u r e for P r ep a r a tion of Bicyclic Su b-
str a tes 3-5. A solution containing mesylate (10-12) in 1:1
DMF-benzene at 0 °C was treated under argon with potas-
sium tert-butoxide (3.0 equiv). The reaction mixture was
stirred for 3 h and then added to 8 volumes of water and 1
volume of additional benzene. The organic layer was washed
with brine and dried over Na2SO4. The solvent was concen-
trated under diminished pressure to afford a residue, which
was purified by flash chromatography on a silica gel column.
(7-Aza bicyclo[2.2.1]h ep t-7-yl)p h osp h on ic Acid Dieth yl
Ester (3). Elution with 9:1 ethyl acetate-petroleum ether
afforded 3 as a colorless oil: yield 1.24 g (74%); Rf 0.71 (9:1
chloroform-methanol); 1H NMR (CDCl3) δ 4.05 (6 H, m),
1.75 (4 H, d, J ) 6.8 Hz), 1.39 (4 H, d, J ) 6.8 Hz), 1.30 (6 H,
t, J ) 7.1 Hz); 13C NMR (CDCl3) δ 62.6 (d, J CP ) 5.8 Hz), 57.8
(d, J CP ) 2.2 Hz), 31.0 (d, J CP ) 5.9 Hz), 16.4 (d, J CP ) 6.8
Hz); 31P NMR (CDCl3) δ 6.46; IR (thin film) νmax 1275, 1256,
1028 cm-1; EIMS m/z (relative intensity) 233 (M+, 28), 204
(100), 148 (90); HRMS calcd for C10H20NO3P 233.1181, found
233.1171.
relative humidity for biotransformation. After 168 h, the cells
were removed from the cultures by vacuum filtration and the
filtrate was extracted with methylene chloride. The combined
organic layer was washed with brine and dried over Na2SO4.
The solvent was concentrated under diminished pressure to
afford a residue which was purified by flash chromatography
on a silica gel column. Elution with 9:1 chloroform-methanol
afforded 13 as a colorless oil: yield 186.3 mg (43%); Rf 0.24
(95:5 chloroform-methanol); [R]25 +2.7° (c ) 1.0, CHCl3); 1H
D
NMR (CDCl3) δ 4.33 (1H, m), 4.04 (4 H, quint, J ) 7.3 Hz),
3.91 (2 H, td, J ) 1.9, 4.8 Hz), 3.52 (1 H, br s), 2.19 (2 H, m),
1.79 (1 H, m), 1.67-1.49 (2 H, series of m), 1.30 (6 H, t, J )
7.1 Hz), 1.04 (1 H, dt, J ) 3.8, 12.3 Hz); 13C NMR (CDCl3) δ
71.7 (d, J CP ) 10.1 Hz), 62.7 (d, J CP ) 6.5 Hz), 61.4, 58.9, 39.7
(d, J CP ) 5.8 Hz), 31.1 (d, J CP ) 4.4 Hz), 21.9 (d, J CP ) 4.4
Hz), 16.3 (d, J CP ) 6.5 Hz); 31P NMR (CDCl3) δ 6.10; IR (thin
film) νmax 3364 (br), 1236, 1024 cm-1; EIMS m/z (relative
intensity) 249 (M+, 20), 205 (86), 112 (100); HRMS calcd for
C
10H20NO4P 249.1130, found 249.1148.
(2-en d o-Hyd r oxy-7-a za bicyclo[2.2.1]h ep t-7-yl)p h osp h o-
n ic Acid Dip h en yl Ester (14) a n d (2-en d o-Hyd r oxy-7-
a za bicyclo[2.2.1]h ep t-7-yl)p h osp h on ic Acid P h en yl, p-
H yd r oxyp h en yl E st er (16). A solution containing (7-
azabicyclo[2.2.1]hept-7-yl)phosphonic acid diphenyl ester 4
(1.00 g, 3.04 mmol) in 1:1 ethanol-DMF (5 mL) was equally
distributed among ten stage II preparative scale liquid cultures
of B. bassiana (prepared as described above). All flasks were
then placed on an orbital shaker at 250 rpm, 29-30 °C, and
50% relative humidity for biotransformation. After 168 h, the
cells were removed from the cultures by vacuum filtration and
the filtrate was extracted with methylene chloride. The
combined organic layer was washed with brine and dried over
Na2SO4. The solvent was concentrated to afford a residue
which was purified by flash chromatography on a silica gel
column. Elution with 9:1 chloroform-methanol afforded 14 as
(7-Aza bicyclo[2.2.1]h ep t-7-yl)ph osp h on ic Acid Dip h en -
yl Ester (4). Elution with 4:6 ethyl acetate-petroleum ether
afforded 4 as a white, crystalline solid: yield 2.06 g (81%); Rf
0.73 (9:1 chloroform-methanol); mp 75-76 °C; 1H NMR
(CDCl3) δ 7.31 (4 H, t, J ) 7.5 Hz), 7.25 (4 H, d, J ) 7.6 Hz),
7.14 (2 H, t, J ) 7.0 Hz), 4.25 (2 H, s), 1.72 (4 H, d, J ) 6.5
Hz), 1.41 (4 H, d, J ) 6.3 Hz); 13C NMR (CDCl3) δ 151.4 (d,
J CP ) 8.0 Hz), 129.7, 124.8, 120.3 (d, J CP ) 5.1 Hz), 58.5 (d,
J CP ) 2.1 Hz), 31.0 (d, J CP ) 5.8 Hz); 31P NMR (CDCl3) δ -4.75;
IR (KBr) νmax 3050, 1279, 1222, 1095 cm-1; EIMS m/z (relative
intensity) 329 (M+, 35), 300 (100), 77 (46). Anal. Calcd for
a pale yellow oil: yield 78 mg (7%); Rf 0.43 (9:1 chloroform-
1
methanol); [R]25 +3.5° (c ) 1.0, CHCl3); H NMR (CDCl3) δ
D
7.31 (4 H, t, J ) 7.8 Hz), 7.22 (4 H, d, J ) 7.6 Hz), 7.15 (2 H,
t, J ) 7.2 Hz), 4.24 (1 H, m), 4.14 (1 H, td, J ) 2.0, 4.7 Hz),
4.09, (1 H, td, J ) 1.6, 4.5 Hz), 2.34 (1 H, s), 2.20 (1 H, m),
2.10 (1 H, m), 1.73 (1 H, m), 1.55 (2 H, m), 1.03 (1 H, dt, J )
3.8, 12.5 Hz); 13C NMR (CDCl3) δ 151.3 (d, J CP ) 6.5 Hz), 129.9,
125.1, 120.3 (dd, J CP ) 3.6, 5.1 Hz), 72.0 (d, J CP ) 9.3 Hz),
C
18H20NO3P: C, 65.65; H, 6.12; N, 4.25. Found: C, 65.64; H,
61.9 (d, J CP ) 2.1 Hz), 59.7 (d, J CP ) 2.2 Hz), 40.1 (d, J CP )
6.26; N, 4.15.
5.1 Hz), 31.3 (d, J CP ) 3.7 Hz), 22.0 (d, J CP ) 3.6 Hz); 31P NMR
(CDCl3) δ -5.07; IR (thin film) νmax 3404 (br), 1591, 1489, 1190,
937 cm-1; EIMS m/z (relative intensity) 345 (M+, 17), 300 (27),
112 (100), 77 (75); HRMS calcd for C18H20NO4P 345.1130,
found 345.1131.
N -(D ip h e n y lp h o s p h in o y l)-7-a za b ic y c lo [2.2.1]h e p -
ta n e (5). Elution with 1:1 ethyl acetate-petroleum ether and
then 7:3 ethyl acetate-petroleum ether afforded 5 as a white,
crystalline solid: yield 882 mg (88%); Rf 0.58 (9:1 chloroform-
methanol); mp 184-185 °C; 1H NMR (CDCl3) δ 7.92 (2 H, dd,
J ) 1.3, 11.6 Hz), 7.90 (2 H, dd, J ) 1.4, 11.6 Hz), 7.44 (6 H,
m), 3.82 (2 H, m), 1.92 (4 H, d, J ) 6.5 Hz), 1.42 (4 H, d, J )
6.9 Hz); 13C NMR (CDCl3) δ 133.6 (d, J ) 131.5 Hz), 132.4 (d,
J CP ) 8.7 Hz), 131.6 (d, J CP ) 2.9 Hz), 128.6 (d, J ) 12.4 Hz),
58.5, 31.5 (d, J CP ) 6.5 Hz); 31P NMR (CDCl3) δ 22.63; IR (KBr)
Further elution with 9:1 chloroform-methanol afforded 16
as a pale yellow oil: yield 403 mg (37%); Rf 0.30 (9:1
chloroform-methanol); [R]25D +4.7° (c ) 1.0, MeOH); 1H NMR
((CD3)2SO) δ 7.39 (2 H, t, J ) 7.8 Hz), 7.22 (3 H, dd, J ) 1.0,
5.2 Hz), 7.02 (2 H, dd, J ) 1.0, 3.5 Hz), 6.73 (2 H, d, J ) 9.0
Hz), 5.03 (1 H, d, J ) 4.1 Hz), 4.01 (1 H, br s), 3.94 (1 H, m),
3.87 (1 H, br s), 2.09 (1 H, m), 1.87 (1 H, m), 1.60-1.30 (4 H,
series of m), 0.89 (1 H, dt, J ) 3.8, 12.1 Hz); 13C NMR ((CD3)2-
SO) δ 154.3, 150.8 (d, J CP ) 7.2 Hz), 142.9 (d, J CP ) 8.0 Hz),
129.8, 124.7, 120.4 (dt, J CP ) 4.4, 82.8 Hz), 115.8, 79.2, 70.5
(d, J CP ) 9.1 Hz), 61.2, 58.7, 30.7 (d, J CP ) 3.6 Hz), 21.5 (d,
J CP ) 3.7 Hz); 31P NMR (CDCl3) δ -4.34; IR (cast film) νmax
3266 (br), 1186, 940 cm-1; EIMS m/z (relative intensity) 361
(M+, 20), 161 (89), 112 (100); HRMS calcd for C18H20NO5P
361.1079, found 361.1088.
2-en do-Hydr oxy-7-(diph en ylph osph in oyl)-7-azabicyclo-
[2.2.1]h ep ta n e (15). A solution containing 7-(diphenylphos-
phinoyl)-7-azabicyclo[2.2.1]heptane 5 (103 mg, 0.35 mmol) in
5:7 ethanol-DMF (1.2 mL) was added to one stage II prepara-
tive scale liquid culture of B. bassiana (prepared as described
above) inside a sterile laminar flow hood. The flask was placed
on an orbital shaker at 250 rpm, 29-30 °C, and 50% relative
humidity for biotransformation. After 168 h, the cells were
removed from the culture by vacuum filtration and the filtrate
was extracted with methylene chloride. The combined organic
layer was washed with brine and dried over Na2SO4. The
ν
max 1438, 1215, 1060 cm-1; EIMS m/z (relative intensity) 297
(M+, 59), 268 (63), 201 (100); HRMS calcd for C18H20NOP
297.1283, found 297.1292.
Gen er a l Biotr a n sfor m a tion P r oced u r e. Details describ-
ing the maintenance of cultures and biotransformation protocol
are provided elsewhere.11 Medium A: 20 g of D-glucose, 5 g of
yeast extract (Difco), 5 g of soybean meal (Victoria Feed Co.),
5 g of NaCl, and 5 g of K2HPO4 per liter of water, pH adjusted
to 5.0 prior to autoclaving. Medium B: 20 g of corn steep liquor
(Sigma), 10 g of D-glucose per liter of water, pH adjusted to
5.0 prior to autoclaving. Media were sterilized for 15 min (125
mL DeLong flasks) or 20 min (1 L DeLong flasks) and allowed
to cool to room temperature prior to inoculation.
(2-en do-Hydr oxy-7-a za bicyclo[2.2.1]h ep t-7-yl)p h osph o-
n ic Acid Dieth yl Ester (13). A solution containing (7-
azabicyclo[2.2.1]hept-7-yl)phosphonic acid diethyl ester 3 (404.0
mg, 1.73 mmol) in ethanol (2 mL) was equally distributed
among four stage II preparative scale liquid cultures of B.
bassiana (prepared as described above). All flasks were then
placed on an orbital shaker at 250 rpm, 29-30 °C, and 50%

6318 J . Org. Chem., Vol. 64, No. 17, 1999
Hemenway and Olivo
solvent was concentrated under diminished pressure to afford
a residue which was purified by flash chromatography on a
silica gel column. Elution with 9:1 chloroform-methanol
afforded 15 as a white solid: yield 38 mg (35%); Rf 0.36 (9:1
NMR (CDCl3, 300 MHz) δ 3.54, 3.50; 19F NMR (CDCl3, 282
MHz) δ -71.78, -71.87.
2-en d o-((R)-R-Met h oxy-R-t r iflu or om et h ylp h en yla ce-
tic ester -7-a za bicyclo[2.2.1]h ep t-7-yl)p h osp h on ic Acid
P h en yl, p-((R)-R-Meth oxy-R-tr iflu or om eth ylp h en yla cetic
ester )p h en yl Ester : Rf 0.61 (95:5 chloroform-methanol); 1H
NMR (CDCl3, 300 MHz) δ 3.68, 3.54, 3.50; 19F NMR (CDCl3,
282 MHz) δ -71.76, -71.87-71.95.
2-en d o-((R)-R-Met h oxy-R-t r iflu or om et h ylp h en yla ce-
tic ester )-7-(d ip h en ylp h osp h in oyl)-7-a za bicyclo[2.2.1]-
h eptan e: Rf 0.33 (95:5 chloroform-methanol); 1H NMR (CDCl3,
300 MHz) δ 3.52, 3.49; 19F NMR (CDCl3, 282 MHz) δ -71.80,
-71.95.
chloroform-methanol); mp 160-161 °C; [R]25 -8.1° (c ) 1.0,
D
1
CHCl3); H NMR (CDCl3) δ 7.88 (2 H, dd, J ) 1.2, 11.8 Hz),
7.86 (2 H, dd, J ) 1.4, 11.7 Hz), 7.44 (6 H, m), 4.60 (1 H, dd,
J ) 4.0, 9.3 Hz), 3.72 (1 H, td, J ) 1.9, 5.1 Hz), 3.66 (1 H, m),
2.36 (2 H, m), 2.25 (1 H, m), 1.96 (1 H, m), 1.78 (1 H, m), 1.58
(1 H, m), 1.09 (1 H, dt, J ) 2.4, 12.4 Hz); 13C NMR (CDCl3) δ
132.9 (d, J CP ) 132.1 Hz), 132.4 (d, J CP ) 16.0 Hz), 132.4, 131.9
(d, J CP ) 3.0 Hz), 128.7 (d, J ) 12.3 Hz), 72.5 (d, J CP ) 7.0
Hz), 62.0, 59.6, 40.5 (d, J CP ) 4.4 Hz), 31.8 (d, J CP ) 6.5 Hz),
22.3 (d, J CP ) 6.6 Hz); 31P NMR (CDCl3) δ 22.87; IR (KBr) νmax
3318 (br), 1439, 1171, 1121 cm-1; EIMS m/z (relative intensity)
313 (M+, 3.4), 201 (44), 112 (100); HRMS calcd for C18H20NO2P
313.1232, found 313.1225.
Ack n ow led gm en t. We thank the following organi-
zations at The University of Iowa for financial support:
The Central Investment Fund for Research Enhance-
ment and The Center for Biocatalysis and Bioprocessing
for a fellowship to M.S.H.
2-en d o-((R)-R-Met h oxy-R-t r iflu or om et h ylp h en yla ce-
tic ester )-7-a za bicyclo[2.2.1]h ep t-7-yl)p h osp h on ic Acid
Dieth yl Ester : Rf 0.42 (95:5 chloroform-methanol); 1H NMR
(CDCl3, 300 MHz) δ 3.55, 3.51; 19F NMR (CDCl3, 282 MHz) δ
-71.83, -71.91.
2-en d o-((R)-R-Met h oxy-R-t r iflu or om et h ylp h en yla ce-
tic ester )-7-a za bicyclo[2.2.1]h ep t-7-yl)p h osp h on ic Acid
Dip h en yl E st er : Rf 0.59 (95:5 chloroform-methanol); 1H
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra for compounds 3-5 and 7-16. This material is
available free of charge via the Internet at http://pubs.acs.org.
J O9904664