α hydroxylation of carboxylic acids with molecular oxygen catalyzed by the α oxidase of peas (Pisum sativum): A novel biocatalytic synthesis of enantiomerically pure (R)-2-hydroxy acids

Article abstract of DOI:10.1021/ja981252r

The substrate selectivities of the α oxidation of saturated, unsaturated, and heteroatom-containing (oxygen, sulfur) carboxylic acids 1 by the enzyme extract of peas (Pisum sativum) indicate that this biotransformation proceeds highly enantioselectively. For the first time, the synthesis of optically pure 2-hydroxy acids 2 has been achieved on the semipreparative scale (1 mmol) by α hydroxylation of long-chain carboxylic acids with molecular oxygen, catalyzed by the α oxidase of peas. For derivatives with sulfur atom in the chain, no sulfoxidation is observed. The functionalities (carbon double and triple bonds, oxygen, and sulfur atoms) must be at least three carbon atoms away from the carboxylic acid group to achieve efficient asymmetric hydroxylation. The absolute configuration of the 2-hydroxy acids 2 was assigned by comparison of the gaschromatographic data with that of authentic reference compounds and by application of the exciton- coupled-circular-dichroism (ECCD) method. This unprecedented asymmetric biocatalytic methodology should be valuable for the preparation of enantiomerically pure (R)-2-hydroxy acids.

Full text of DOI:10.1021/ja981252r

11044
J. Am. Chem. Soc. 1998, 120, 11044-11048
R Hydroxylation of Carboxylic Acids with Molecular Oxygen
Catalyzed by the R Oxidase of Peas (Pisum satiVum): A Novel
Biocatalytic Synthesis of Enantiomerically Pure (R)-2-Hydroxy Acids
Waldemar Adam,† Wilhelm Boland,§ Jenny Hartmann-Schreier,‡ Hans-Ulrich Humpf,‡
Michael Lazarus,*,‡ Alexander Saffert,‡ Chantu R. Saha-Mo1ller,† and Peter Schreier‡
Contribution from the Institutes of Organic Chemistry and of Pharmacy and Food Chemistry, UniVersity of
Wu¨rzburg, Am Hubland, D-97074 Wu¨rzburg, Germany, and the Max-Planck Institute of Chemical Ecology,
Tatzendpromenade 1a, D-07745 Jena, Germany
ReceiVed April 14, 1998
Abstract: The substrate selectivities of the R oxidation of saturated, unsaturated, and heteroatom-containing
(oxygen, sulfur) carboxylic acids 1 by the enzyme extract of peas (Pisum satiVum) indicate that this
biotransformation proceeds highly enantioselectively. For the first time, the synthesis of optically pure 2-hydroxy
acids 2 has been achieved on the semipreparative scale (1 mmol) by R hydroxylation of long-chain carboxylic
acids with molecular oxygen, catalyzed by the R oxidase of peas. For derivatives with sulfur atom in the
chain, no sulfoxidation is observed. The functionalities (carbon double and triple bonds, oxygen, and sulfur
atoms) must be at least three carbon atoms away from the carboxylic acid group to achieve efficient asymmetric
hydroxylation. The absolute configuration of the 2-hydroxy acids 2 was assigned by comparison of the gas-
chromatographic data with that of authentic reference compounds and by application of the exciton-coupled-
circular-dichroism (ECCD) method. This unprecedented asymmetric biocatalytic methodology should be
valuable for the preparation of enantiomerically pure (R)-2-hydroxy acids.
Introduction
Scheme 1. R Oxidation of Fatty Acids in Higher Plants
The R oxidation of fatty acids is known for higher plants
such as pea leaves (Pisum satiVum),1 germinating peanuts
(Arachis hypogaea),2 cucumbers (Cucumis satiVus),3 and po-
tatoes (Solanum tuberosum),4 as well as for simple organisms
such as marine green algae (UlVa pertusa).5 In higher plants,
2-hydroxy fatty acids are formed in the oxidative lipid metabo-
lism by R oxidation of the corresponding acids.2,6
The mechanism of this biochemical reaction was worked out
by Shine and Stumpf 2 in the seventies (Scheme 1). They have
postulated that the flavoprotein-catalyzed oxidation of the fatty
acid leads to an intermediary R-hydroperoxy acid, which
preferentially decarboxylates to the corresponding aldehyde
(Scheme 1, path B) in competition with reduction to the
2-hydroxy acid (Scheme 1, path A). Presumably, the 2-hydro-
peroxy acid is first converted to an intermediary R-peroxy
lactone, which decomposes very fast to CO2 and the aldehyde.
Evidence for the R-peroxy lactone has been inferred by the fact
that chlorophyll could be chemically excited when present in
this R-oxidation process.6,7 While the 2-hydroxy acid accum-
ulates, the aldehyde is oxidized by an NAD+-dependent
aldehyde dehydrogenase to the next lower homologous fatty
acid, which in turn functions as a substrate for the R oxidation.2,6
In the case of hexadecanoic (palmitic) acid, the (R)-2-hydroxy-
hexadecanoic acid was formed enantioselectively.8 Unfortu-
nately, the membrane-bound R oxidase has not yet been isolated
in pure form and, therefore, its structure elucidation is still
pending.
† Institute of Organic Chemistry, University of Wu¨rzburg.
§ Max-Planck Institute of Chemical Ecology.
‡ Institute of Pharmacy and Food Chemistry, University of Wu¨rzburg.
(1) Hitchcock, C.; James, A. T. Biochim. Biophys. Acta 1966, 116, 413-
424.
(2) Shine, W. E.; Stumpf, P. K. Arch. Biochem. Biophys. 1974, 162,
147-157.
Chiral R-hydroxy acids are important building blocks for
the synthesis of optically active glycols,9a halo esters,9b and
epoxides.9c For this purpose, several chemical10 and enzymatic11
(3) Galliard, T.; Matthew, J. A. Biochim. Biophys. Acta 1976, 424, 26-
35.
(4) Laties, G. G.; Hoelle, C. Phytochemistry 1967, 6, 49-57.
(5) Kajiwara, T.; Yoshikawa, H.; Saruwatari, T.; Hatanaka, A.; Kawai,
T.; Ishihara, M.; Tsuneya, T. Phytochemistry 1988, 27, 1643-1645.
(6) (a) Salim-Hanna, M.; Campa, A.; Cilento, G. Photochem. Photobiol.
1987, 45, 849-854. (b) Campa, A.; Salim-Hanna, M.; Cilento, G.
Photochem. Photobiol. 1989, 49, 349-354.
(7) Schuster, G. B. Acc. Chem. Res. 1979, 12, 366-373.
(8) (a) Morris, L. J.; Hitchcock, C. Eur. J. Biochem. 1968, 4, 146-148.
(b) Hitchcock, C.; Rose, A. Biochem. J. 1971, 125, 1155-1156. (c)
Markovetz, A. J.; Stumpf, P. K.; Hammarstro¨m, S. Lipids 1972, 7, 159-
164.
10.1021/ja981252r CCC: $15.00 © 1998 American Chemical Society
Published on Web 10/14/1998

R Hydroxylation of Carboxylic Acids
J. Am. Chem. Soc., Vol. 120, No. 43, 1998 11045
Table 1. R Oxidation of Carboxylic Acids 1 with the Crude
Homogenate of Young Pea Leaves (Method A) and the Crude
Extract of Germinating Peas (Method B)a
Scheme 2. R Hydroxylation of Carboxylic Acids with
Molecular Oxygen Catalyzed by the R Oxidase from Peas
(Pisum SatiVum)
product
substrate
(mmol)
time convn
ratio
ee (%)c
(R)-2
entry
1
method
A
(h)
(%)
(%)b 2:3
1a
6
41
31:69
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99g
>99
>99
>99
(0.02)
2
3
1ad
B
B
A
A
A
A
A
A
A
A
A
B
B
B
47
71
22
22
23
23
22
23
22
23
23
23
23
24
100
100
57
99:1
(0.1)
1ad
(0.5)
1b
(0.04)
1d
(0.05)
1g
(0.07)
1h
(0.07)
1i
(0.03)
1j
(0.03)
1m
(0.05)
1o
(0.06)
1q
(0.03)
1r
(0.2)
1r
80:20e
45:55
f
4
5
40
6
10
20:80
17:83
24:76
16:84
65:35
49:51
70:30
99:1
7
67
8
86
methods have been reported previously on the synthesis of
optically active R-hydroxy acids, whereas the biocatalytic
techniques utilize oxyfunctionalized substrates. Prior to our
efforts, nothing was known on the synthesis of optically active
2-hydroxy acids by the enzymatic CH-insertion from readily
available carboxylic acids.
9
22
10
11
12
13
14
15
<10
87
To assess the scope of the enzymatic R oxidation for the
preparation of enantiomerically pure 2-hydroxy acids, we have
studied in detail the R-oxidation system of peas (Scheme 2). In
this context, we have recently reported our results on the
substrate selectivities for the enantioselective R hydroxylation
of saturated carboxylic acids with molecular oxygen catalyzed
by the R-oxidation-enzyme system of young pea leaves.12
Because of the low enzyme activity in the pea leaves, the R
oxidation with the crude homogenate was only possible on the
analytical scale and the aldehyde 3 was produced preferentially.
Herein, we present the first enantioselective R hydroxylation
of functionalized carboxylic acids with molecular oxygen on
the semipreparative scale (1 mmol), catalyzed by the R oxidases
of germinating peas. This novel biocatalytic method affords
exclusively enantiomerically pure (R)-2-hydroxy acids from
carboxylic acids, in particular long-chain fatty acids, when the
enzymatic autoxidation is conducted in the presence of tin(II)
chloride as reducing agent.
17
100
100
100
74:26
99:1
(0.2)
1s
(0.02)
a Method A: 0.2 M phosphate buffer (pH 6.0), 0.1% Triton X-100.
Method B: 0.1 M Tris-HCl (pH 6.0). b The product distribution,
normalized to 100% conversion, was determined by GC analysis; error
limits (2%. c The ee values were determined by GC analysis after
esterification with the Mosher reagent; error limits (2%. d SnCl2 (entry
2: 1.0 equiv; entry 3: 0.5 equiv) was added to the solution. e At long
reaction times, the aldehyde 3 is transformed by oxido-reductases in
the crude enzyme preparation to the corresponding alcohol 4 and
carboxylic acid 5 (cf. Figure 1). f Only the 2-hydroxy acid was detected
as product of the R oxidation. g The ee values were determined by GC
analysis after esterification with (-)-menthyl chloroformate; error limits
(2%.
Results and Discussion
the enantiomeric excess of the 2-hydroxy carboxylic acids 2a-
s, was assessed by gas chromatography. In Table 1, the
optimized reaction times are given, beyond which no further
conversion of the carboxylic acids 1a-s was observed.
The substrate selectivity studies of the biocatalytic R oxidation
of functionalized carboxylic acids were carried out under
optimized conditions12 with the crude homogenate of young pea
leaves in 0.2 M phosphate buffer (pH 6.0) with Triton X-100
as emulsifier (Method A). The results are given in Table 1,
wherein the conversion of the carboxylic acids 1a-s to the
2-hydroxy acids 2a-s and the next lower aldehyde 3, as well as
The R oxidation of the unsaturated undec-10-enoic acid 1b
and the cis- and trans-oleic acids (1i and 1j) by the crude
homogenate yielded exclusively the corresponding (R)-2-
hydroxy acid 2 and the next lower aldehyde 3, with higher
preference for the latter (Table 1, entries 4, 8, and 9). It was,
therefore, of interest to investigate the influence of the carbon
double bond in close proximity to the carboxylic acid func-
tionality. As shown in Table 1 (entries 6 and 7), the diaster-
eomeric pair cis- and trans-hexadec-5-enoic acids (1g and 1h)
is oxidized to the 2-hydroxy acids 2g and 2h and the corre-
sponding aldehydes 3 in ratios of 20:80 and 17:83 at 10% and
67% conversion, but the cis- and trans-hexadec-4-enoic acid
pair 1e,f (not shown in Table 1) was not accepted by the pea-
leaf oxidase. Moreover, while the trans-dec-4-enoic acid (1d)
is a substrate (Table 1, entry 5) for this enzyme to afford
the trans-2-hydroxydec-4-enoic acid (2d), its geometrical
isomer cis-dec-4-enoic acid (1c) is not converted (not shown
in Table 1).
(9) (a) Prelog, V.; Wilhelm, M.; Bright, D. B. HelV. Chim. Acta 1954,
37, 221-224. (b) Lee, J. B.; Downie, I. M. Tetrahedron 1967, 23, 359-
363. (c) Mori, K.; Takigawa, T.; Matsuo, T. Tetrahedron 1979, 35, 933-
940.
(10) (a) Evans, D. A.; Morrissey, M. M.; Dorow, R. L. J. Am. Chem.
Soc. 1985, 107, 4346-4348. (b) Corey, E. J.; Link, J. O.; Shao, Y.
Tetrahedron Lett. 1992, 33, 3435-3438.
(11) (a) Wong, C.-H.; Matos, J. R. J. Org. Chem. 1985, 50, 1992-1994.
(b) Nakamura, K.; Inoue, K.; Ushio, K.; Oka, S.; Ohno, A. J. Org. Chem.
1988, 53, 2589-2593. (c) Effenberger, F. Angew. Chem. 1994, 106, 1609-
1619. (d) Sugai T.; Ohta, H. Tetrahedron Lett. 1991, 32, 7063-7064. (e)
Adam, W.; Lazarus, M.; Boss, B.; Saha-Mo¨ller, C. R.; Humpf, H.-U.;
Schreier, P. J. Org. Chem. 1997, 62, 7841-7843. (f) Adam, W.; Fell, R.
T.; Hoch, U.; Saha-Mo¨ller, C. R.; Schreier, P. Tetrahedron: Asymmetry
1995, 6, 1047-1050.
(12) Adam, W.; Lazarus, M.; Saha-Mo¨ller, C. R.; Schreier, P. Tetrahe-
dron: Asymmetry 1997, 7, 2287-2292.

11046 J. Am. Chem. Soc., Vol. 120, No. 43, 1998
Adam et al.
Scheme 3. Selective R Oxidation of the Prochiral
R Methylenic Group
Similar results were obtained for the R oxidation of alkynoic
acids. While the dec-4-ynoic acid (1k) and hexadec-4-ynoic
acid (1l) were not accepted as substrates by the R-oxidation
enzyme (not shown in Table 1), the hexadec-5-ynoic acid (1m)
was converted to the 2-hydroxy acid 2m and the aldehyde 3m
(Table 1, entry 10). In the case of the alkyloxy derivatives of
the propionic and butyric acid, the 3-octyloxypropanoic acid
(1n) was not oxidized (not shown in Table 1), but the
4-decyloxybutanoic acid (1o) was transformed to the enantio-
merically pure (R)-2-hydroxy acid 2o and the aldehyde 3o in a
ratio of 49:51 at 87% conversion (Table 1, entry 11). Although
sebacic acid (1p) resists R oxidation by this enzyme system, its
methyl ester 1q is R-hydroxylated (Table 1, entry 12), albeit at
a significantly diminished reaction rate. Thus, these results show
that fatty acids with less than three carbon atoms between the
carboxylic acid group and functionalities such as a triple or
double bond and also a heteroatom are generally not accepted
by this enzymatic R-oxidation system. Furthermore, more
hydrophilic carboxylic acids with short alkyl chains12 or the
dicarboxylic acids 1p and 1q represent very poor substrates.
A more detailed study was conducted with myristic acid (1a),
one of the best substrates for this enzyme system. Thus, its
R-oxidase-catalyzed hydroxylation with crude homogenate at
pH 6 led to the enantiomerically pure (R)-2-hydroxymyristic
acid (2a) and the aldehyde 3a in a ratio of 31:69 (Table 1, entry
1) at 41% conversion.12 Since the R-oxidase activity of this
crude homogenate (grown for 14 days, Method A) was quite
low and the optically active (R)-2-hydroxy acids 2 only
accessible on the analytical scale, the crude enzyme extract of
germinating peas (germinated for 3 days, Method B) was
employed.
The results in Table 1 (entries 2 and 3) show that this enzyme
preparation exhibits a significantly higher R-oxidase activity
toward fatty acid 1a. Nevertheless, to achieve its complete
conversion to the 2-hydroxy acid 2a, the reaction conditions
had to be optimized for this model substrate (cf. Supporting
Information, Table 2). Since the R oxidation is known6,13 to
proceed through a 2-hydroperoxy acid intermediate (Scheme
1), reaction conditions which favor the reduction over decar-
boxylation were required to increase the yield of the 2-hydroxy
acid. Several reducing additives were tried out for this purpose.
While cysteine or the glutathione peroxidase (GPX)/glutathione
(GSH) system did not help, fortunately, the R oxidation yielded
exclusively the 2-hydroxy acid 2a in the presence of tin(II)
chloride, which was successfully utilized in the in-situ reduction
of fatty acid hydroperoxides formed by lipoxygenase-catalyzed
autoxidation.14 Under optimized reaction conditions, the myris-
tic acid (1a) was completely converted to the enantiomerically
pure (R)-2-hydroxy acid 2a (Table 1, entry 2). These results
demonstrate that the R oxidase of pea autoxidizes selectively
the C-HR bond of the prochiral R methylenic group in
carboxylic acids (Scheme 3). Thus, the observed enantiose-
Figure 1. Product distribution, determined by GC analysis, for the R
oxidation of myristic acid (1a) with the crude extract of germinating
peas.
lectivity of the R oxidation does not originate from the
asymmetric reduction of the intermediary hydroperoxy acid
through peroxidase activity in the crude homogenate of germi-
nating peas.
Unquestionably, a significant step forward was accomplished
in developing this asymmetric R oxidation of long-chain
carboxylic acids into a useful preparative synthesis of optically
active 2-hydroxy acids. Indeed, to illustrate the synthetic value
of this novel biotechnological methodology, this R oxidation
was conducted on 0.5 mmol (100 mg) of the fatty acid 1a (Table
1, entry 3; cf. Supporting Information, Table 2). After about 3
days of reaction time, the resulting 2-hydroxymyristic acid (2a)
was obtained enantiomerically pure and isolated in 46% yield.
The homologous aldehyde 3a was formed as the minor pro-
duct, which was transformed at long reaction times (3 days) by
oxido reductases in the crude homogenate to the corresponding
alcohol 4a and carboxylic acid 5a, as confirmed by GC analysis
(Figure 1).
Also the 12-thiamyristic (1r) and 9-thiapalmitic (1s) acids
were treated with the crude extract of germinating peas (Table
1, entries 13-15) to examine whether such thioether functional-
ity survives in view of its ease of sulfoxidation.15 Amazingly,
on the analytical scale, these thia-substituted acids were
exclusively and completely converted to the enantiomerically
pure (R)-2-hydroxy acids 2r and 2s (entries 13 and 15). In fact,
this R oxidation of the 12-thiamyristic acid (1r) on the
semipreparative scale (ca. 50 mg, 0.2 mmol) yielded preferen-
tially the enantiomerically pure 2-hydroxy acid 2r at 100%
conversion (entry 14); not even traces of sulfoxidation were
noted.
The enantiomeric excess of the 2-hydroxy acids 2 was
determined by gas chromatography on chiral columns after
esterification of their methyl esters with either (+)-R-methoxy-
R-(trifluoromethyl)phenylacetyl chloride (Mosher reagent)16 or
(-)-menthyl chloroformate.17 The elution order of the diaster-
eomeric derivatives of the 2-hydroxy acids 2a,d,i,j,o,q was
ascertained on an achiral stationary phase by comparing the gas-
chromatographic data with that of the authentic reference
compounds.11e,18,19 The configurations of the 2-hydroxy acids
2b,g,h,m were assigned accordingly. For the determination of
the configuration of the 2-hydroxy-12-thiamyristic acid (2r),
we have employed the exciton-coupled-circular-dichroism (EC-
(15) Kagan, H. B. In Catalytic Asymmetric Synthesis; Ojima, I., Ed.;
VCH Publishers: New York, 1993; pp 203-226.
(16) Dale, J. A.; Dull, D. L.; Mosher, H. S. J. Org. Chem. 1969, 34,
2543-2549.
(17) Rees, L.; Suckling, C. J.; Wood, H. C. S. J. Chem. Soc., Chem.
Commun. 1987, 470-472.
(18) Adam, W.; Lazarus, M.; Schmerder, A.; Humpf, H.-U.; Saha-Mo¨ller,
C. R.; Schreier, P. Eur. J. Org. Chem. 1998, 2013-2018.
(19) Hamberg, M. Anal. Biochem. 1971, 43, 515-526.
(13) Kajiwara, T.; Matsui, K.; Akakabe, Y. In Biotechnology for
ImproVed Foods and FlaVours; Takeoaka, G. R., Teranishi, R., Williams,
P. J., Kobayashi, A., Eds.; American Chemical Society: Washington, 1996;
pp 146-166.
(14) Koljak, R.; Boutaud, O.; Shieh, B.-H.; Samel, N.; Brash, A. R.
Science 1997, 277, 1994-1996.

R Hydroxylation of Carboxylic Acids
J. Am. Chem. Soc., Vol. 120, No. 43, 1998 11047
Scheme 4. Functionalization of the 2-Hydroxy Acid (R)-2r to the Corresponding Bichromophoric Derivative (R)-6r for
Configurational Analysis
are not converted by the enzyme. Despite these limitations,
the R oxidation of long-chain carboxylic acids provides a
convenient synthesis of enantiomerically pure (R)-2-hydroxy
acids. Under optimized reaction conditions (pH 6 and SnCl2
as reductant), the R oxidation by the crude extract of germinating
peas produces R-enantioselectively the 2-hydroxy acid 2, in
which the reduction of the intermediary 2-hydroperoxy acid is
favored over its decomposition to CO2 and to the aldehyde 3.
In this context, the recently reported23 purification and structure
elucidation of a cucumber enzyme with high R-oxidation activity
should be instrumental in understanding the mechanism of
R-oxidase action. The biotechnological production of such
enzymes in large amounts should foster the practical utility of
this unprecedented biocatalytic method.
Experimental Section
Procedure for the Enzymatic r Oxidation of Carboxylic Acids
1 by Molecular Oxygen. Method A: To facilitate the dissolution of
the solid carboxylic acids in the aqueous medium, the particular
substrate was first taken up in ethyl ether (1 mL), the solvent was
Figure 2. CD and UV spectra of the bichromophoric diester (R)-6r in
acetonitrile (1-cm cell); the bold lines represent the direction of the
transition dipoles.
evaporated under reduced pressure (20 °C, 17 Torr), 2 mL of phosphate
buffer (pH 6), which contained 0.1% Triton X-100, was added to the
residue, and the mixture was sonicated for 1 min. The liquid carboxylic
acids were directly dissolved in the phosphate buffer (pH 6) and 0.1%
Triton X-100. The crude homogenate was prepared by homogenizing
10-15 g of pea leaves (var. satiVum, grown for 14 days) with 150 mL
of 0.2 M phosphate buffer and 0.1% Triton X-100 in a blender for
45 s. The aqueous carboxylic acid solution and the crude enzyme
homogenate were stirred together for several hours at 4 °C, while a
slow stream of oxygen gas was passed continually through the reaction
mixture. Subsequently, the insoluble materials were removed by
filtration, and the filtrate was acidified with 6 N hydrochloric acid
(pH 3) and extracted with ethyl ether (3 × 75 mL). The combined
organic phases were dried over Na2SO4, and the solvent was evaporated
under reduced pressure (20 °C, 17 Torr). The free acids 1 and 2 were
converted to their methyl esters with diazomethane. After determination
of the amount of conversion and the product distribution by GC and
GC-MS analysis, the crude reaction mixture was submitted to column
chromatography (silica gel, 0.032-0.062 mesh, 7:3 mixture of petro-
leum ether/ethyl ether) and the methyl 2-hydroxy ester was isolated
in pure form. The enantiomeric excess (ee) of the methyl 2-hydroxy
esters was determined by quantitative gas chromatography after
derivatization with (S)-(+)-R-methoxy-R-trifluoromethylphenylacetyl
chloride (Mosher reagent)16 or (-)-menthyl chloroformate.17
CD)20 method established for acyclic R-hydroxy acids.21 After
derivatization (Scheme 4) of the enantiomerically pure 2-hy-
droxy acid 2r (cf. Table 1, entry 14) to the corresponding 9′-
methylanthryl (R)-2-(2′′-naphthoyloxy)-12-thiatetradecanoic acid
(6r), the CD and UV22 spectra of (R)-6r were recorded (Figure
2). A positive split CD curve was obtained, with extrema at
254 (∆ꢀ ) +16.6) and 237 nm (∆ꢀ ) -27.8) and an amplitude
A of +44.4. This positive CD couplet suggests the R config-
uration.21
In summary, our results show that the R-oxidation enzyme
system of peas (Pisum satiVum) asymmetrically R-hydroxylates
a broad variety of fatty acids on the semipreparative scale. As
substrates serve saturated fatty acids with 7 to 16 carbon atoms12
and the unsaturated fatty acids 1b, 1d, 1g-j, and 1m, as well as
the heteroatom-containing (oxygen, sulfur) carboxylic acids 1o,
1q, 1r and 1s. However, when the triple and double bond or
heteroatom are proximate to the carboxylic acid functionality
(less than three carbon atoms), the R hydroxylation of such
carboxylic acids does not take place. Also substrates without
a substantially hydrophobic moiety, as is the case for the short-
chain alkanoic acids12 and the dicarboxylic acids 1p and 1q,
Method B: The carboxylic acid solution was prepared as described
in Method A, but instead of the phosphate buffer, the acids were
dissolved in 2 M tris(hydroxymethyl)aminoethane (Tris-HCl, pH 10.3).
The crude extract was prepared by homogenizing 20 g of germinating
peas (germinated for 3 days) with 100 mL of 0.1 M Tris-HCl
(pH 7.5) in a mortar. Subsequently, the crude homogenate was
centrifuged at 9000 g for 7 min. After the aqueous carboxylic acid
solution was added to the supernatant (15 mg protein/mL), the resulting
(20) Nakanishi, K.; Berova, N. In Circular Dichroism. Principles and
Applications; Nakanishi, K., Berova, N., Woody, R. W., Eds; VCH
Publishers: New York, 1994; pp 361-398.
(21) (a) Gimple, O.; Schreier, P.; Humpf, H.-U. Tetrahedron: Asymmetry
1997, 8, 11-14. (b) Ho¨r, K.; Gimple, O.; Schreier, P.; Humpf, H.-U.
J. Org. Chem. 1998, 63, 322-325.
(22) ꢀ254 ) 140 000 M-1 cm-1 for the methylanthryl chromophore (1-
cm cell, CH3CN).
(23) Andersen Borge, G. I.; Slinde, E.; Nilsson, A. Biochim. Biophys.
Acta 1997, 1344, 47-58.

11048 J. Am. Chem. Soc., Vol. 120, No. 43, 1998
Adam et al.
reaction mixture was adjusted to pH 6, blended with 3 mL of 1 M
phosphate buffer (pH 6), and stirred at 4 °C under 0.4 MPa O2 in an
Amicon Ultrafiltration Cell (Model 8200), in which the filtration
membrane was substituted by a Teflon sheet. Aliquots of 5 mL were
removed and worked up to monitor the progress of substrate conversion.
For this purpose, the solution was acidified with 6 N hydrochloric acid
(pH 3) and extracted with ethyl ether (3 × 5 mL). The combined
organic phases were dried over Na2SO4 and the solvent was evaporated
under reduced pressure (20 °C, 17 Torr). The acids 1 and 2 were
converted to their methyl esters by treatment with diazomethane and
submitted to GC and GC-MS analysis. The enantiomeric excess (ee)
was determined as described above.
and the combined organic layers were dried over Na2SO4. The solvent
was removed (20 °C, 17 Torr), the products were submitted to column
chromatography (silica gel, 0.032-0.062 mesh, 80:20:1 mixture of
petroleum ether/ethyl acetate/acetic acid), and the 2-hydroxymyristic
acid (1a) was isolated in pure form in 46% yield. A sample (1 mg) of
the purified 2-hydroxy acid 2a was methylated with diazomethane for
analysis by capillary gas chromatography-mass spectrometry. (R)-
2a: [R]20 -2.7 (c 1.5, CHCl3) (lit.24 [R]25 ) -3.1 (c 1.0, CHCl3)).
D
D
Methyl ester of 2a: HRGC-MS, m/z (%) 199 (26), 127 (11), 125 (17),
111 (39), 103 (15), 97 (74), 90 (41), 83 (88), 81 (23), 71 (32), 69
(100), 67 (24), 57 (76), 55 (82), 43 (62), 41 (56).
Scale-up of the r Oxidation of Carboxylic Acid 1a in the Presence
of Molecular Oxygen. The aqueous carboxylic acid solution was
prepared as described under Method B, and the crude extract was
obtained by homogenizing 100 g of germinating peas (germinated for
3 days) with 400 mL of 0.1 M Tris-HCl (pH 7.5) in a mortar.
Subsequently, the crude homogenate was centrifuged at 9000 g for 7
min. After the aqueous carboxylic acid solution and SnCl2 (0.5 equiv)
were added to the supernatant, the pH of the resulting reaction mixture
was adjusted to 6, blended with 12 mL of 1 M phosphate buffer (pH
6), and stirred at room temperature (ca. 20 °C) under 0.4 MPa O2 in
an Amicon Ultrafiltration Cell (Model 8400), in which the membrane
filter was replaced by a Teflon sheet. Aliquots (5 mL) were removed
and worked up as described under Method B to monitor the progress
of substrate conversion. After 71 h of reaction time the conversion
was complete, the mixture was extracted with ethyl ether (3 × 75 mL),
Acknowledgment. This work was supported by the Bay-
erische Forschungsstiftung (Bayerischer Forschungsverbund
Katalyse-FORKAT), the Deutsche Forschungsgemeinschaft
(SFB 347, Selektive Reaktionen Metall-aktivierter Moleku¨le),
and the Fonds der Chemischen Industrie. J.H.-Sch. thanks the
University of Wu¨rzburg for a fellowship (HSP II).
Supporting Information Available: Experimental Section
and Table 2 (8 pages, print/PDF). See any current masthead
page for ordering information and Web access instructions.
JA981252R
(24) Horn, D. H. S.; Hougen, F. W.; von Rudloff, E.; Sutton, D. A.
J. Chem. Soc. 1954, 177-180.