Chiral auxiliaries as docking/protecting groups in biohydroxylation: The hydroxylation of enantiopure spirooxazolidines derived from cyclopentanone using Beauveria bassiana ATCC 7159

Article abstract of DOI:10.1002/1099-0690(200012)2000:23<3835::AID-EJOC3835>3.0.CO;2-E

The aim of this work was to explore the scope and limitations of chiral docking/protecting groups as chiral auxiliaries in the biohydroxylation of unactivated methylene groups. As a model compound, cyclopentanone 1 was reacted with a range of enantiomerically pure amino alcohols 2a-n as well as 7a and b, varying substituents R¹ and R². The resulting chiral spirooxazolidines 3a-n as well as 8a and b were exposed to the fungus Beauveria bassiana ATCC 7159 and the resultant hydroxylated products were characterised. Introducing chirality into the substrate before the fermentation was found to have a major effect on the course of the biohydroxylation relative to the achiral analogue 3a (Table 1, entry 1). The nature of R¹/R₂ influenced both the product yield and the optical purity of the products (e.g. Table 1, entry 2). In addition, the absolute configuration of the final product 6 could be dictated solely by the nature of the docking/protecting group used (compare entry 8 with entry 9). Concerning the chain length of R¹/R₂, it was found that hydroxylation only took place in the cyclopentane ring when the heterocyclic ring was substituted with a methyl, ethyl or isopropyl (entries 2-5, 8, 9, 15, and 16). With increasing chain length, where R¹/R₂ are propyl, isobutyl or sec-butyl groups, a mixture of products was obtained in which the hydroxyl group was either on the cyclopentane ring or on the side-chain (entries 10-14).

Full text of DOI:10.1002/1099-0690(200012)2000:23<3835::AID-EJOC3835>3.0.CO;2-E

FULL PAPER
Chiral Auxiliaries as Docking/Protecting Groups in Biohydroxylation: The
Hydroxylation of Enantiopure Spirooxazolidines Derived from Cyclopentanone
Using Beauveria bassiana ATCC 7159
Anna de Raadt,*[a] Barbara Fetz,[a] Herfried Griengl,*[a] Markus Florian Klingler,[a]
[a]
[a]
[a]
[a]
[a]
´
Irene Kopper, Birgit Krenn, Dieter Franz Münzer, Rene Georg Ott, Peter Plachota,
Hans Jörg Weber,[a] Gerhard Braunegg,[b] Winfried Mosler,[c] and Robert Saf[d]
Keywords: Biohydroxylation / Biotransformations / Chiral auxiliaries / Asymmetric synthesis / Ketones
The aim of this work was to explore the scope and limitations
the optical purity of the products (e.g. Table 1, entry 2). In
of chiral docking/protecting groups as chiral auxiliaries in addition, the absolute configuration of the final product 6
the biohydroxylation of unactivated methylene groups. As a
model compound, cyclopentanone 1 was reacted with a
range of enantiomerically pure amino alcohols 2a−n as well
as 7a and b, varying substituents R1 and R2. The resulting
could be dictated solely by the nature of the docking/pro-
tecting group used (compare entry 8 with entry 9). Con-
cerning the chain length of R1/R2, it was found that hy-
droxylation only took place in the cyclopentane ring when
chiral spirooxazolidines 3a−n as well as 8a and b were ex- the heterocyclic ring was substituted with a methyl, ethyl or
posed to the fungus Beauveria bassiana ATCC 7159 and the
resultant hydroxylated products were characterised. Introdu-
cing chirality into the substrate before the fermentation was
isopropyl (entries 2−5, 8, 9, 15, and 16). With increasing chain
length, where R1/R2 are propyl, isobutyl or sec-butyl groups,
a mixture of products was obtained in which the hydroxyl
found to have a major effect on the course of the biohydroxyl- group was either on the cyclopentane ring or on the side-
ation relative to the achiral analogue 3a (Table 1, entry 1).
The nature of R1/R2 influenced both the product yield and
chain (entries 10−14).
Introduction
important terpenes, applications in general synthetic chem-
istry are in relative infancy because of a number of draw-
backs. Despite proposed models,[5Ϫ7] problems are experi-
enced with the predictability of the hydroxylation position
and not all functional groups remain intact upon exposure
to fermentation conditions because they are susceptible to
undesired reactions such as reduction or oxidation.[8,9]
The chemical hydroxylation[1,2] of unactivated CϪH
bonds in organic compounds, in particular secondary and
primary carbon atoms, remains an obstacle in classical syn-
thetic chemistry. Bioconversion processes[3] for such diffi-
cult transformations represent a valuable alternative to
‘‘mainstream’’ preparative chemistry. Although biohydroxy-
lations[4] performed by whole cells are well established in the
production of medicinally valuable steroids and industrially
To develop a general approach which could be used by
organic chemists for the preparation of intermediates useful
in synthesis, the concept of docking/protecting groups was
devised.[9Ϫ12] The aim of this concept was not only to pro-
tect vulnerable functional groups in the substrate, but also
to promote and direct the course of the hydroxylation.[9Ϫ12]
Ease of substrate handling (UV activity for TLC detection,
modification of polarity and volatility) was also envisaged
as a further advantage of this approach. This concept has
been successfully applied to carboxylic acids,[9,12] alco-
hols,[9] aldehydes,[13] and ketones[9] which were hydroxylated
by whole cell systems as the corresponding benzoxazoles,
isosaccharine derivatives, N-benzoylated oxazolidines and
N-benzoylated spirooxazolidines, respectively.
[a]
Spezialforschungsbereich F01 Biokatalyse, Institut für
Organische Chemie der Technischen Universität Graz,
Stremayrgasse 16, 8010 Graz, Austria
Fax: (internat.) ϩ 43-316/873-8740
E-mail: sekretariat@orgc.tu-graz.ac.at
Institut für Biotechnologie der Technischen Universität Graz,
[b]
Petersgasse 10, 8010 Graz, Austria
Fax: (internat.) ϩ 43-316/873-8412
E-mail: braunegg@biote.tu-graz.ac.at
[c]
Institut für Physikalische Chemie der Karl-Franzens-
Universität,
Heinrichstrasse 28, 8010 Graz, Austria
Fax: (internat.) ϩ 43-316/380-5483
E-mail: winfried.mosler@kfunigraz.ac.at
[d]
Institut für Chemische Technologie Organischer Stoffe der
In particular, for the hydroxylation of ketone substrates,
it was found that the well-known fungus Beauveria bassiana
ATCC 7159[4a,14] was particularly suited to hydroxylate N-
benzoylspirooxazolidine derivatives.[9] These substrates
were easily formed by a simple two-step, one-pot reaction.
For example, cyclopentanone 1 was treated with amino eth-
Technischen Universität Graz,
Stremayrgasse 16, 8010 Graz, Austria
Fax: (internat.) ϩ 43-316/873-8959
E-mail: saf@ictos.tu-graz.ac.at
Supporting information for this article is available on the
WWW under http://www.wiley-vch.de/home/eurjoc or from
the author.
Eur. J. Org. Chem. 2000, 3835Ϫ3847
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
1434Ϫ193X/00/1223Ϫ3835 $ 17.50ϩ.50/0
3835

A. de Raadt, H. Griengl et al.
FULL PAPER
anol 2a to give a spirooxazolidine which was subsequently improve the enantioselectivity of lipase-catalysed hydrolysis
benzoylated in situ with benzoyl chloride to afford 3a. and transesterification.[24,25] Bearing this in mind, it was en-
However, although 3a led to hydroxylated compound 4a in visaged that the use of a chiral amino alcohol instead of
reasonable isolated yield, optical purity was far from being simple amino ethanol for preparing spiroxazolidine derivat-
useful for synthetic purposes (Table 1, entry 1).[9] This result ives would have a favourable effect on the course of the
is in agreement with the literature because many other ex- biohydroxylation with respect to the optical purities of the
amples of moderate stereoselectivity have been reported[4,15] products. In addition, it was hoped that it might be possible
for the hydroxylating microorganism Beauveria bassiana. to obtain either configuration of the alcohols, depending on
However, despite this general trend, a few highly selective the configuration of the chiral auxiliary. We wish to report
biohydroxylations (80Ϫ90% optical purities) have also been our investigations concerning the use of chiral amino alco-
observed[7,16,17] for this fungus. This encouraged us to at- hols as docking/protecting groups for the biohydroxylation
tempt to improve the selectivity of the hydroxylation un- of cyclic ketones.
der consideration.
All attempts to find an alternative docking/protecting
Results and Discussion
group for ketones were unsuccessful because selectivity was
not improved.[11,18] Consequently, employing spirooxazolid-
ine derivatives to apply the docking/protecting group prin-
ciple to ketones was considered to be the best strategy. In
addition, the use of other microorganisms, for example
Cunninghamella blakesleeana DSM 1906 and Bacillus mega-
terium DSM 32, was not found to improve the overall yield
and the optical purity of product 4a.[19]
Methods to optimise the given substrate structure, some-
times coined ‘‘substrate engineering’’,[20Ϫ22] were consid-
ered as a way of achieving high yields and selectivities. One
form of substrate engineering which has enjoyed good suc-
cess[23] in the field of organic synthesis is the use of chiral
To examine the scope and limitations of chiral docking/
protecting groups in biohydroxylation, the model ketone
cyclopentanone 1 was systematically reacted with a range
of chiral amino alcohols, namely 2bϪ2n as well as 7a and
7b. In this manner, the effect of subtle substrate structural
changes Ϫ that is changes of R1 and R2 with respect to
chain length and structure Ϫ on the course of the biohyd-
roxylation could be examined.
Simple and Unbranched Side-Chain Substrates
Commercially available (2R)-2-amino-1-propanol 2b
auxiliaries.[23] It is well-known that the introduction of a (98% ee) was treated with cyclopentanone 1 to give crystal-
stereogenic centre into the starting material can improve the line spirooxazolidine 3b in good isolated yield (75%).[9] Sub-
stereoselectivity of a chemical transformation. However, strate 3b (Table 1, entry 2) was then incubated with Beau-
this approach to increase selectivity has been reported only veria bassiana ATCC 7159. Satisfyingly, crystalline product
occasionally in the biotransformation field, for example to 4b was obtained in 84% isolated yield as a mixture of two
Table 1. Influence of the docking/protecting group on the outcome of the biohydroxylation
Entry
Substance
3
4
5
6[a]
yield (%)[b]
yield (%)[b]
de (%)[c]
yield (%)[b]
de (%)[c]
config.
yield (%)[b]
ee (%)[d]
1
a
b
c
d
e
f
63
75
89
76
71
76
94
81
75
83
84
63
52
84
60
40
90
50
81
47
Ϫ
73
85[e]
74
75
71
Ϫ
n.d.
89
33
75
45
Ϫ
R
R
R
R
R
Ϫ
Ϫ
R
S
R
Ϫ
Ϫ
Ϫ
Ϫ
43
61
74
77
71
Ϫ
40
84
29
76
53
Ϫ
2
84[e]
23
3
4
82
5
53[e]
0[f]
6
7
g
h
i
0[f]
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
8
42[e]
38[e]
71
20
17
Ϫ
Ϫ
Ϫ
77
80
65
Ϫ
Ϫ
Ϫ
80
33
13
Ϫ
Ϫ
Ϫ
68
49
65
Ϫ
Ϫ
Ϫ
78
20
13
Ϫ
Ϫ
Ϫ
9
10
11
12
13
14
j
54[e][g]
[g]
k
l
0[h]
[i]
m
n
[j]
Ϫ
Ϫ
Ϫ
Ϫ
Ϫ
Entry
Substance
8
9
10
6[a]
yield (%)[b]
yield (%)[b]
de (%)[c]
83
53
yield (%)[b]
de (%)[c]
89
73
config.
R
R
yield (%)[b]
ee (%)[d]
91
71
15
16
a
b
50
39
71
20
41
53
82
73
[a] The configuration, yield, and ee of benzyloxy ketone 6 is given as it was observed when performing the biohydroxylation on compounds
3aϪ3n as well as 8a and 8b. Ϫ [b] Isolated yields only. If unchanged substrate was also recovered, the amount was taken into consideration
[c]
[d]
when calculating yield. Ϫ de was calculated with respect to the major stereoisomer and determined by NMR and HPLC. Ϫ ee was
[e]
[f]
calculated with respect tot he major enantiomer and determined by chiral GC. Ϫ Major isomer was crystalline. Ϫ Only unchanged
[g]
starting was recovered. Ϫ For 3j: besides products 4j, additional products were obtained as shown in Scheme 6. For 3k: similar to 3j
[h]
a complex mixture was observed and this was not further investigated. Ϫ
Although the substrate was consumed, product formation
was not observed. Ϫ Here, hydroxylation occurred solely on R2, see Scheme 6. Ϫ A complex mixture was afforded which was not
[i]
[j]
further investigated.
3836
Eur. J. Org. Chem. 2000, 3835Ϫ3847

Chiral Auxiliaries as Docking/Protecting Groups in Biohydroxylation
FULL PAPER
Scheme 1. Variation of the docking/protecting group for the biohydroxylation of cyclopentanone 1 with Beauveria bassiana ATCC 7159.
The configuration of the stereogenic centre marked with the asterisk (*) is given in Table 1. Reagents and conditions: (i) amino alcohol
2 or 7, K2CO3, CH2Cl2, 20 °C, 24 h then BzCl, 20 °C, 24 h; (ii) Beauveria bassiana ATCC 7159; (iii) BnBr, NaH, THF/DMF, 20 °C;
(iv) IR 120 (Hϩ, cat), CH3CN, 20 °C
chromatographically very similar isomers (de 90%, as deter-
mined by NMR and HPLC). The diastereomeric excess of
this mixture could be further improved by recrystallisation.
As previously observed with achiral compound 3a, hy-
droxylation occurred on the cyclopentane ring (Scheme 1)
Scheme 2. Conversion of alcohol 4b to ketone 11
of 3b. This conclusion was based on NMR experiments
conducted with alcohol 4b and ketone 11, the latter having
been synthesised from the former (Scheme 2).[26] Spectro-
scopic data from ketone 11 indicated that functionalisation
had taken place on carbon atom 7[27] as the pair of protons
attached to carbon atom 6 clearly gave rise to two doublets
(δ ϭ 3.50 and 2.40; J ϭ 18.2 Hz). Before docking/protecting
group removal, which was carried out under mild acidic
conditions, compound 4b was derivatised in order to pre-
vent possible elimination of the alcohol as well as to ease
detection (TLC) of the released ketone. For this purpose,
the benzyl group was chosen because of its UV activity,
acid stability as well as the fact that the resulting ketone (6)
could also be prepared chemically as either enantiomer,[9]
this attribute being very valuable for the assignment of the
configuration. Benzylation of 4b under standard condi-
tions[28] afforded crystalline 5b in good yield (85% yield,
Scheme 3. Recycling of the chiral docking/protecting group. Re-
89% de as determined by NMR). Removal of the docking/
agents and conditions: (i) Beauveria bassiana ATCC 7159; (ii) BnBr,
protecting group was effected smoothly with [Hϩ] exchange
resin (IR 120) in acetonitrile at room temperature to give
ketone 6 (61% yield). It should be noted at this point that
the cleaved docking/protecting group 12 can be recycled to
obtain additional amounts of starting material 3b as shown
NaH, THF/DMF, 20 °C; (iii) IR 120 (Hϩ, cat), CH3CN, 20 °C;
(iv) cyclopentanone 1, benzene, reflux, pyridinium tosylate,
molecular sieves
Once ketone 6 had been obtained, it was possible to es-
in Scheme 3 (depicted for 4b MAJ). This is an important tablish the configuration of the newly formed stereogenic
criterion for the efficiency of a chiral auxiliary.[23]
centre. Isolated ketone 6 was compared (GC) with chemic-
Eur. J. Org. Chem. 2000, 3835Ϫ3847
3837

A. de Raadt, H. Griengl et al.
FULL PAPER
ally synthesised[29] (S)-3-benzyloxycyclopentanone (6) and
found to have the (R)-configuration with an ee of 84%.
An additional advantage of the chiral docking/protecting
group was evident upon X-ray analysis of the hydroxylation
products afforded from 3b. After chromatography and crys-
tallisation, suitable crystals for both the minor (as the alco-
hol, 4b MIN) and major (as the benzylated derivative, 5b
MAJ) isomers could be obtained (Figure 1). Because the
configuration of the stereogenic centre in the docking/pro-
tecting group was already known, the configuration at car-
bon atom 7 could be confirmed and, more importantly, the
absolute configuration of spiro-carbon atom 5 could also be
evaluated. Determining the configuration of carbon atom 5
was deemed to be especially important because this know-
ledge could give insight into the stereochemical course of
the biohydroxylation. It should also be mentioned at this
point that upon removal of the docking/protecting group,
this stereogenic centre is destroyed and so this information
is lost. As can be seen more clearly from Scheme 4, hy-
droxylation anti or syn to the nitrogen moiety can give ke-
tone 6 with the same configuration, thereby making the anti
or syn ‘‘mode’’ of hydroxylation indistinguishable. NOE
Scheme 4. Hydroxylation of substrate 3b syn or anti to the nitrogen
atom. Reagents and conditions: Hydroxylation step; (a) overall
substitution of Ha; (b) overall substitution of Hb; (c) overall substi-
measurements failed to reveal the absolute configuration of tution of Hc; (d) overall substitution of Hd; (i) BnBr, NaH, THF/
this compound because of the large distance between the
DMF, 20 °C; (ii) IR 120 (Hϩ, cat), CH3CN, 20 °C
docking/protecting group of known chirality and the newly
introduced stereogenic centre. Consequently, from XRD found to be superior with respect to the isolated product
crystal-structure data, it could be concluded that hy- yield and to the diastereomeric excess. Interestingly, for sub-
droxylation had proceeded anti to the nitrogen moiety in strates 3f and 3g, where phenyl groups were attached to the
both isomers. Indeed, as many examples of such hy- heterocyclic ring, hydroxylation was not observed for either
droxylations anti to the nitrogen moiety can be found in the enantiomer and only unchanged starting material was reco-
literature when employing Beauveria bassiana as the hy- vered. These observations could be accounted for by the
droxylating microorganism, this observation was in full low solubility of these substrates in the fermentation me-
agreement with previous findings.[4a]
dium or by the unsuitability of this bulky docking/pro-
tecting group for this transformation.
Regarding general trends, it is interesting to note that dif-
ferences in product yield and diastereomeric excess can be
seen when comparing the enantiomeric substrate pairs. As
depicted in Scheme 1, substrates where carbon atom 2 has
the (S)-configuration or carbon atom 3 the (R)-configura-
tion, for example R1 ϭ methyl or ethyl, afforded hy-
droxylated products in higher isolated yield and superior
Figure 1. Configuration of compounds 4b MIN and 5b MAJ as
determined by X-ray crystal-structure analysis
In summary, compared with the achiral analogue 3a as diastereomeric excess (4b, 84% yield, 90% de; 4d, 82% yield,
starting material, the isolated product yield (4a 60% c.f. 4b 81% de; 9a, 71% yield, 83% de) than the respective enanti-
84%) and diastereomeric purity (4a 40% c.f. 4b 90% de) omer, i.e. R2 ϭ methyl or ethyl (4c, 23% yield, 50% de; 4e,
were markedly improved by the use of chiral auxiliary 2b.
53% yield, 47% de; 9b, 20% yield, 40% de). Evidently, the
In order to examine the scope and limitations of these existing stereogenic centre in the respective substrate had a
chiral docking/protecting groups, substrates 3cϪ3n as well notable influence on the outcome of the biohydroxylation.
as 8a and 8b were also prepared. As can be seen from The absolute stereochemistry of a substrate affecting the
Table 1, all compounds could be synthesised in good yields selectivity of the biohydroxylation has been previously ob-
as previously described for 3a and 3b.[9] The fermentations served with Beauveria bassiana by Furstoss and co-
were then carried out under the same conditions as for 3b workers.[16] Employing an enantiomeric pair of α-pinene de-
and the hydroxylated products were isolated and charac- rivatives, it was found that the site of hydroxylation was
terised (NMR, HPLC, GC) as previously described.
entirely controlled by the absolute stereochemistry of the
As can be concluded from Table 1, hydroxylation oc- substrate, in other words, each enantiomer gave rise to a
curred solely on the cyclopentane ring when the heterocyc- different product. In addition, unlike the spirooxazolidine
lic ring was substituted with either a methyl (entries 2, 3, derivatives, the hydroxylation proceeded with high selectiv-
15, and 16), ethyl (entries 4 and 5) or isopropyl (entries 8 ity for both enantiomers and the two respective products
and 9) group. Nonetheless, employing substrate 3b was still were obtained with high optical purity (85Ϫ90%).
3838
Eur. J. Org. Chem. 2000, 3835Ϫ3847

Chiral Auxiliaries as Docking/Protecting Groups in Biohydroxylation
FULL PAPER
The major influence of one stereogenic centre on the not be grown. In Figure 2, the X-ray crystal structures of
product yield and the diastereomeric excess would suggest all isomers are depicted.
these substrates to be interesting for molecular modelling
studies and such investigations could lead to the refinement
of existing hydroxylation models.[5Ϫ7] The accuracy of an
active-site model is strongly dependent on the knowledge
that the reaction is catalysed by either one enzyme or by a
number of closely related isozymes.[14] However, recent
Figure 2. Configuration of compounds 4h MIN, 4h MAJ, and 4i
studies conducted by Holland and co-workers[14] suggest
MAJ as determined by X-ray crystal-structure analysis
that Beauveria bassiana exhibits up to at least four distinct
hydroxylase enzyme activities. A consequence of this obser-
vation is that in order to obtain a reliable ‘‘active-site
model’’ for these spirooxazolidine substrates, further in-
vestigations into the nature of the hydroxylating enzyme/s
would be important. Efforts in this context are currently
in progress.
An interesting point is the fact that 4i MAJ is the enanti-
omer of 4h MAJ. This observation is reflected by the similar
melting points (4h MAJ; M.p. 127.5Ϫ128.5 °C and 4i MAJ;
M.p. 127.0Ϫ128.0 °C), optical rotations (4i MAJ; [α]D20
ϭ
Ϫ98.6 and 4h MAJ; [α]2D0 ϭ ϩ93.5) and NMR spectroscopic
data (Experimental Section) acquired for these compounds.
Upon comparison of the experimental data obtained for 4h
MIN with 4i MIN, for which an X-ray crystal structure
could not be obtained, it is not unreasonable to assume that
these compounds are also an enantiomeric pair (4h MIN;
m.p. 169.5Ϫ170.5 °C, [α]2D0 ϭ Ϫ82.6 and 4i MIN; m.p.
174.0Ϫ174.5 °C, [α]2D0 ϭ ϩ79.6). The discrepancies evident
between the melting points and optical rotations of these
enantiomeric pairs could be explained by differences in
their respective enantiomeric purities. However, because
this parameter could not be evaluated (HPLC), full evid-
ence for this proposal cannot be provided.
Branched Side-Chain Substrates
Upon examination of entries 8 and 9, it is clear that the
isopropyl substrates 3h and 3i provided very interesting re-
sults. Depending on the configuration of the docking/pro-
tecting group used, either the (S)- or (R)-ketone 6 was ob-
tained (Scheme 5). While (R)-3h afforded (R)-ketone 6 (78%
ee), the (S)-enantiomer 3i furnished (S)-ketone 6 (20% ee).
Being able to choose, at will, the configuration of the intro-
duced hydroxyl moiety by selecting the appropriate dock-
ing/protecting group has significant preparative potential
and will be investigated further by this group.
At first glance, it is clear from Figure 1 and 2 that regard-
less of the configuration of the hydroxyl group and the
docking/protecting group, hydroxylation always occurred
anti to the nitrogen moiety. This observation was also made
for 4b MAJ and 4b MIN. Owing to the fact that experi-
mental results support the view that 4i MIN is the enanti-
omer of 4h MIN, it is reasonable to assume that hy-
droxylation also proceeded anti to the nitrogen atom in this
compound. As mentioned previously, this finding is in full
accordance with the literature.[4a]
Concerning the general stereochemical course of the hy-
droxylation, for this set of compounds it seems that hy-
droxylation always occurred from the same side of the mo-
lecule. A similar observation was reported by Palmer et
al.[30] employing the same microorganism to hydroxylate a
lactam derivative. In addition, we found the same outcome
for the benzoxazole substrates,[12] although another micro-
Scheme 5. Synthesis of (3R)- or (3S)-benzyloxycyclopentanone 6 organism was employed in this case (Cunninghamella
from cyclopentanone 1. Reagents and conditions: (i) amino alcohol
blakesleeana DSM 1906). However, as mentioned previ-
2h or 2i, K2CO3, CH2Cl2, 20 °C, 24 h then BzCl, 20 °C, 24 h;
ously, until it is established that only one enzyme or closely
related isozymes are responsible for the production of these
hydroxylated products, any conclusions concerning the
course of the hydroxylation, although tempting, are uncer-
tain.
(ii) Beauveria bassiana ATCC 7159; (iii) BnBr, NaH, THF/DMF,
20 °C; (iv) IR 120 (Hϩ, cat), CH3CN, 20 °C
An insight into the possible course of the hydroxylation
was also provided by alcohols 4h and 4i. Similar to the hy-
droxylated products obtained from 3b, column chromato-
graphy and recrystallisation furnished crystals for both iso-
mers (4h MAJ and 4h MIN) and these were suitable for X-
Long Side-Chain Substrates
Increasing the chain length of R1/R2 had a pronounced
ray crystal-structure analysis. In addition, an X-ray crystal adverse effect on the regioselectivity of the hydroxylation,
structure for the major isomer of product 4i (4i MAJ) could as can be seen from Table 1 (entries 10Ϫ14) and Scheme 6.
also be obtained. Unfortunately, crystals of the minor iso- Regardless of the enantiomer employed, propyl-substituted
mer of 4i suitable for X-ray crystal structure analysis could substrates 3j and 3k (Table 1, entries 10 and 11, respect-
Eur. J. Org. Chem. 2000, 3835Ϫ3847
3839

A. de Raadt, H. Griengl et al.
FULL PAPER
ively) furnished complex mixtures after exposure to Beau- dominantly metabolised through general pathways.[32] Con-
veria bassiana. Taking this result into account, only one de- cerning the (R)-enantiomer 3l (Table 1, entry 12), this com-
rivative was investigated in detail, namely 3j. After exposure pound was totally metabolised by Beauveria bassiana and
to Beauveria bassiana, a number of products were isolated products could not be detected (TLC).
from the fermentation broth by column chromatography.
It was also of interest to determine if the addition of a
As the main product, the expected derivative 4j was af- second stereogenic centre into the docking/protecting group
forded in moderate isolated yield (54%) and very poor de would have an effect on the course of the biohydroxylation.
(17%). The major (4j MAJ) and the minor isomer (4j MIN, To this end, diastereoisomer 3n was prepared and subjected
a white crystalline solid) could be separated by a combina- to Beauveria bassiana in the usual manner. As can be seen
tion of chromatography and crystallisation. For 4j MIN, from Table 1 (entry 14), only a complex mixture of products
the absolute configuration was elucidated by X-ray crystal- was afforded in low yield after exposure to this fungus and
structure analysis (Figure 3). As expected, hydroxylation this was no longer investigated.
had again taken place anti to the nitrogen moiety. A minor
The results disclosed above suggest that extending the
product (13, isolated yield 1.5%) was also obtained which side-chain R1 beyond a certain length reduces the regiose-
was tentatively assigned as having the structure depicted in lectivity of the hydroxylation considerably, even to the point
Scheme 6, in which the penultimate carbon atom of the that either mixtures are afforded, the substrate is completely
docking/protecting group had been hydroxylated [(ω Ϫ 1)- metabolised or only side-chain hydroxylation takes place.
hydroxylation]. Investigations concerning the configuration Consequently, this was not investigated further. Existing hy-
of this newly introduced stereogenic centre are currently in droxylation models[5Ϫ7] for Beauveria bassiana state that the
progress. The other products which were also isolated have distance between the carbon which is hydroxylated and the
not been identified to date owing to the small amounts in- carbonyl oxygen of a benzamide or carboxyl oxygen of a
˚
volved and to the presence of impurities. However, it can carbamate group is approximately 5.5 A. It has already
be said that evidence for hydroxylation on the terminal car- been mentioned that these models have to be used with cau-
bon atom (ω-hydroxylation) could not be found (NMR).
tion because it is not known which enzymes are responsible
for a particular conversion. However, in the case of these
long side-chain substrates, it is not unreasonable to assume
˚
that two modes of binding which satisfy the 5.5 A distance
criterion are possible: one binding mode where hy-
droxylation takes place on the cyclopentane ring and an-
other where hydroxylation takes place on the side-chain of
the docking/protecting group.
Conclusion
Scheme 6. Loss of hydroxylation regioselectivity upon extension of
side-chain length
The results presented show that docking/protecting
groups can be successfully applied as chiral auxiliaries to
increase the diastereomeric excess of the hydroxylated prod-
ucts obtained. More importantly, employing docking/pro-
tecting groups 2h and 2i, the absolute configuration of the
hydroxylation product 6 could be chosen. In addition,
yields of hydroxylated products could be increased and
product characterisation with NMR and X-ray crystal-
structure analysis was simplified.
Figure 3. Configuration of compound 4j MIN as determined by
X-ray crystal-structure analysis
Homologation of the side-chain by an additional carbon
atom to give the iso-butyl derivative 3m (Table 1, entry 13)
further enhanced this effect to such an extent that the sole
product observed was syrupy ω-hydroxylated 14 (13%
yield). As the hydroxylation had generated a new ste-
reogenic centre, it was necessary to establish the absolute
configuration of this compound by X-ray crystal-structure
analysis. Esterification of 14 with (1S)-camphanic acid pro-
duced a crystalline compound[31] suitable for this method.
As indicated in Scheme 6, the newly formed centre was
found to have the (R)-configuration. Because unchanged
starting material 3m was not recovered from this trans-
Experimental Section
General Methods: All chemicals were purchased from either Ald-
rich or Fluka. When required, chemicals and solvents were purified
according to Perrin and Armarego.[33] All chiral amino alcohols,
apart from 2i, 2j, and 2k which were prepared from the respective
amino acids,[34] were commercially available. Ϫ Optical rotations
were measured on a DIP-370 Digital Polarimeter (Japan Spectro-
scopic Co., Ltd). Ϫ Melting points (uncorrected) were determined
in open capillaries using a Büchi 530. Ϫ H and 13C NMR spectra
were recorded on either a Gemini 200 (Varian) or MSL 300
(Bruker). HETCOR, DEPT and COSY experiments were carried
1
formation, it can be concluded that substrate 3m was pre- out as required. CDCl3 was used as solvent and as internal stand-
3840 Eur. J. Org. Chem. 2000, 3835Ϫ3847

Chiral Auxiliaries as Docking/Protecting Groups in Biohydroxylation
FULL PAPER
ard unless otherwise stated. Before use, the CDCl3 was filtered
through a short plug of basic alumina to remove traces of acid.
ately (filtrate: 3 ϫ 300 mL ethyl acetate per litre; biomass: 3 ϫ
150 mL). All organic phases were combined, dried over sodium
The minor isomer is shown in italics. Ϫ Mass spectra (EI, 70 eV) sulfate, and evaporated under reduced pressure at 35 °C to yield a
were recorded on a Kratos Profile HV-4 double-focussing magnetic
sector instrument equipped with direct insertion (DI). Relative in-
tensities are given in brackets. Ϫ Chiral HPLC was determined with
crude product. This was then purified by column chromatography
employing mixtures of petroleum ether/ethyl acetate unless other-
wise stated. Yields were calculated after having taken into consid-
a JASCO system containing pump 880-PU, UV-detector 875-UV eration the amount of substrate and product lost through sample
(detection at 238 nm), and AXXIOM Model 727 chromatography
software. The chiral column used was a CHIRALCEL OD-H un-
less otherwise stated (flow rate: 0.50 mL/min, eluent: n-heptane/2-
propanol, 7:3 unless otherwise stated). For an improved separation,
the column was cooled to 10 °C. Ϫ GC was performed with a HP
5890 series II plus equipped with a HP 5 (25m) and a FID. Chiral
GC was measured on a Lipodex E (MachereyϪNagel). Ϫ LC was
performed on Silica gel 60 (Merck, 70Ϫ230 mesh) using mixtures
of ethyl acetate and petroleum ether unless otherwise stated. Ϫ
TLC was performed on Silica gel 60 F254 aluminium plates
(Merck) and compounds detected with UV (254 nm) and spraying
with either reagent A (5% vanillin in concentrated H2SO4) or re-
agent B (10% H2SO4, 10% (NH4)6Mo7O24 · 4 H2O, and 0.8%
Ce(SO4)2 · 4 H2O in water). The TLC plates were then developed
on a hot plate. Unless otherwise stated, mixtures of petroleum
ether/ethyl acetate were used as eluent. Ϫ Solvents needed for
recrystallisation were filtered through basic alumina (ALDRICH,
Basic Brockmann I, 150 mesh) prior to use.
taking and the amount of starting material recovered after chroma-
tography.
General Procedure. ؊ Synthesis of 3b: In a manner similar to that
described by Saavedra,[35] potassium carbonate (3.680 g,
26.6 mmol) was suspended in dry dichloromethane (10 mL) in a
round-bottomed flask fitted with a drying tube. To this mixture,
cyclopentanone 1 (1.800 g, 21.4 mmol) and (2R)-2-amino-1-pro-
panol 3a (1.000 g, 13.3 mmol) were added and the mixture was
stirred at room temperature for 24 h. Subsequently, the heterogen-
eous reaction mixture was cooled (ice bath) and benzoyl chloride
(1.870 g, 13.3 mmol) slowly added. After the addition, the mixture
was allowed to reach room temperature. After 24 h, the mixture
was filtered and the filtrate diluted with dichloromethane (100 mL).
This was then quickly washed with aqueous HCl (5%, 1 ϫ 100 mL),
saturated aqueous NaHCO3 (1 ϫ 100 mL) as well as water (2 ϫ
100 mL) and the organic phase was dried over Na2SO4. The pale
yellow solution was filtered and the filtrate was concentrated down
under reduced pressure to give an orange oil. Column chromato-
graphy (petroleum ether/ethyl acetate, 10:1) was then used to isolate
the title product 3b (2.450 g, 75% yield, for additional experimental
data please see below) as a pale yellow solid.
Microorganism and Medium: The fungus Beauveria bassiana ATCC
7159 (DSM 1344) was obtained from DSM (Braunschweig, Ger-
many). Stock cultures of the organism were maintained on PD
(Difco) agar slants (per litre: 24 g potato-dextrose broth and 15 g
agar), stored at 4 °C and subcultured every 4 weeks at room tem-
perature. Medium E was employed for the fermentations. Medium
E consisted of (per litre) 5 g of malt extract (Merck), 10 g of glu-
cose, 5 g of peptone from meat (Merck), 2 g of yeast extract
(Oxoid) and 2 g of KH2PO4.
General Procedure for Docking/Protecting Group Recycling. ؊ Syn-
thesis of 3b: In a round-bottomed flask, cyclopentanone 1 (1.180 g,
14.0 mmol) was dissolved in dry benzene (100 mL) and (2R)-2-
benzoylamino-1-propanol 12[36] (1.940 g, 10.8 mmol) as well as pyr-
idinium tosylate (0.810 g, 3.24 mmol) added. The mixture was
heated at reflux after a dropping funnel containing a small plug of
˚
glass wool and molecular sieves (4 A) as well as a reflux condenser
General Procedure. ؊ Fermentation Conditions and Product Isola-
tion: Stage I cultures (70 mL) were inoculated with 1 cm2 of a Petri-
dish culture (1Ϫ2 weeks old) and incubated for 72 h in 300 mL
baffled shaking flasks at 25 °C. Stage II cultures were inoculated
(10%) aseptically with stage I culture and the fermentations were
performed either in a Biostat M fermentor (Braun, 1.5 L-glass ves-
sel, agitation was provided by 3 Rushton rotors and kept at
400 rpm) or in a Bioengineering AG model 1523 (16 L-stainless
steel vessel with a working volume of 11 L, agitation was provided
by 2 Rushton rotors and kept at 250Ϫ400 rpm). For both fer-
mentors, the pH was measured with a combined glass electrode
(Ingold, Urdorf, Switzerland) and kept constant at pH 7.0. Tem-
perature was determined with a Pt-100 sensor and maintained at 25
°C. Dissolved oxygen was measured with a polarimetric electrode
(Ingold). At the beginning of the stationary phase (generally after
32 h of growth) the substrate was added aseptically (usually
0.4918 mmol/L, the induction phase) after it had been dissolved in
minimal amounts of EtOH. The second substrate addition (usually
2.459 mmol/L) occurred at the late stationary phase (generally after
an additional 15 h). Typically, 60 to 160 h after the first addition
of substrate, which is termed as the ‘‘total fermentation time’’, the
hydroxylation was complete (TLC, GC). The concentrations of
products and substrates were determined by gas chromatography.
Samples were taken by aseptically removing 5 mL of the fermenta-
tion broth and extracting with ethyl acetate (2 mL). The organic
phase was then measured directly without drying.
had been fitted to the round-bottomed flask. After 24 h, the mix-
ture was set aside to cool, diethyl ether (100 mL) was added and the
mixture was filtered. The filtrate was then washed with saturated
aqueous NaHCO3 (1 ϫ 200 mL) as well as saturated aqueous NaCl
(1 ϫ 200 mL) and the organic phase was dried with Na2SO4. The
solution was filtered and the filtrate was concentrated under re-
duced pressure to give a syrup. Column chromatography (cyclohex-
ane/ethyl acetate, 20:1) was then used to isolate the title compound
3b (1.110 g, 42% yield, for additional experimental data see below)
as a pale yellow solid.
General Procedure for the Removal of the Docking/Protecting
Group. ؊ Synthesis of Ketone 6: Derivative 5b (60 mg) was dis-
solved in acetonitrile (2 mL) and IR 120[Hϩ] (pre-washed twice
with MeOH and once with water) was slowly added with vigorous
stirring until a pH of 5Ϫ6 was reached (pH paper). Stirring was
continued at room temperature until TLC had indicated that start-
ing material was no longer present. Filtration of the reaction mix-
ture to remove the ion-exchange resin and concentration of the
filtrate under reduced pressure furnished a residue which was sub-
sequently purified with column chromatography (petroleum ether/
ethyl acetate, 6:1) to afford ketone 6 (21.3 mg, 61% yield, for addi-
tional experimental details see Table 1).
Substrates 3a؊3n and 8a, 8b
Achiral Substrate 3a: The general procedure gave the title com-
pound 3a from 2a (10.000 g) as a white solid (23.857 g, 63% yield).
Ϫ M.p. 60.0Ϫ61.0 °C (from hexane /ethyl acetate). Ϫ 1H NMR:
Immediately after the fermentation was complete, the culture broth
was filtered and both the filtrate and biomass were extracted separ-
Eur. J. Org. Chem. 2000, 3835Ϫ3847
3841

A. de Raadt, H. Griengl et al.
δ ϭ 1.72 [m, 4 H, 8(8Ј)-H, 7(7Ј)-H], 1.91 & 2.54 [2 ϫ br. s, 2 ϫ 2 solid (0.210 g, 94% yield) which slightly discoloured upon expose
FULL PAPER
H, 6(6Ј)-H, 9(9Ј)-H], 3.52 & 3.88 [2 ϫ t, 2 ϫ 2 H, 2(2Ј)-H, 3(3Ј)-
to light and warmth. Ϫ M.p. 128.0Ϫ131.0 °C (from petroleum
H, J2,3 ϭ 6.1 Hz], 7.38 (m, 5 H, COPh). Ϫ 13C NMR: δ ϭ 24.9, ether/ethyl acetate). Ϫ [α]2D0 ϭ ϩ185.8 (c ϭ 0.6, CH2Cl2). Ϫ 1H
35.3 (C-6, C-7, C-8, C-9), 49.0 (C-3), 63.9 (C-2), 104.9 (C-5), 126.8, NMR: δ ϭ 1.64Ϫ2.19 [m, 6 H, 6-H, 7(7Ј)-H, 8(8Ј)-H, 9-H], 2.61 &
128.5, 130.0, 138.1, (COPh), 167.8 (COPh).
2.86 (2 ϫ m, 2 ϫ 1 H, 6Ј-H, 9Ј-H), 3.82 (dd, 1 H, 2-H, J2,2Ј ϭ
9.0 Hz, J2,3 ϭ 4.0 Hz), 4.25 (dd, 1 H, 2Ј-H, J2Ј,3 ϭ 6.4 Hz), 4.80
(dd, 1 H, 3-H), 6.86Ϫ7.28 (m, 10 H, Ph, COPh). Ϫ 13C NMR: δ ϭ
24.9, 25.0 (C-7, C-8), 35.2, 35.8 (C-6, C-9), 62.9 (C-3), 71.9 (C-2),
106.1 (C-5), 126.1, 126.4, 127.5, 128.0, 128.4, 128.5, 129.1, 138.0,
141.3 (COPh, Ph), 169.2 (COPh).
(3R)-Me Substrate 3b: The general procedure furnished substrate
3b from 2b as a pale yellow solid (2.450 g, 75% yield). Ϫ M.p.
65.5Ϫ67.5 °C (from petroleum ether/ethyl acetate). Ϫ [α]2D0 ϭ Ϫ79.8
(c ϭ 2.1, CH2Cl2). Ϫ 1H NMR: δ ϭ 0.95 (d, 3 H, CH3, J ϭ
6.5 Hz,), 1.64Ϫ1.98 [m, 6 H, 6-H, 7(7Ј)-H, 8(8Ј)-H, 9-H], 2.37Ϫ2.71
[2 ϫ br. m, 2 H, 6Ј-H, 9Ј-H], 3.60 (m, 1 H, 2-H), 4.00 (m, 2 H, 2Ј-
H, 3-H), 7.40 (s, 5 H, COPh). Ϫ 13C NMR: δ ϭ 20.1 (CH3), 24.7,
24.8 (C-7, C-8), 35.0, 36.5 (C-6, C-9), 54.1 (C-3), 70.0 (C-2), 105.0
(C-5), 126.2, 128.5, 129.4, 138.2, (COPh), 168.1 (COPh).
(3R)-iPr Substrate 3h: The title compound was synthesised from
2h (5.184 g) according to the general procedure to furnish a white
crystalline solid (11.159 g, 81% yield). Ϫ M.p. 79.5Ϫ80.5 °C (from
petroleum ether/ethyl acetate). Ϫ [α]2D0 ϭ Ϫ93.9 (c ϭ 1.2, CH2Cl2).
Ϫ
1H NMR: δ ϭ 0.62 & 0.80 [2 ϫ d, 2 ϫ 3 H, CH(CH3)2, J ϭ
(3S)-Me Substrate 3c: The title substance was prepared from 2c
(1.000 g) according to the general procedure above to give a pale
yellow solid (2.920 g, 89% yield). Ϫ M.p. 65.5Ϫ67.5 °C (from petro-
leum ether/ethyl acetate). Ϫ [α]2D0 ϭ ϩ76.4 (c ϭ 2.7, CH2Cl2). Ϫ
1H NMR: δ ϭ 0.93 (m, 3 H, CH3), 1.57Ϫ2.03 [m, 6 H, 6-H, 7(7Ј)-
H, 8(8Ј)-H, 9-H], 2.32Ϫ2.71 (2 ϫ br. m, 2 H, 6Ј-H, 9Ј-H), 3.57 (m,
1 H, 2-H), 3.98 (m, 2 H, 2Ј-H, 3-H), 7.38 (s, 5 H, COPh). Ϫ 13C
NMR: δ ϭ 20.1 (CH3), 24.7, 24.8 (C-7, C-8), 35.0, 36.5 (C-6, C-
9), 54.1 (C-3), 70.0 (C-2), 105.0 (C-5), 126.2, 128.5, 129.4, 138.2,
(COPh), 168.0 (COPh).
7.1 Hz], 1.43Ϫ2.02 [m,
7 H, 6-H, 7(7Ј)-H, 8(8Ј)-H, 9-H,
CH(CH3)2], 2.39 & 2.65 (2 ϫ m, 2 ϫ 1 H, 6Ј-H, 9Ј-H), 3.89 [m, 3
H, 2(2Ј)-H, 3-H], 7.40 (s, 5 H, COPh). Ϫ 13C NMR: δ ϭ 16.3, 19.4
[CH(CH3)2], 24.8, 25.0 (C-7, C-8), 30.0 [CH(CH3)2] 35.2, 36.2 (C-
6, C-9), 62.9 (C-3), 64.6 (C-2), 105.3 (C-5), 126.5, 128.4, 129.5,
138.4 (COPh), 168.6 (COPh).
(3S)-iPr Substrate 3i: Substrate 3i was made from 2i[34] (5.600 g)
according to the general procedure to give a white solid (11.189 g,
75% yield). Ϫ M.p. 79.5Ϫ80.0 °C (from petroleum ether). Ϫ [α]2D0
ϭ
1
ϩ95.7 (c ϭ 1.1, CH2Cl2). Ϫ H NMR: δ ϭ 0.62 & 0.79 (2 ϫ d, 2
ϫ 3 H, CH(CH3)2, J ϭ 7.1 Hz), 1.43Ϫ2.02 [m, 7 H, 6-H, 7(7Ј)-H,
8(8Ј)-H, 9-H, CH(CH3)2], 2.39 & 2.65 (2 ϫ m, 2 ϫ 1 H, 6Ј-H, 9Ј-
H), 3.88 [m, 3 H, 2(2Ј)-H, 3-H], 7.39 (s, 5 H, COPh). Ϫ 13C NMR:
δ ϭ 16.3, 19.4 [CH(CH3)2], 24.8, 25.0 (C-7, C-8), 30.0 [CH(CH3)2],
35.2, 36.2 (C-6, C-9), 62.9 (C-3), 64.6 (C-2), 105.3 (C-5), 126.5,
128.4, 129.5, 138.4 (COPh), 168.5 (COPh).
(3R)-Et Substrate 3d: The title compound was prepared from 2d
(1.009 g) as described above in the general procedure to afford a
white waxy solid (2.240 g, 76% yield). Ϫ M.p. 48.0Ϫ49.0 °C (from
petroleum ether/ethyl acetate). Ϫ [α]2D0 ϭ Ϫ81.1 (c ϭ 1.4, CH2Cl2).
Ϫ
1H NMR: δ ϭ 0.65 (t, 3 H, CH2CH3, J ϭ 7.4 Hz), 1.33 (m,
2 H, CH2CH3), 1.60Ϫ2.02 [m, 6 H, 6-H, 7(7Ј)-H, 8(8Ј)-H, 9-H],
2.31Ϫ2.71 (2 ϫ br. m, 2 H, 6Ј-H, 9Ј-H), 3.76 (dd, 1 H, 2-H, J2,2Ј
ϭ
(3R)-Pr Substrate 3j: The title compound was prepared from 2j[34]
(3.099 g) according to the general procedure to furnish a white solid
(6.833 g, 83% yield). Ϫ M.p. 77.5Ϫ78.5 °C (from petroleum ether).
8.3 Hz, J2,3 ϭ 2.0 Hz), 3.85 (m, 1 H, 3-H), 3.95 (dd, 1 H, 2Ј-H,
J2Ј,3 ϭ 5.4 Hz), 7.40 (s, 5 H, COPh). Ϫ 13C NMR: δ ϭ 9.9
(CH2CH3), 24.8, 24.8, 26.5 (C-7, C-8, CH2CH3), 35.0, 36.4 (C-6,
C-9), 59.6 (C-3), 67.3 (C-2), 104.7 (C-5), 126.3, 128.4, 129.4, 137.8
(COPh), 167.7 (COPh).
1
Ϫ [α]2D0 ϭ Ϫ100.7 (c ϭ 1.3, CH2Cl2). Ϫ H NMR: δ ϭ 0.61 (t, 3
H, CH2CH2CH3,
J ϭ 7.1 Hz), 0.83Ϫ1.52 (2 ϫ m, 4 H,
CH2CH2CH3), 1.61Ϫ2.04 [m, 6 H, 6-H, 7(7Ј)-H, 8(8Ј)-H, 9-H],
2.44 & 2.63 (2 ϫ m, 2 ϫ 1 H, 6Ј-H, 9Ј-H), 3.74 (m, 1 H, 2-H), 3.92
(m, 2 H, 2Ј-H, 3-H), 7.39 (s, 5 H, COPh). Ϫ 13C NMR: δ ϭ 13.4
(CH2CH2CH3), 18.8 (CH2CH2CH3), 24.7, 24.8 (C-7, C-8), 34.9,
35.7, 36.4 (CH2CH2CH3, C-6, C-9), 58.1 (C-3), 67.6 (C-2), 104.8
(C-5), 126.3, 128.4, 129.4, 138.3 (COPh), 168.1 (COPh).
(3S)-Et Substrate 3e: Substrate 3e was prepared from 2e (1.025 g)
according to the general procedure to give a white waxy solid
(2.129 g, 71% yield). Ϫ M.p. 49.0Ϫ50.0 °C (from petroleum ether/
ethyl acetate). Ϫ [α]2D0 ϭ ϩ82.3 (c ϭ 1.5, CH2Cl2). Ϫ 1H NMR:
δ ϭ 0.63 (t, 3 H, CH2CH3, J ϭ 7.4 Hz), 1.31 (m, 2 H, CH2CH3),
1.60Ϫ2.01 [m, 6 H, 6-H, 7(7Ј)-H, 8(8Ј)-H, 9-H], 2.31Ϫ2.71 (2 ϫ br.
m, 2 H, 6Ј-H, 9Ј-H), 3.75 (dd, 1 H, 2-H, J2,2Ј ϭ 8.3 Hz, J2,3
ϭ
(3S)-Pr Substrate 3k: The title compound was prepared from 2k[34]
(2.859 g) according to the general procedure to furnish a white solid
(6.396 g, 84% yield). Ϫ M.p. 76.5Ϫ78.0 °C (from petroleum ether).
Ϫ [α]2D0 ϭ ϩ98.4 (c ϭ 1.1, CH2Cl2). Ϫ 1H NMR: δ ϭ 0.61 (t, 3 H,
CH2CH2CH3, J ϭ 7.1 Hz), 0.83Ϫ1.52 (2 ϫ m, 4 H, CH2CH2CH3),
1.61Ϫ2.04 [m, 6 H, 6-H, 7(7Ј)-H, 8(8Ј)-H, 9-H], 2.44 & 2.64 [2 ϫ
m, 2 ϫ 1 H, 6Ј-H, 9Ј-H], 3.74 (m, 1 H, 2-H), 3.92 (m, 2 H, 2Ј-H,
3-H), 7.39 (s, 5 H, COPh). Ϫ 13C NMR: δ ϭ 13.4 (CH2CH2CH3),
18.8 (CH2CH2CH3), 24.7, 24.8 (C-7, C-8), 35.0, 35.7, 36.4
(CH2CH2CH3, C-6, C-9), 58.1 (C-3), 67.6 (C-2), 104.8 (C-5), 126.3,
128.4, 129.4, 138.3 (COPh), 168.1 (COPh).
2.0 Hz), 3.85 (m, 1 H, 3-H), 3.95 (dd, 1 H, 2Ј-H, J2Ј,3 ϭ 5.5 Hz),
7.39 (s, 5 H, COPh). Ϫ 13C NMR: δ ϭ 9.9 (CH2CH3), 24.8, 24.8,
26.4 (C-7, C-8, CH2CH3), 35.0, 36.3 (C-6, C-9), 59.6 (C-3), 67.3 (C-
2), 104.9 (C-5), 126.3, 128.4, 129.4, 138.3 (COPh), 168.2 (COPh).
(3R)-Ph Substrate 3f: Substrate 3f was synthesised from 2f (0.100 g)
following the general procedure above to afford a white solid
(0.170 g, 76% yield) which slightly discoloured upon expose to light
and warmth. Ϫ M.p. 131.0Ϫ133.0 °C (from petroleum ether/ethyl
acetate). Ϫ [α]2D0 ϭ Ϫ189.1 (c ϭ 1.5, CH2Cl2). Ϫ 1H NMR: δ ϭ
1.70Ϫ2.14 [m, 6 H, 6-H, 7(7Ј)-H, 8(8Ј)-H, 9-H], 2.60 & 2.84 (2 ϫ
m, 2 ϫ 1 H, 6Ј-H, 9Ј-H), 3.82 (dd, 1 H, 2-H, J2,2Ј ϭ 9.0 Hz, J2,3
ϭ
(3R)-iBu Substrate 3l: Substrate 3l was prepared from 2l (0.351 g)
using the general procedure above to give a white crystalline solid
(0.620 g, 72% yield). Ϫ M.p. 107.0Ϫ109.0 °C (from cyclohexane/
4.0 Hz), 4.25 (dd, 1 H, 2Ј-H, J2Ј,3 ϭ 6.4 Hz), 4.80 (dd, 1 H, 3-H),
6.87Ϫ7.28 (m, 10 H, Ph, COPh). Ϫ 13C NMR: δ ϭ 24.9, 25.0 (C-
7, C-8), 35.2, 35.8 (C-6, C-9), 62.9 (C-3), 71.9 (C-2), 106.1 (C-5),
126.1, 126.4, 127.5, 128.0, 128.4, 128.5, 129.1, 138.0, 141.3 (COPh,
Ph), 169.1 (COPh).
1
ethyl acetate). Ϫ [α]2D0 ϭ Ϫ100.0 (c ϭ 1.2, CH2Cl2). Ϫ H NMR:
δ ϭ 0.02 & 0.37 [2 ϫ m, 2 ϫ 3 H, CH2CH(CH3)2], 0.65 [m, 1
H, CH2CH(CH3)2], 0.92 [m, 1 H, CH2CH(CH3)2], 1.18 [m, 1 H,
CH2CH(CH3)2], 1.27Ϫ1.73 [2 ϫ m, 6 H, 6-H, 7(7Ј)-H, 8(8Ј)-H, 9-
H], 2.12 & 2.33 (2 ϫ m, 2 H, 6Ј-H, 9Ј-H), 3.39 (m, 1 H, 2-H) 3.60
(3S)-Ph Substrate 3g: Substrate 3g was synthesised from 2g
(0.100 g) following the general procedure above to afford a white
3842
Eur. J. Org. Chem. 2000, 3835Ϫ3847

Chiral Auxiliaries as Docking/Protecting Groups in Biohydroxylation
FULL PAPER
(m, 2 H, 2Ј-H, 3-H), 7.08 (s, 5 H, COPh). Ϫ 13C NMR: δ ϭ 20.6,
1.96, 2.28 & 2.56 [4 ϫ m, 6 H, 6-H, 8(8Ј)-H, 9(9Ј)-H, OH], 2.85
23.5 [CH2CH(CH3)2], 24.7, 24.8, 25.3 [C-7, C-8, CH2CH(CH3)2], (dd, 1 H, 6Ј-H, J ϭ 5.9 and 14.1 Hz), 3.53 & 3.92 [2 ϫ t, 2 ϫ 2 H,
35.0, 36.5 (C-6, C-9), 43.0 [CH2CH(CH3)2], 57.1 (C-3), 67.8 (C-2),
104.5 (C-5), 126.3, 128.4, 129.3, 138.2 (COPh), 167.9 (COPh).
2(2Ј)-H, 3(3Ј)-H, J2,3 ϭ 6.1 Hz], 4.49 (br. s, 1 H, 7-H), 7.36Ϫ7.49
(m, 5 H, COPh). Ϫ 13C NMR: 33.8, 34.7 (C-8, C-9), 43.7 (C-6),
48.7 (C-3), 64.2 (C-2), 72.9 (C-7), 103.8 (C-5), 126.7, 128.6, 130.2,
137.6 (COPh), 169.0 (COPh).
(3S)-iBu Substrate 3m: Compound 3m was made from 2m (7.000 g)
as described in the general procedure to give a white crystalline
compound (12.200 g, 71% yield). Ϫ M.p. 104.0Ϫ106.0 °C (from
cyclohexane/ethyl acetate). Ϫ [α]2D0 ϭ ϩ99.0 (c ϭ 1.2, CH2Cl2). Ϫ
1H NMR: δ ϭ 0.02 & 0.36 [2 ϫ d, 2 ϫ 3 H, CH2CH(CH3)2, J ϭ
(3R)-Me Hydroxylated Product 4b: Treating substrate 3b (Biostat
M: first substrate addition, 0.110 g after 14 h; second substrate ad-
dition, 0.441 g after 11 h) with Beauveria bassiana as given above
in the general procedure, furnished (total fermentation time 30 h)
a mixture of isomers as a white solid (0.410 g, 84% yield, de ϭ 90%
as determined by NMR and HPLC). The isomers could be separ-
ated by column chromatography combined with recrystallisation.
6.4 Hz], 0.69 [m,
1 H, CH2CH(CH3)2], 0.89 [m, 1 H,
CH2CH(CH3)2], 1.17 [m, 1 H, CH2CH(CH3)2], 1.31Ϫ1.75 [2 ϫ m,
6 H, 6-H, 7(7Ј)-H, 8(8Ј)-H, 9-H], 2.12 & 2.35 (2 ϫ m, 2 H, 6Ј-H,
9Ј-H), 3.40 (br. d, 1 H, 2-H, J2,2Ј ϭ 7.3 Hz), 3.60 (br. d, 2 H, 2Ј-H,
3-H), 7.07 (s, 5 H, COPh). Ϫ 13C NMR: δ ϭ 20.8, 23.6
[CH2CH(CH3)2], 24.7, 24.9, 25.4 [C-7, C-8, CH2CH(CH3)2], 34.9,
36.7 (C-6, C-9), 43.1 [CH2CH(CH3)2], 57.3 (C-3), 67.9 (C-2), 104.7
(C-5), 126.5, 128.5, 129.5, 138.4 (COPh), 168.2 (COPh).
4b MAJ: M.p. 106Ϫ108 °C (from petroleum ether/ethyl acetate). Ϫ
1
[α]2D0 ϭ Ϫ79.6 (c ϭ 1.5, CH2Cl2). Ϫ H NMR (assigned with the
aid of HETCOR experiments): δ ϭ 0.93 (d, 3 H, Me, J3,Me
ϭ
5.7 Hz), 1.77 (br. d, 1 H, 6-H, J6,6Ј ϭ 13.9 Hz), 1.84Ϫ2.41 (m, 4 H,
8-H, 9-H, 9Ј-H or 8Ј-H, OH), 2.57Ϫ2.69 (m, 1 H, 8Ј-H or 9Ј-H),
(3S)-sBu Substrate 3n: Diastereoisomer 3n was prepared from 2n
(0.400 g) as described by the general procedure above to give a
white crystalline solid (0.820 g, 84% yield). Ϫ M.p. 66.0Ϫ70.0 °C
(from cyclohexane/ethyl acetate). Ϫ [α]2D0 ϭ ϩ99.6 (c ϭ 1.2,
CH2Cl2). Ϫ 1H NMR: δ ϭ 0.5 [t, 3 H, CH(CH3)CH2CH3, J ϭ
7.2 Hz], 0.76 [d, 3 H, CH(CH3)CH2CH3, J ϭ 7.0 Hz], 0.90 [m, 2
H, CH(CH3)CH2CH3], 1.18 [br. m, 1 H, CH(CH3)CH2CH3],
1.55Ϫ1.97 [m, 6 H, 6-H, 7(7Ј)-H, 8(8Ј)-H, 9-H], 2.35 & 2.62 (2 ϫ
m, 2 H, 6Ј-H, 9Ј-H), 3.82 [m, 2 H, 2(2Ј)-H], 3.97 [m, 1 H, 3-H],
7.36 (s, 5 H, COPh). Ϫ 13C NMR: δ ϭ 11.7 [CH(CH3)CH2CH3],
13.5 [CH(CH3)CH2CH3], 24.7, 25.0 (C-7, C-8), 26.6
2.75 (dd, 1 H, 6Ј-H, J6Ј,7 ϭ 5.7 Hz), 3.63 (dd, 1 H, 2-H, J2,3
ϭ
5.1 Hz, J2,2Ј ϭ 11.3 Hz), 3.98 (m, 2 H, 2Ј-H, 3-H), 4.42 (br. s, 1 H,
7-H), 7.37 (s, 5 H, COPh). Ϫ 13C NMR (assigned with the aid of
HETCOR and DEPT experiments): δ ϭ 20.1 (Me), 34.6, 34.7 (C-
8, C-9), 43.1 (C-6), 54.0 (C-3), 70.3 (C-2), 72.9 (C-7), 104.0 (C-5),
126.2, 128.6, 129.6, 137.7 (COPh), 168.2 (COPh).
4b MIN: M.p. 86.5Ϫ88.0 °C (from n-hexane/CH2Cl2). Ϫ [α]2D0
ϭ
Ϫ37.9 (c ϭ 1.8, THF). Ϫ 1H NMR (assigned with the aid of
HETCOR experiments): δ ϭ 0.93 (d, 3 H, Me, J3,Me ϭ 6.3 Hz),
1.73Ϫ2.05, 2.12Ϫ2.57 (2 ϫ br. m, 6 H, 6-H, 8(8Ј)-H, 9(9Ј)-H, OH),
2.92 (dd, 1 H, 6Ј-H, J6Ј,7 ϭ 6.1 Hz, J6,6Ј ϭ 14.0 Hz), 3.62 (m, 1 H,
2-H), 4.01 (m, 2 H, 2Ј-H, 3-H), 4.48 (br. s, 1 H, 7-H), 7.41 (s, 5 H,
COPh). Ϫ 13C NMR (assigned with the aid of HETCOR and
DEPT experiments): δ ϭ 20.1 (Me), 33.6, 34.4 (C-8, C-9), 44.8 (C-
6), 54.0 (C-3), 70.3 (C-2), 72.8 (C-7), 103.8 (C-5), 126.2, 128.6,
129.6, 137.8 (COPh), 168.3 (COPh).
[CH(CH3)CH2CH3],
35.2,
35.9
(C-6,
C-9),
36.8
[CH(CH3)CH2CH3], 61.0 (C-3), 64.2 (C-2), 105.3 (C-5), 126.3,
128.4, 129.4, 138.3 (COPh), 168.4 (COPh).
(2S)-Me Substrate 8a: Compound 8a was prepared from 7a
(1.000 g) as described in the general procedure above to furnish a
pale yellow oil (1.630 g, 50% yield). Ϫ [α]2D0 ϭ ϩ120.6 (c ϭ 2.2,
CH2Cl2). Ϫ 1H NMR: δ ϭ 1.27 (d, 3 H, CH3, J ϭ 5.9 Hz),
1.59Ϫ2.03 (m, 6 H, 6-H, 7(7Ј)-H, 8(8Ј)-H, 9-H], 2.35Ϫ2.75 (2 ϫ
br. m, 2 H, 6Ј-H, 9Ј-H), 3.19 (dd, 1 H, 3-H, J2,3Ј ϭ J3,3Ј ϭ 9.5 Hz),
3.45 (dd, 1 H, 3Ј-H, J2,3Ј ϭ 5.2 Hz), 4.04 (m, 1 H, 2-H), 7.33Ϫ7.51
(m, 5 H, COPh). Ϫ 13C NMR: δ ϭ 17.5 (CH3), 24.5, 25.2 (C-7, C-
8), 35.2, 36.1 (C-6, C-9), 55.6 (C-3), 70.8 (C-2), 105.0 (C-5), 126.6,
128.4, 129.8, 137.9 (COPh), 167.4 (COPh).
(3S)-Me Hydroxylated Product 4c: Substrate 3c (Biostat M: first
substrate addition, 0.114 g after 14 h; second substrate addition,
0.563 g after 12 h) was hydroxylated by Beauveria bassiana (total
fermentation time 50 h) as described in the general procedure to
give unchanged starting material (0.400 g) and a pale yellow syrup
(0.050 g, 23% yield, de ϭ 50% as determined by HPLC and NMR)
1
after chromatography. Ϫ H NMR (minor isomer in italics): δ ϭ
(2R)-Me Substrate 8b: Substrate 8b was made from 7b (1.000 g)
following the general procedure given above to afford a pale yellow
oil (1.260 g, 39% yield). Ϫ [α]2D0 ϭ Ϫ123.5 (c ϭ 2.3, CH2Cl2). Ϫ 1H
NMR: δ ϭ 1.27 (d, 3 H, CH3, J ϭ 5.9 Hz), 1.56Ϫ2.05 (m, 6 H, 6-
H, 7(7Ј)-H, 8(8Ј)-H, 9-H], 2.36Ϫ2.75 (2 ϫ br. m, 2 H, 6Ј-H, 9Ј-H),
3.20 (dd, 1 H, 3-H, J2,3Ј ϭ J3,3Ј ϭ 9.5 Hz), 3.45 (dd, 1 H, 3Ј-H,
J2,3Ј ϭ 5.2 Hz), 4.03 (m, 1 H, 2-H), 7.33Ϫ7.51 (m, 5 H, COPh). Ϫ
13C NMR: δ ϭ 17.5 (CH3), 24.5, 25.2 (C-7, C-8), 35.2, 36.1 (C-6,
C-9), 55.6 (C-3), 70.8 (C-2), 105.0 (C-5), 126.6, 128.4, 129.8, 137.9
(COPh), 167.4 (COPh).
0.94 (m, 3 H, Me), 1.74Ϫ2.70 [m, 6 H, 6-H, 8(8Ј)-H, 9(9Ј)-H, OH],
2.76 & 3.01 (2 ϫ dd, 1 H, ratio: 1:3, 6Ј-H, J6Ј,7 ϭ 5.9 Hz, J6,6Ј
ϭ
14.0 Hz), 3.63 (m, 1 H, 2-H), 4.02 (m, 2 H, 2Ј-H, 3-H), 4.47 (br. s,
1 H, 7-H), 7.38 (s, 5 H, COPh). Ϫ 13C NMR (minor isomer in
italics): δ ϭ 20.1, 20.3 (Me), 33.8, 34.6, 34.7, 34.9 (C-8, C-9), 43.4,
45.0 (C-6), 54.2 (C-3), 70.5 (C-2), 73.0 (C-7), 104.0, 104.2 (C-5),
126.4, 128.7, 129.8, 138.1 (COPh), 168.5 (COPh).
(3R)-Et Hydroxylated Product 4d: Substrate 3d (Biostat M: first
substrate addition, 0.156 g after 23 h, second substrate addition,
0.661 g after 12 h) was subjected to Beauveria bassiana (total fer-
mentation time 165 h) to afford a pale yellow syrup (0.560 g, 82%
Hydroxylated Products 4a؊4e, 4h؊4k as well as 9a, 9b and 13؊15
1
Hydroxylated Product 4a: Employing 4 ϫ 1 L shaking flasks
(250 mL medium per flask) for the Stage II culture, substrate 3a
(0.400 g of substrate was added in one portion after 48 h of growth)
was subjected (total fermentation time 48 h) to Beauveria bassiana
to give a syrupy product, after chromatography, as a mixture of
two isomers (0.256 g, 60% yield, chiral GC of 4a and an NMR of
yield, de ϭ 81% as determined by HPLC). Ϫ H NMR: δ ϭ 0.64
(t, 3 H, CH2CH3, J ϭ 7.4 Hz), 1.08Ϫ1.49 (br. m, 2 H, CH2CH3),
1.72Ϫ2.40 & 2.57Ϫ3.15 [2 ϫ m, 4 H & 3 H, 6(6Ј)-H, 8(8Ј)-H, 9(9Ј)-
H, OH], 3.74Ϫ4.08 [m, 3 H, 2(2Ј)-H, 3-H], 4.47 (br. m, 1 H, 7-H),
7.40 (s, 5 H, COPh) Ϫ 13C NMR (minor isomer in italics): δ ϭ 9.9
(CH2CH3), 26.3, 26.4 (CH2CH3), 33.6, 34.5 (C-8, C-9), 43.1, 44.7
(C-6), 59.4 (C-3), 67.5, 67.6 (C-2), 72.7, 72.8 (C-7), 103.9 (C-5),
the corresponding Mosher ester[12c] indicated an isomeric ratio of
1
2.3:1). Ϫ [α]2D0 ϭ Ϫ2.9 (c ϭ 2.3, CH2Cl2). Ϫ H NMR: δ ϭ 1.80, 126.2, 128.4, 128.4, 128.5, 129.7, 137.7 (COPh), 168.3 (COPh).
Eur. J. Org. Chem. 2000, 3835Ϫ3847
3843

A. de Raadt, H. Griengl et al.
FULL PAPER
(3S)-Et Hydroxylated Product 4e: Substrate 3e (Bioengineering AG
model 1523: after 33 h 1.274 g was added for induction and, after
an additional 18 h, 6.388 g was added) was subjected to a culture
of Beauveria bassiana (total fermentation time 96 h) to furnish a
syrup (4.220 g, 53% yield, de ϭ 47% as determined by HPLC). Ϫ
1H NMR: δ ϭ 0.62 (t, 3 H, CH2CH3, J ϭ 7.5 Hz), 1.29 (br. m, 2
H, CH2CH3), 1.72Ϫ2.69 [m, 6 H, 6-H, 8(8Ј)-H, 9(9Ј)-H, OH],
2.76 & 2.97 (2 ϫ dd, 1 H, 6Ј-H, J6,6Ј ϭ 13.8 Hz, J6Ј,7 ϭ 5.8 Hz),
3.74Ϫ4.03 [m, 3 H, 2(2Ј)-H, 3-H], 4.49 (br. m, 1 H, 7-H), 7.40 (s,
5 H, COPh). Ϫ 13C NMR (minor isomer in italics): δ ϭ 9.8
(CH2CH3), 26.3, 26.5 (CH2CH3), 33.6, 34.5, 34.5 (C-8, C-9), 43.0,
44.7 (C-6), 59.4 (C-3), 67.6, 67.6 (C-2), 72.9, 73.1 (C-7), 103.7,
103.9 (C-5), 126.2, 127.0, 128.5, 129.7, 137.8 (COPh), 168.4
(COPh).
CH(CH3)2], 1.66Ϫ2.42 [m, 5 H, 6-H, 8Ј-H, 9Ј-H, 8-H or 9-H, OH],
2.71 [m, 2 H, 6Ј-H, 8-H or 9-H], 3.93 [s, 3 H, 2(2Ј)-H, 3-H], 4.44 (
br. s, 1 H, 7-H), 7.40 (s, 5 H, COPh). Ϫ 13C NMR: δ ϭ 16.4, 19.5
[CH(CH3)2], 30.1 [CH(CH3)2], 34.2, 34.4 (C-8, C-9), 42.9 (C-6),
62.8 (C-3), 65.0 (C-2), 73.2 (C-7), 104.4 (C-5), 126.4, 128.5, 129.7,
137.9 (COPh), 168.3 (COPh).
4i MIN: M.p. 174.0Ϫ174.5 °C (from petroleum ether/EtOH). Ϫ
1
[α]2D0 ϭ ϩ79.6 (c ϭ 1.1, CH2Cl2). Ϫ H NMR: δ ϭ 0.60 & 0.77 [2
ϫ d, 2 ϫ 3 H, CH(CH3)2, J ϭ 6.9 Hz], 1.50 [br. m, 1 H, CH(CH3)2],
1.71Ϫ2.58 [4 ϫ m, 6 H, 6-H, 8(8Ј)-H, 9(9Ј)-H, OH], 2.98 (dd, 1 H,
6Ј-H, J6Ј,6 ϭ 13.9 Hz, J6Ј,7 ϭ 5.9 Hz), 3.91 [m, 3 H, 2(2Ј)-H, 3-H],
4.49 (br. s, 1 H, 7-H), 7.39 (s, 5 H, COPh). Ϫ 13C NMR: δ ϭ 16.3,
19.4 [CH(CH3)2], 29.8 [CH(CH3)2], 33.8, 34.6 (C-8, C-9), 44.5 (C-
6), 62.8 (C-3), 64.9 (C-2), 72.9 (C-7), 104.0 (C-5), 126.4, 128.5,
129.8, 137.9 (COPh), 168.8 (COPh).
The main isomer (4e MAJ) of this mixture was isolated by column
chromatography as a crystalline solid. Ϫ M.p. 127Ϫ129 °C (from
petroleum ether/ethyl acetate). Ϫ [α]2D0 ϭ ϩ67.9 (c ϭ 1.2, CH2Cl2).
(3R)-Pr Hydroxylated Product 4j: Subjecting substrate 3j (Bioengi-
neering AG model 1523: after 31 h, 0.930 g was added for induc-
tion and, after an additional 17 h, 3.967 g was added) to Beauveria
bassiana as described in the general procedure (total fermentation
time 169 h) furnished a dark brown syrup (4.949 g) after extraction.
Column chromatography afforded the following products: 4j
(2.753 g, 54%) and 13 (0.075 g, 1.5%). In addition, two other prod-
ucts (11 mg and 20 mg) were also isolated and could not be identi-
fied owing to the presence of impurities and to the small amounts
involved. Repeated chromatography of 4j and subsequent recrys-
tallisation furnished the individual isomers.
1
Ϫ H NMR: δ ϭ 0.63 (t, 3 H, CH2CH3, J ϭ 7.4 Hz), 1.31 (br. m,
2 H, CH2CH3), 1.72Ϫ2.61 [3 ϫ m, 6 H, 6-H, 8(8Ј)-H, 9(9Ј)-H, OH],
2.96 (dd, 1 H, 6Ј-H, J6,6Ј ϭ 14.0 Hz, J6Ј,7 ϭ 6.0 Hz), 3.74Ϫ4.07 [m,
3 H, 2(2Ј)-H, 3-H], 4.49 (br. m, 1 H, 7-H), 7.42 (s, 5 H, COPh). Ϫ
13C NMR: δ ϭ 9.9 (CH2CH3), 26.3 (CH2CH3), 33.6, 34.5 (C-8, C-
9), 44.7 (C-6), 59.4 (C-3), 67.6 (C-2), 72.9 (C-7), 103.7 (C-5), 126.2,
128.5, 129.7, 137.5 (COPh), 168.6 (COPh).
(3R)-iPr Hydroxylated Product 4h: Subjecting substrate 3h (Biostat
M: after 32 h 0.144 g were added for induction and, after an addi-
tional 16 h, 0.736 g were added) to Beauveria bassiana as described
in the general procedure (total fermentation time 93 h) furnished
the title compound as a pale yellow solid (0.354 g, 42%). The indi-
vidual isomers were separated by chromatography combined with
recrystallisation.
1
4j MAJ: Syrup. Ϫ [α]2D0 ϭ Ϫ96.4 (c ϭ 1.7, CH2Cl2). Ϫ H NMR:
δ ϭ 0.61 (t, 3 H, CH2CH2CH3, J ϭ 7.0 Hz), 0.82Ϫ1.55 (m, 4 H,
CH2CH2CH3), 1.79 (br. d, 1 H, 6-H, J6,6Ј ϭ 13.9 Hz), 1.85Ϫ2.11,
2.30 & 2.66 [3 ϫ m, 3 H, 2 H & 1 H, 6-H, 8(8Ј)-H, 9(9Ј)-H, OH],
2.76 (dd, 1 H, 6Ј-H, J6Ј,7 ϭ 5.7 Hz), 3.89 [m, 3 H, 2(2Ј)-H, 3-H],
4.45 (br. s, 1 H, 7-H), 7.39 (s, 5 H, COPh). Ϫ 13C NMR: δ ϭ 13.4
4h MAJ: M.p. 127.5Ϫ128.5 °C (from petroleum ether/CH2Cl2). Ϫ
1
[α]2D0 ϭ Ϫ98.6 (c ϭ 1.2, CH2Cl2). Ϫ H NMR: δ ϭ 0.60 & 0.78 [2 (CH2CH2CH3), 18.8 (CH2CH2CH3), 34.5, 35.6 (C-8, C-9,
ϫ d, 2 ϫ 3 H, CH(CH3)2, J ϭ 6.9 Hz], 1.52 [br. m, 1 H, CH(CH3)2], CH2CH2CH3), 43.0 (C-6), 58.0 (C-3), 68.0 (C-2), 73.0 (C-7), 103.9
1.72Ϫ2.44 [3 ϫ m, 5 H, 6-H, 8Ј-H, 9Ј-H, 8-H or 9-H, OH], 2.70 (C-5), 126.3, 128.5, 129.6, 137.7 (COPh), 168.2 (COPh).
[m, 2 H, 6Ј-H, 8-H or 9-H], 3.91 [s, 3 H, 2(2Ј)-H, 3-H], 4.44 ( br.
4j MIN: White crystalline solid. Ϫ M.p. 116.5Ϫ117.0 °C (from pet-
s, 1 H, 7-H), 7.40 (s, 5 H, COPh). Ϫ 13C NMR: δ ϭ 16.4, 19.5
roleum ether/diethyl ether). Ϫ [α]2D0 ϭ Ϫ82.3 (c ϭ 0.9, CH2Cl2). Ϫ
[CH(CH3)2], 30.1 [CH(CH3)2], 34.2, 34.4 (C-8, C-9), 43.0 (C-6),
62.8 (C-3), 65.0 (C-2), 73.1 (C-7), 104.3 (C-5), 126.4, 128.5, 129.7,
137.9 (COPh), 168.5 (COPh).
1H NMR: δ ϭ 0.60 (t, 3 H, CH2CH2CH3, J ϭ 7.0 Hz), 0.81Ϫ1.51,
(m, 4 H, CH2CH2CH3), 1.63Ϫ2.13, 2.27 & 2.51 [3 ϫ m, 4 H, 1 H,
1 H, 6-H, 8(8Ј)-H, 9(9Ј)-H, OH], 2.96 (dd, 1 H, 6Ј-H, J6,6Ј
ϭ
4h MIN: M.p. 169.5Ϫ170.5 °C (from petroleum ether/EtOH). Ϫ
14.1 Hz, J6Ј,7 ϭ 6.0 Hz), 3.78 (dd, 1 H, 2 or 3-H, J ϭ 1.6 Hz, J ϭ
8.2 Hz), 3.95 (m, 2 H, 2Ј-H, 3 or 2-H), 4.50 (m, 1 H, 7-H), 7.42 (s,
1
[α]2D0 ϭ Ϫ82.6 (c ϭ 0.8, CH2Cl2). Ϫ H NMR: δ ϭ 0.61 & 0.77 [2
ϫ d, 2 ϫ 3 H, CH(CH3)2, J ϭ 6.9 Hz], 1.51 [br. m, 1 H, CH(CH3)2], 5 H, COPh). Ϫ 13C NMR: δ ϭ 13.4 (CH2CH2CH3), 18.8
1.71Ϫ2.58 [4 ϫ m, 6 H, 6-H, 8(8Ј)-H, 9(9Ј)-H, OH], 2.98 (dd, 1 H, (CH2CH2CH3), 33.5, 34.5, 35.7 (C-8, C-9, CH2CH2CH3), 44.7 (C-
6Ј-H, J6,6Ј ϭ 13.9 Hz, J6Ј,7 ϭ 5.9 Hz), 3.91 [m, 3 H, 2(2Ј)-H, 3-H],
6), 58.0 (C-3), 68.0 (C-2), 72.9 (C-7), 103.6 (C-5), 126.3, 128.5,
4.50 (br. s, 1 H, 7-H), 7.40 (s, 5 H, COPh). Ϫ 13C NMR: δ ϭ16.3, 129.6, 137.8 (COPh), 168.3 (COPh).
19.4 [CH(CH3)2], 29.9 [CH(CH3)2], 33.8, 34.6 (C-8, C-9), 44.5 (C-
(3S)-Pr Hydroxylated Products 4k: Subjecting substrate 3k (Bioen-
6), 62.8 (C-3), 64.9 (C-2), 72.9 (C-7), 104.0 (C-5), 126.4, 128.5,
gineering AG model 1523: after 24.0 h, 1.259 g was added for in-
duction and, after an additional 17.0 h, 4.400 g was added) to Be-
auveria bassiana as described in the general procedure (total fer-
mentation time 88.0 h) furnished a dark brown syrup (9.583 g) after
extraction. TLC indicated that a complex mixture had been ob-
tained and this was not further investigated.
129.8, 137.9 (COPh), 168.8 (COPh).
(3S)-iPr Hydroxylated Product 4i: Subjecting substrate 3i (Bioengi-
neering AG model 1523: after 26 h, 1.141 g was added for induc-
tion and, after an additional 14 h, 5.626 g was added) to Beauveria
bassiana as described in the general procedure (total fermentation
time 96 h) furnished the title compound as a pale yellow solid and
as a mixture of isomers (2.678 g, 38%). The individual isomers
could be separated by chromatography combined with recrystallis-
ation.
(2S)-Me Hydroxylated Product 9a: Treating substrate 8a (Biostat
M: first substrate addition, 0.109 g after 15 h; second substrate ad-
dition, 0.170 g after 10 h) with Beauveria bassiana as given above
in the general procedure, furnished (total fermentation time 49 h)
a mixture of isomers as a pale yellow oil (0.160 g, 71% yield, de ϭ
83% as determined by NMR and HPLC). Ϫ [α]2D0 ϭ ϩ87.8 (c ϭ
1.6, CH2Cl2). Ϫ 1H NMR (minor isomer given in italics): δ ϭ 1.26
4i MAJ: M.p. 127.0Ϫ128.0 °C (from petroleum ether/diethyl ether).
1
Ϫ [α]2D0 ϭ ϩ93.5 (c ϭ 0.9, CH2Cl2). Ϫ H NMR: δ ϭ 0.61 & 0.80
[2 ϫ d, 2 ϫ 3 H, CH(CH3)2, J ϭ 7.0 Hz], 1.52 [br. m, 1 H,
3844
Eur. J. Org. Chem. 2000, 3835Ϫ3847

Chiral Auxiliaries as Docking/Protecting Groups in Biohydroxylation
(d, 3 H, CH3, J ϭ 6.0 Hz), 1.69Ϫ2.37 & 2.53Ϫ2.69 [2 ϫ m, 4 H & was filtered and the filtrate concentrated down under reduced pres-
FULL PAPER
1 H, 6-H, 8(8Ј)-H, 9(9Ј)-H], 2.76 & 2.93 (2 ϫ dd, 1 H, ratio: 11:1,
6Ј-H, J ϭ 13.9 Hz, J ϭ 5.9 Hz), 3.19 (dd, 1 H, 3-H, J2,3
sure to give a syrup. Column chromatography (cyclohexane/ethyl
acetate, 15:1) was then used to isolate 15 as colourless crystals
ϭ
6,6Ј
6Ј,7
J3,3Ј ϭ 9.6 Hz), 3.45 (dd, 1 H, 3Ј-H, J3Ј,2 ϭ 5.3 Hz), 4.06 (m, 1 H, (0.150 g, 68% yield). Ϫ M.p. 127.0Ϫ128.0 °C (from cyclohexane/
1
2-H), 4.46 (m, 1 H, 7-H), 5.15 (br. s, 1 H, OH), 7.42 (m, 5 H, diethyl ether). Ϫ [α]2D0 ϭ ϩ79.1 (c ϭ 1.45, CH2Cl2). Ϫ H NMR:
COPh). Ϫ 13C NMR: δ ϭ 17.4 (CH3), 34.2 (C-8, C-9), 43.3 (C-6),
δ ϭ 0.36 [d, 3 H, 12-H, J11,12 ϭ 5.3 Hz], 0.91, 1.01, 1.09 [3 ϫ s, 3
55.3 (C-3), 71.4 (C-2), 73.3 (C-7), 104.0 (C-5), 126.6, 128.4, 130.1, ϫ 3 H, 22-H, C(CH3)2], 1.15Ϫ2.07 [br. m, 13 H, 6-H, 7(7Ј)-H,
137.3 (COPh), 167.7 (COPh).
8(8Ј)-H, 9-H, 10(10Ј)-H, 11-H, 20(20Ј)-H, 21(21Ј)-H], 2.39 & 2.64
(2 ϫ m, 2 H, 6Ј-H, 9Ј-H), 3.62Ϫ3.97 [m, 5 H, 2(2Ј)-H, 3-H, 13(13Ј)-
H], 7.44 (m, 5 H, COPh). Ϫ 13C NMR: δ ϭ 9.7 (C-22), 15.2 (C-
12), 16.8, 18.1 [C(CH3)2], 24.6, 24.8 (C-7, C-8), 28.9, 29.9 (C-20,
C-21), 30.7 (C-11), 34.8, 36.6 (C-6, C-9), 37.2 (C-10), 54.1, 54.8 [C-
19, C(CH3)2], 56.6 (C-3), 67.3 (C-2), 70.0 (C-13), 91.1 (C-16), 104.0
(C-5), 126.4, 128.4, 129.9, 137.8 (COPh), 167.4, 171.2, 171.3
(COPh, C-15, C-18).
(2R)-Me Hydroxylated Product 9b: Treating substrate 8b (Biostat
M: first substrate addition, 0.110 g after 17 h; second substrate ad-
dition, 0.550 g after 14 h) with Beauveria bassiana as given above
in the general procedure, furnished (total fermentation time 70 h)
unchanged starting material (0.280 g) and a mixture of isomers as
a pale yellow syrup (0.040 g, 20% yield, de ϭ 53% as determined
by NMR). Ϫ 1H NMR (minor isomer in italics): δ ϭ 1.23 (d, 3 H,
CH3, J ϭ 5.9 Hz), 1.68Ϫ2.56 [m, 5 H, 6-H, 8(8Ј)-H, 9(9Ј)-H],
Derivatised Products 5a؊5e, 5h؊5j as well as 10a and 10b
2.73 & 2.90 (2 ϫ dd, 1 H, ratio: 1:3.3, 6Ј-H, J
ϭ 14.0 Hz,
6,6Ј
Derivatised Product 5a: Benzylation of alcohol 4a (0.140 g) under
standard conditions[28] afforded the title compound 5a as a pale
yellow syrup after column chromatography (0.140 g, 73% yield). Ϫ
[α]2D0 ϭ Ϫ10.5 (c ϭ 3.5, CH2Cl2). Ϫ 1H NMR: δ ϭ 1.95 &
2.21Ϫ2.63 [2 ϫ m, 5 H, 6-H, 8(8Ј)-H, 9(9Ј)-H], 2.87 (dd, 1 H, 6Ј-
H, J ϭ 7.1 and 14.2 Hz), 3.53 & 3.93 [2 ϫ m, 2 ϫ 2 H, 2(2Ј)-H,
3(3Ј)-H], 4.37 (m, 1 H, 7-H), 4.55 (s, 2 H, CH2Ph), 7.20Ϫ7.56 (m,
10 H, COPh, CH2Ph). Ϫ 13C NMR: δ ϭ 31.7, 34.0, (C-8, C-9),
41.9 (C-6), 48.6 (C-3), 64.2 (C-2), 71.0 (CH2Ph), 79.2 (C-7), 102.9
(C-5), 126.7, 127.5, 127.8, 128.4, 128.5, 130.0, 137.5, 138.9 (CH2Ph,
COPh,), 167.7 (COPh).
J6Ј,7 ϭ 6.3 Hz), 3.17 (dd, 1 H, 3-H, J2,3 ϭ J3,3Ј ϭ 9.7 Hz), 3.43
(dd, 1 H, 3Ј-H, J3Ј,2 ϭ 5.3 Hz), 4.03 (m, 1 H, 2-H), 4.46 (m, 1 H,
7-H), 5.04 (br. s, 1 H, OH), 7.39 (m, 5 H, COPh). Ϫ 13C
NMR(minor isomer in italics): δ ϭ 17.4 (CH3), 33.8, 34.6 (C-8, C-
9), 43.2, 44.5 (C-6), 55.3 (C-3), 71.4 (C-2), 72.2, 73.3 (C-7), 103.5,
104.0 (C-5), 126.6, 128.4, 130.2, 137.2 (COPh), 167.8 (COPh).
Hydroxylated Product 13: White crystalline solid.
Ϫ M.p.
115.5Ϫ116.5 °C (from petroleum ether/diethyl ether). Ϫ [α]2D0
ϭ
Ϫ91.5 (c ϭ 0.8, CH2Cl2). Ϫ 1H NMR: δ ϭ 0.97 [d, 3 H,
CH2CH(OH)CH3, J ϭ 6.2 Hz], 1.13Ϫ2.03 [4 ϫ br. m, 9 H,
CH2CH(OH)CH3, 6-H, 7(7Ј)-H, 8(8Ј)-H, 9-H], 2.37 & 2.68 [2 ϫ
br. m, 2 ϫ 1 H, 6Ј-H, 9Ј-H], 3.65, 3.92 & 4.17 [3 ϫ m, 4 H,
CH2CH(OH)CH3, 2(2Ј)-H, 3-H], 7.40 (s, 5 H, COPh). Ϫ 13C NMR
(3R)-Me Derivatised Product 5b: Benzylation of alcohol 4b[28]
(0.070 g) under standard conditions and column chromatography
furnished the title compound 5b as a white solid (0.080 g, 85%
yield, 89% de NMR). Chromatography and recrystallisation
(CH2Cl2/petroleum ether) furnished 5b MAJ as fine colourless
needles which were suitable for X-ray crystallographic analysis. Ϫ
M.p. 85.0Ϫ86.0 °C . Ϫ [α]2D0 ϭ Ϫ75.2 (c ϭ 3.0, CH2Cl2). Ϫ 1H
NMR: (isomeric mixture, minor isomer in italics): δ ϭ 0.95 (d, 3
H, CH3, J ϭ 6.6 Hz), 1.83Ϫ2.67 [m, 5 H, 6-H, 8(8Ј)-H, 9(9Ј)-H],
(minor isomer in italics):
δ ϭ 23.4, 23.8, 24.5, 24.6, 24.9
[CH2CH(OH)CH3, C-7, C-8], 35.0, 35.0, 36.3 (C-6, C-9), 42.6
[CH2CH(OH)CH3], 55.8 (C-3), 64.6, 68.0, 68.7 [CH2CH(OH)CH3,
C-2], 104.5, 105.1 (C-5), 126.2, 126.6, 128.5, 129.5, 129.6, 138.0
(COPh), 168.0 (COPh).
Hydroxylated Product 14: Substrate 3m (Biostat M: 0.760 g total;
after 24 h of growth, 0.150 g was added for induction and, after
an additional 12.5 h, 0.610 g was added to the fermentation) was
subjected to a culture of Beauveria bassiana (total fermentation
time 99 h) to afford a pale yellow oil, 14, after work up and chro-
matography (0.085 g, 13% yield). Ϫ [α]2D0 ϭ ϩ81.4 (c ϭ 1.1,
CH2Cl2). Ϫ 1H NMR: δ ϭ 0.03 [d, 3 H, CH2CH(CH3)CH2OH,
J ϭ 5.3 Hz], 0.62 [m, 1 H, CH2CH(CH3)CH2OH], 0.98 [m, 1 H,
CH2CH(CH3)CH2OH], 1.30Ϫ1.67 [br. m, 7 H, 6-H, 7(7Ј)-H, 8(8Ј)-
H, 9-H, CH2CH(CH3)CH2OH], 2.11Ϫ2.32 ( 2 ϫ m, 2 ϫ 1 H, 6Ј-
H, 9Ј-H), 2.87 [d, 2 H, CH2CH(CH3)CH2OH, J ϭ 6.2 Hz], 3.42
(br. d, 1 H, 2-H, J2,2Ј ϭ 7.2 Hz), 3.63 (m, 2 H, 2Ј-H, 3-H), 7.09 (s,
5 H, COPh). Ϫ 13C NMR: δ ϭ 15.3 [CH2CH(CH3)CH2OH], 24.8,
24.9 (C-7, C-8), 33.3 [CH2CH(CH3)CH2OH], 35.0, 36.7 (C-6, C-
9), 37.7 [CH2CH(CH3)CH2OH], 57.1 (C-3), 67.8, 68.0 [C-2,
CH2CH(CH3)CH2OH], 104.9 (C-5), 126.5, 128.6, 129.6, 138.3
(COPh), 168.3 (COPh).
2.77 & 2.96 (2 ϫ dd, 1 H, ratio: 18:1, 6Ј-H, J6Ј,7 ϭ 6.7 Hz, J6Ј,6
ϭ
14.4 Hz,), 3.64 (m, 1 H, 2-H), 4.03 (m, 2 H, 2Ј-H, 3-H), 4.33 (br.
s, 1 H, 7-H), 4.54 (s, 2 H, CH2Ph), 7.34 (m, 10 H, COPh, CH2Ph).
Ϫ
13C NMR: δ ϭ 20.1 (CH3), 31.7, 35.3 (C-8, C-9), 41.9 (C-6),
54.0 (C-3), 70.4, 70.9 (C-2, CH2Ph), 79.2 (C-7), 103.3 (C-5), 126.3,
127.5, 127.7, 128.4, 128.4, 129.7, 137.9, 138.9 (CH2Ph, COPh,),
168.2 (COPh).
(3S)-Me Derivatised Product 5c: Benzylation of alcohol 4c (0.030 g)
under standard conditions[28] and column chromatography fur-
nished the title compound 5c as a clear syrup (0.030 g, 74% yield,
33% de NMR). Ϫ 1H NMR (minor isomer in italics): δ ϭ 0.96 (m,
3 H, CH3), 1.79Ϫ2.66 [m, 5 H, 6-H, 8(8Ј)-H, 9(9Ј)-H], 2.78 & 2.96
(2 ϫ dd, 1 H, ratio: 1:2, 6Ј-H, J6Ј,7 ϭ 7.3 Hz, J6,6Ј ϭ 14.4 Hz), 3.56
(m, 1 H, 2-H), 4.03 (m, 2 H, 2Ј-H, 3-H), 4.33 (m, 1 H, 7-H), 4.54
(m, 1 H, CH2Ph), 7.34 (m, 10 H, COPh, CH2Ph). Ϫ 13C NMR
(minor isomer in italics): δ ϭ 20.1 (CH3), 31.5, 31.7, 33.9, 34.1 (C-
8, C-9), 41.9, 43.0 (C-6), 54.0, 54.1 (C-3), 70.3, 70.4, 70.9, 71.1 (C-
2, CH2Ph), 79.1, 79.2 (C-7), 103.0 (C-5), 126.2, 126.2, 127.4, 127.8,
128.3, 128.5, 129.6, 129.7, 138.0, 138.8 (COPh, CH2Ph), 168.3
(COPh).
Camphanoate Derivative 15:[31] Compound 14 (0.140 g, 0.46 mmol)
was dissolved in dry CH2Cl2 (10 mL) and pyridine (0.040 g,
0.51 mmol) as well as catalytic amounts of 4-dimethylaminopyrid-
ine added. After the mixture had been stirred at room temperature
for 15 min, (Ϫ)-camphanic chloride (0.110 g, 0.51 mmol) was ad- (3R)-Et Derivatised Product 5d: Benzylation of alcohol 4d (0.120 g,
ded and stirring continued for 48 h. The reaction mixture was then
isomeric mixture) under standard conditions[28] and column chro-
matography afforded the title derivative as a pale yellow syrup
washed with aqueous HCl (5%, 1 ϫ 30 mL), saturated aqueous
NaHCO3 (1 ϫ 30 mL) as well as saturated aqueous NaCl (1 ϫ (0.120 g, 75% yield, de ϭ 75% as determined by NMR). Ϫ 1H
30 mL) and the organic phase was dried with Na2SO4. The solution
NMR (minor isomer in italics): δ ϭ 0.64 (t, 3 H, CH2CH3, J ϭ
Eur. J. Org. Chem. 2000, 3835Ϫ3847
3845

A. de Raadt, H. Griengl et al.
7.4 Hz), 1.20Ϫ1.43 (br. m, 2 H, CH2CH3), 1.84Ϫ2.09 & 2.20Ϫ2.65 CH2CH2CH3), 1.55Ϫ2.58 [m, 5 H, 6-H, 8(8Ј)-H, 9(9Ј)-H], 2.74 &
FULL PAPER
[2 ϫ m, 5 H, 6-H, 8(8Ј)-H, 9(9Ј)-H], 2.76 & 2.98 (2 ϫ dd, 1 H,
ratio: 7:1, 6Ј-H, J6Ј,7 ϭ 6.9 Hz, J6,6Ј ϭ 14.1 Hz), 3.79 (dd, 1 H, 2-
2.97 (2 ϫ dd, 1 H, ratio: 1.3:1, 6Ј-H, J6Ј,7 ϭ 6.9 Hz, J6,6Ј
ϭ
14.2 Hz), 3.80 [m, 3 H, 2(2Ј)-H, 3-H], 4.23 (br. s, 1 H, 7-H), 4.47
H, J2,2Ј ϭ 8.6 Hz, J2,3 ϭ 2.6 Hz), 3.84 (m, 1 H, 3-H), 3.99 (dd, 1 H, (m, 2 H, CH2Ph), 7.12Ϫ7.45 (m, 10 H, CH2Ph, COPh). Ϫ 13C
2Ј-H, J2Ј,3 ϭ 5.5 Hz), 4.33 (br. s, 1 H, 7-H), 4.55 (m, 2 H, CH2Ph), NMR (major isomer only):
δ ϭ 13.4 (CH2CH2CH3), 18.7
7.22Ϫ7.47 (m, 10 H, CH2Ph, COPh). Ϫ 13C NMR (minor isomer (CH2CH2CH3), 31.6, 35.1, 35.7 (C-8, C-9, CH2CH2CH3), 41.8 (C-
in italics): δ ϭ 9.9 (CH2CH3), 26.5, 31.8, 34.0, 35.3, (C-8, C-9, 6), 57.9 (C-3), 68.1 (C-2), 70.9 (CH2Ph), 79.3 (C-7), 103.2 (C-5),
CH2CH3), 42.1, 43.0 (C-6), 59.6, 59.7 (C-3), 67.8, 67.9 (C-2), 71.0,
126.3, 127.4, 127.7, 128.4, 128.5, 129.6, 137.9, 138.9 (CH2Ph,
71.3 (CH2Ph), 79.3, 79.6 (C-7), 103.5 (C-5), 126.5, 127.6, 127.8, COPh), 168.4 (COPh).
128.5, 128.7, 129.7, 129.8, 138.2, 139.2 (CH2Ph, COPh), 168.4
(2S)-Me Derivatised Product 10a: Subjecting alcohol 9a (0.090 g)
(COPh).
to the standard benzylation procedure[28] furnished the title com-
pound as a pale yellow oil (0.050 g, 41% yield, de ϭ 89% as deter-
mined by NMR). Ϫ [α]2D0 ϭ ϩ62.7 (c ϭ 2.7, CH2Cl2). Ϫ 1H NMR:
(minor isomer in italics): δ ϭ 1.31 (d, 3 H, CH3, J ϭ 5.9 Hz),
1.84Ϫ2.39 & 2.52Ϫ2.73 [2 ϫ m, 4 H & 1 H, 6-H, 8(8Ј)-H, 9(9Ј)-
H], 2.81 & 2.96 ( 2 ϫ dd, 1 H, ratio: 18:1, 6Ј-H, J6Ј,7 ϭ 7.0 Hz,
J6,6Ј ϭ 13.8 Hz), 3.22 (dd, 1 H, 3-H, J2,3 ϭ J3,3Ј ϭ 9.6 Hz), 3.47
(dd, 1 H, 3Ј-H, J2,3Ј ϭ 9.6 Hz), 4.06 (m, 1 H, 2-H), 4.38 (m, 1 H,
7-H), 4.55 (s, 2 H, CH2Ph), 7.21Ϫ7.57 (m, 10 H, CH2Ph, COPh).
(3S)-Et Derivatised Product 5e: The title compound was furnished
from alcohol 4e (0.179 g, isomeric mixture) after benzylation under
standard conditions[28] as a pale yellow oil (0.169 g, 71% yield, de ϭ
45% as determined by NMR). Ϫ 1H NMR (minor isomer in it-
alics): δ ϭ 0.64 (t, 3 H, CH2CH3, J ϭ 7.4 Hz), 1.13Ϫ1.50 (br. m,
2 H, CH2CH3), 1.66Ϫ2.66 [m, 5 H, 6-H, 8(8Ј)-H, 9(9Ј)-H], 2.74 &
2.97 (2 ϫ dd, 1 H, ratio: 1:2.8, 6Ј-H, J6Ј,7 ϭ 7.2 Hz, J6,6Ј
ϭ
14.1 Hz), 3.54Ϫ4.05 [m, 3 H, 2(2Ј)-H, 3-H], 4.35 (br. m, 1 H, 7-H),
4.56 (m, 2 H, CH2Ph), 7.21Ϫ7.50 (m, 10 H, CH2Ph, COPh). Ϫ 13C
NMR (minor isomer in italics): δ ϭ 9.8, 9.9 (CH2CH3), 26.4, 31.6,
31.6, 33.8, 35.1 (C-8, C-9, CH2CH3), 41.9, 42.8 (C-6), 59.4, 59.5
(C-3), 67.6, 67.7 (C-2), 70.9, 71.1 (CH2Ph), 79.1, 79.3 (C-7), 102.9
(C-5), 126.3, 127.4, 127.8, 128.4, 128.5, 129.6, 138.0, 138.9 (CH2Ph,
COPh), 168.3 (COPh).
Ϫ
13C NMR: δ ϭ 17.6 (CH3), 31.5, 34.9 (C-8, C-9), 42.3 (C-6),
55.5 (C-3), 70.9, 71.1 (C-2, CH2Ph), 79.8 (C-7), 103.2 (C-5), 126.8,
127.0, 127.5, 127.8, 128.4, 128.4, 128.8, 130.1, 137.6, 139.0 (CH2Ph,
COPh), 167.6 (COPh).
(2R)-Me Derivatised Product 10b: Subjecting alcohol 9b (0.070 g)
to the standard benzylation procedure[28] furnished the title com-
pound as a pale yellow oil (0.050 g, 53% yield, de ϭ 73% as deter-
(3R)-iPr Derivatised Product 5h: The title compound was obtained
from 4h (0.153 g, isomeric mixture) after benzylation under stand-
ard conditions[28] as a pale yellow oil (0.155 g, 77% yield, de ϭ 80%
1
mined by NMR). Ϫ H NMR: (minor isomer in italics): δ ϭ 1.31
(d, 3 H, CH3, J ϭ 6.0 Hz), 1.77Ϫ2.70 [2 ϫ m, 5 H, 6-H, 8(8Ј)-H,
9(9Ј)-H], 2.81 & 2.94 (2 ϫ dd, 1 H, ratio: 1:6.3, 6Ј-H, J6Ј,7 ϭ 7.7 Hz,
J6,6Ј ϭ 14.1 Hz), 3.22 (dd, 1 H, 3-H, J2,3 ϭ J3,3Ј ϭ 9.5 Hz), 3.48
(dd, 1 H, 3Ј-H, J2,3Ј ϭ 5.2 Hz), 4.08 (m, 1 H, 2-H), 4.40 (m, 1 H,
7-H), 4.54 (s, 2 H, CH2Ph), 7.21Ϫ7.57 (m, 10 H, CH2Ph, COPh).
1
as indicated by NMR). Ϫ H NMR (minor isomer in italics): δ ϭ
0.62 & 0.79 [2 ϫ d, 2 ϫ 3 H, CH(CH3)2, J ϭ 6.9 Hz], 1.51 [br. m,
1 H, CH(CH3)2], 1.82Ϫ2.64 [m, 5 H, 6-H, 8(8Ј)-H, 9(9Ј)-H], 2.71 &
3.03 (2 ϫ dd, 1 H, 6Ј-H, ratio: 9:1, J6Ј,7 ϭ 6.5 Hz, J6,6Ј ϭ 13.9 Hz),
3.92 [m, 3 H, 2(2Ј)-H, 3-H], 4.31 (br. m, 1 H, 7-H), 4.54 (s, 2 H,
CH2Ph), 7.21Ϫ7.60 (m, 10 H, COPh, CH2Ph). Ϫ 13C NMR (minor
isomer in italics): δ ϭ 16.3, 16.4, 19.3, 19.4 [CH(CH3)2], 29.7, 29.8,
31.5, 31.7, 34.8 [C-8, C-9, CH(CH3)2], 42.1, 42.7 (C-6), 62.7, 62.8
(C-3), 65.0 (C-2), 70.9, 71.1 (CH2Ph), 79.0, 79.4 (C-7), 103.7 (C-5),
126.4, 127.0, 127.4, 127.6, 127.7, 127.8, 127.9, 128.1, 128.4, 128.5,
128.6, 138.0, 139.0 (minor isomer not assigned, COPh, CH2Ph),
168.6 (COPh).
Ϫ
13C NMR: δ ϭ 17.5 (CH3), 32.1, 33.9 (C-8, C-9), 42.8 (C-6),
55.3 (C-3), 71.3, 71.3 (C-2, CH2Ph), 78.8 (C-7), 102.7 (C-5), 126.7,
126.7, 127.4, 127.8, 128.4, 128.4, 130.1, 137.5, 139.0 (CH2Ph,
COPh), 167.5 (COPh).
Final Product Ketone 6
(R/S)-Ketone 6: The title compound was prepared from derivatised
products 5aϪe, 5hϪj, 10a and 10b as described in the general pro-
cedure. Please refer to Table 1 for isolated yields. 1H NMR spectro-
scopic data were in agreement with reported values.[37] The respect-
ive ee values were determined by chiral GC and the configuration
established by comparison with reference compound (3S)-ketone 6
(results are listed in Table 1).
(3S)-iPr Derivatised Product 5i: The title compound was obtained
from 4i (0.190 g, isomeric mixture) after benzylation under stand-
ard conditions[28] as a pale yellow oil (0.199 g, 80% yield, de ϭ 33%
1
as indicated by NMR). Ϫ H NMR (minor isomer in italics): δ ϭ
0.62 & 0.79 [2 ϫ d, 2 ϫ 3 H, CH(CH3)2, J ϭ 6.9 Hz], 1.51 [br. m,
1 H, CH(CH3)2], 1.72Ϫ2.64 [m, 5 H, 6-H, 8(8Ј)-H, 9(9Ј)-H], 2.71 &
3.03 (2 ϫ dd, 1 H, 6Ј-H, ratio: 2:1, J6Ј,7 ϭ 6.6 Hz, J6,6Ј ϭ 13.9 Hz),
3.92 [m, 3 H, 2(2Ј)-H, 3-H], 4.32 (br. m, 1 H, 7-H), 4.55 (s, 2 H,
CH2Ph), 7.21Ϫ7.65 (m, 10 H, COPh, CH2Ph). Ϫ 13C NMR (minor
isomer in italics): δ ϭ 16.4, 16.6, 19.6, 19.7 [CH(CH3)2], 30.0, 30.3,
31.7, 31.8, 34.1, 35.0 [C-8, C-9, CH(CH3)2], 42.3, 42.8 (C-6), 62.9,
63.0 (C-3), 65.1, 65.2 (C-2), 71.1, 71.3 (CH2Ph), 79.2, 79.6 (C-7),
103.3, 103.9 (C-5), 126.6, 126.9, 127.1, 127.2, 127.5, 127.5, 127.6,
127.7, 127.8, 127.8, 127.9, 127.9, 128.1, 128.2, 128.5, 128.6, 128.7,
138.2, 139.1 (minor isomer not assigned, COPh, CH2Ph), 168.7,
168.8 (COPh).
(3S)-Ketone 6: This chiral GC reference compound was prepared
from (1S,4R)-4-benzyloxy-2-cyclopenten-1-ol as described in the
supporting information.[29]
Acknowledgments
We wish to thank Professor K. Mereiter for conducting an XRD
structural measurement, M. Bauer for performing the elemental
analyses, Ing. C. Illaszewicz for the measurement of NMR spectra
as well as M. Kaube, J. Kern, and W. Schmidt for their helpful
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