R. Ramu et al. / Tetrahedron Letters 52 (2011) 2950–2953
2951
Negative controls of E. coli without the AlkBGT expression dis-
played no evidence of bioconversion of these substrates. Aside
from 1-octanol, there was no detectable octyl aldehyde or acid ob-
served during the operations of the E. coli–AlkB whole-cell system
using n-octane as the substrate. These results indicate that further
oxidation exerted by an alcohol dehydrogenase could be neglected
in this recombinant catalysis system.2,3
As an illustration of the utility of our E. coli–AlkB whole-cell sys-
tem as a synthetic tool, the enzymatic chemical conversion of 1,1-,
2,2-, 3,3- and 4,4-difluorooctanes to their corresponding terminal
alcohols was accomplished as the major products in excellent yields
8.34 min, respectively, in the GC. These minor products corre-
sponded to the proximal terminal alcohols. To identify these prod-
ucts, we synthesized 2,2-, 3,3- and 4,4-difluorooctan-1-ols to serve
as authentic standards (Scheme 4).
In order to accomplish the highly efficient synthesis of 2,2-, 3,3-
and 4,4-difluorooctan-1-ols (33, 17 and 18), we have developed a
synthetic strategy that uses 1,2-, 1,3- and 1,4-octanediols (21ꢀ23)
as the starting materials. The synthetic procedure was straight-for-
ward, involving only four synthetic steps in good yields (Scheme 4).
1,2-Octanediol 21 is commercially available (Acros). To achieve
the synthesis of diol 22, methyl 3-oxooctanoate was synthesized
by coupling hexanoyl chloride with meldrum’s acid.16 After the
reduction of methyl 3-oxooctanoate via LiAlH4, we obtained the oc-
and with the desired selectivity at the
x-position. Thus, we have
developed a method to systematically synthesize variable regio-
selective gem-difluorinated octan-1-ols. In addition, other minor
products, which were otherwise difficult to prepare in the labora-
tory, were obtained from these chemoenzymatic conversions.
We have carried out gem-difluorination of the octane deriva-
tives employing the fluorination reagent, Deoxo-FluorTM [bis(2-
methoxyethyl)aminosulfur trifluoride] (Matrix Scientific) in
CH2Cl2.10 1-Octanal and 2-, 3-, 4-octanones (1ꢀ4), were used as
the starting material to produce 1,1-, 2,2-, 3,3-, and 4,4-difluorooc-
tanes (5ꢀ8) in 43ꢀ80% yields (Scheme 1). All the products were
monitored by GC or GCMSD at 120 °C under isothermal conditions
(tR = 5.04, 4.87, 4.91, and 4.90 min for 5ꢀ8, respectively). 1,1,1-Tri-
fluorooctane 19 was synthesized by a modified sulfinatodehalo-
genation system.11–14
When the E. coli–AlkB whole-cell system (1.0 mL, O.D.600 = 30)
was treated with 5ꢀ8, gas chromatographic separation at 120 °C iso-
thermal) revealed distal CꢀH activation at the terminal methyl car-
bon. The products 9ꢀ12 appeared at retention times 9.36, 8.39, 8.69,
and 8.47 min, respectively. The higher retention times observed for
the products reflected their increased polarity after the aliphatic
hydroxylation. To determine the regio-selectivity of the hydroxyl-
ation, we have recorded both the 1H NMR and 13C NMR spectra of
(R)-O-acetylmandelic acid derivatives of the corresponding alcohol
obtained by esterification of fluorinated octanols with (R)-2-acet-
oxy-2-phenylethanoate in the presence of dicyclohexylcarbodiim-
ide (DCC) and a catalytic amount of 4-dimethylaminopyridine
(DMAP) (Scheme 2). These spectral data indicated that the major
oxidation had occurred at the omega position (Supplementary data).
GC intensities of the various products were used to quantify
conversion yields. Following a 3 h whole-cell catalysis, GC analysis
yielded conversions of 43%, 68%, 40%, and 43% for 9, 10, 11 and 12,
respectively. Under similar catalytic conditions, the yield for the
production of 20 from 19 was 27% (Scheme 3). The relatively lower
yield of 20 versus 9ꢀ12 may just reflect the longer bond length and
van der Waals radius of the CꢀF bond (1.35 and 1.47 Å) compared
with the CꢀH bond (1.09 and 1.20 Å).15 Perhaps, the size of the
pocket is restricting the entry of the substrate into the protein. A
similar behavior has been also observed in the recent study of
P450 BM-3.11 Interestingly, when n-octane and 1,7-octandiene
were used as substrates in the AlkB whole-cell catalysis, the con-
version yields were relatively poor, 1.2% and 1.1%, respectively,
after a 3 h incubation.
tane-1,3-diol 22. The diol 23 was obtained by the reduction of c-
octanoic lactone by LiAlH4. After protection of the primary alcohol
by a benzyl group, 24ꢀ26 were readily oxidized to their correspond-
ing ketones 27ꢀ29 (Scheme 4). gem-Difluorination was then accom-
plished by subjecting 27ꢀ29 to Deoxo-FluorTM. After deprotection
by heterogeneous hydrogenation on Pd/C, we obtained the target
2,2-, 3,3-, and 4,4-difluorooctan-1-ol 33, 17 and 18, respectively, in
21%, 11% and 15% overall yield.
Compounds 17 and 18 provided authentic standards for the
identification of the minor products derived from the oxidation
of 7 and 8 in the AlkB whole-cell system (Scheme 3). The product
ratio of 6,6-difluorooctan-1-ol to 3,3-difluorooctan-1-ol was
7.6:1.0 after a 3 h conversion of 7, and their GC yields were 40%
and 5.2%, respectively. In the case of 8, the product ratio of 5,5-
difluorooctan-1-ol to 4,4-difluorooctan-1-ol was 7.5:1.0, and their
GC yields are 43% and 5.7%, respectively. We could not obtain a
comparable GC signal for the proximal product 33 upon the treat-
ment of substrate 6 by the whole-cell catalysis. A similar outcome
was obtained for fluorinated substrate 5. The structures of the
proximal products were also established from the spectral data
(1H, 13C, 19F NMR, EI-MS and HR-ESI-MS in Supplementary data).
The substantially higher yields of oxidation of the gem-difluo-
rooctanes relative to the n-octane by the AlkB whole-cell system
reflect the higher kcat and lower KM expected for the fluoro-hydro-
carbons in AlkB. Similar observations have previously been re-
ported by this laboratory for the oxidation of fluorooctanes
versus n-octane by cytochrome P450 BM-3.11 The greater electro-
static and van der Waals interactions between the fluorinated sub-
strates and the enzyme pocket introduced by the –CF2– and CF3–
enhance the binding affinity and presumably KM. Structural and
electronic factors lower the activation free energy and accelerate
11,17
kcat
.
Other factors include the greater membrane permeability
of the fluorinated substrates,7,9b which might come into play dur-
ing prolonged catalysis, although theoretical calculations of
water–octanol partition co-efficients suggest that the permeability
of fluorinated octanes across the cell membranes are similar to that
of n-octane (Table S2, Supplementary data).7c In any case, we can
conclude that the hydrophobic pocket of AlkB is more readily ac-
cessed by the fluorinated octanes than the corresponding alcohols,
because we do not observe any diol formation during n-octane oxi-
dation by the E. coli–AlkB whole-cell system.
When 3,3-difluorooctane 7 and 4,4-difluorooctane 8 were acti-
vated by AlkB, we observed minor products at tR = 7.76 and
In conclusion, we have developed an efficient procedure for the
preparation of gem-difluorinated octan-1-ols starting from gem-
difluorooctanes. Enzymatic conversion of gem-difluorooctanes by
AlkB in the E. coli whole-cell system yielded regio-selective
hydroxylation at the primary carbon in 40ꢀ68% yield. The high
throughputs of the hydroxylated products with the fluorine sub-
stituents suggest that this system offers a reasonable approach to-
ward green conversions of the gem-difluorooctanes to the
difluorinated octan-1-ols. The availability of these gem-difluorooc-
tan-1-ols should facilitate the straightforward synthesis of many
interesting gem-difluorooctan-1-ol-derived compounds via
aliphatic chain extension and further functionalization, a strategy
Deoxo-FluorTM
H
H
n
H
H
m
m
O
n
F F
CH2Cl2
5, m = 0; n = 4
6, m = 1; n = 3
7, m = 2; n = 2
8, m = 3; n = 1
1, m = 0; n = 4
2, m = 1; n = 3
3, m = 2; n = 2
4, m = 3; n = 1
Scheme 1. Preparation of 1,1-, 2,2-, 3,3- and 4,4-difluorooctanes.
Ramu, Ravirala
