Soybean lipoxygenase and horseradish peroxidase catalysed asymmetric oxidation-reduction sequence for the synthesis of chiral (Z,E) diene-diols

Article abstract of DOI:10.1055/s-2001-14907

Unnatural synthetic substrates with a properly spaced prosthetic modifier having non-ionic hydroxy terminus undergoes soybean lipoxygenase catalysed asymmetric peroxidation followed by horseradish peroxidase induced reduction affords (Z, E)-dienediol in high enantiomeric excess.

Full text of DOI:10.1055/s-2001-14907

LETTER
787
Soybean Lipoxygenase and Horseradish Peroxidase Catalysed Asymmetric
Oxidation-Reduction Sequence for the Synthesis of Chiral (Z,E) Diene-Diols
Jhillu S. Yadav,* S. Nanda, A. Bhaskar Rao
Organic Division, Indian Institute of Chemical Technology, Hyderabad, 500 007, India
Fax +91-40-7170512; E-mail: yadav@iict.ap.nic.in
Received 22 March 2001
Abstract: Unnatural synthetic substrates with a properly spaced
prosthetic modifier having non-ionic hydroxy terminus undergoes
soybean lipoxygenase catalysed asymmetric peroxidation followed
by horseradish peroxidase induced reduction affords (Z, E)-diene-
diol in high enantiomeric excess.
Key words: oxygenation, soybean lipoxygenase, horseradish per-
oxidase, asymmetric synthesis
Lipoxygenases (linoleate: oxygen oxidoreductase) are a
group of nonheme iron containing dioxygenases catalys-
ing the addition of molecular oxygen to (1Z, 4Z) polyun-
saturated fatty acids in a regio and stereospecific way to
form the peroxy compounds.1 The peroxides are then re-
Scheme 1 Chiral (Z,E) diene-diol obtained from 1a-1c through
double enzymatic reaction sequence.
Reagent and Conditions: a) SBLO, O2, 0 °C, 0.2 M sodium borate
duced conveniently to the corresponding hydroxy deriva-
tives by several methods as reported earlier.2 Previous
studies of modified substrates with soybean lipoxygenase buffer (pH = 9); b) HRP, 0.1 M phosphate buffer (pH = 6); c) KOH,
MeOH, r.t., 12 h.
reveals that the essential structural requirements are a sin-
gle -6(Z,Z)-1,4-pentadienyl moiety and an appropriately
spaced proximal carboxyl group as characterised by the
natural substrate linoleic acid.3 However research from
different groups4 has established that a carboxylic group
or a charged head group in a fatty acid analogue is not an
essential structural requirement for binding and catalysis
in soybean lipoxygenase oxidations. Substrates having a
non-ionic hydroxy terminus can recognise the enzyme
(soybean lipoxygense) very well. On the other hand per-
oxidases are heme containing enzymes which catalyse the
reduction of hydrogen peroxide and organic hydroperox-
Scheme 2 Reagent and conditions: a) LiNH2, THF-HMPA (6:1),
C5H11Br, 70%; b) H2, Lindlar catalyst, 95%; c) I2, TPP, imidazole,
CH3CN-Et2O (1:3), 85%; d) TPP, CH3CN, 60 °C, 48 h. e) n-BuLi,
THF-HMPA (10:1), 0 °C, 2 h, MeOH, TsOH, 80%.
ides in the presence of structurally diverse electron - do-
nating substrates.5 Reports on enantioselective reactions
catalysed by horseradish peroxidase are rather scarce.6
Herein we wish to report the synthesis of (Z,E) diene-diol
(6a-6c) in high enantiomeric excess through a consecutive
soybean lipoxygenase and horseradish peroxidase cataly-
sed oxidation-reduction sequence (Scheme 1). Synthesis
of the substrates (1a-1c) is described as follows (Scheme
2 and 3).
All the dienealcohols (10a-10c) had > 95% (Z,Z) geome-
try as determined by GC. The small amount of (Z,E) iso-
mer could not be easily separated form the (Z,Z) dienol,
hence the mixtures of isomers were used for subsequent
enzymatic reaction.
For the enzymatic reaction partially soluble substrates 1a-
1c (1 mmol) were taken up in 40 mL of borate buffer pH
9.0 at 0 °C with 150 mg of SBLO7 and incubated in the
presence of oxygen with vigorous stirring, followed by
reduction of the intermediate hydroperoxide with HRP in
Scheme 3 Reagent and conditions: a) NaH, PMB-Br, n-Bu4N+I,
80%; b) CrO3, H2SO4 (8 N); c) DCC, DMAP, 10a-10c, r.t.; d) DDQ,
DCM-H2O (20:1).
Synlett 2001 No. 6, 787–788 ISSN 0936-5214 © Thieme Stuttgart · New York

788
J. S. Yadav et al.
LETTER
Table 2 Regiospecificity for SBLO-Catalysed Oxidation on
Changing the Distal Unit.
presence of phosphate buffer pH 6.0 at 25 °C. Finally re-
moval of the prosthetic group affords (Z,E) diene-diol 6a-
6c9 in (50-60%) isolated yield (Table 1).
Table 1 SBLO-HRP-Catalysed Oxidation-Reduction Sequence of
1a-1c
References and Notes
†
IICT Communication No. 4560.
(1) Veldink, G.A.; Vliegenthart, J.F.G. Substrates and products of
lipoxygenase catalysis. In, Studies in natural products
chemistry; Vol. 9, Structure and chemistry (part-B),
Amsterdam, Elsevier Science Publishers BV 1991, pp. 559-
598.
a) Values were determined by normal-phase HPLC at 235 nm; b) All
rotations were measured as solution in chloroform, c = 1; c) S : R ra-
tios were determined by chiral HPLC (Diacel, Chiral OD column. He-
xane: isopropanol, 9:1).
(2) a) Datcheva, K.V.; Kiss, K.; Solomon, L.; Kyler, K.S. J. Am.
Chem. Soc. 1991, 113, 270. b) Martini, D.; Buono, G.; Iacazio,
G. J. Org. Chem. 1996, 61, 9062
(3) a) Gunstone, F.D. Comprehensive Organic Chemistry;
Barton, D.H.R.; Ollis, W.B.; Halsam, E. Eds. Pregamon press;
New York, 1979, pp, 587-632. b) Zhang, P.; Kyler, K.S. J.
Am. Chem. Soc. 1989, 111, 9241.
(4) a) Brash, A.R.; Ingram, C.D.; Harris, T.M. Biochemistry
1987, 26, 5645. b) Eskola, J.; Lassko, S. Biochim. Biophys.
Acta 1983, 751, 305. c) Novak, M.J. Bioorg. Med. Chem. Lett.
1999, 9, 31. d) Nanda, S.; Rao, A.B.; Yadav, J.S.
(Unpublished results)
The primary purpose of the prosthetic modifier is to sup-
ply the missing structural features needed for enzymatic
recognition and binding. It has also strong influence on
the regiochemical outcome of the reaction by its high hy-
drophobic content when compared to natural substrate li-
noleic acid. Linoleic acid when subjected to oxygenation
with SBLO under the same reaction condition the ob-
served regioisomeric ratio was 90:10.8 But in all the above
three cases regioselection are excellent (98:2).
(5) Adam, W.; Lazarus, M.; Saha-Möller, C.R.; Weichold, O.;
Hoch, U.; Häring, D.; Schreier, P. Biotransformations with
peroxidases. In Advances in Biochemical Engineering &
Biotechnology; Faber, K.; Ed; Springer: Berlin, 1998; vol. 63.
(6) a) Ozaki, S.I.; Oritzde Montellano, P.R. J. Am. Chem. Soc.
1994, 116, 4487. b) Adam, W.; Lazarus, M.; Hoch, U.; Korb,
M.N.; Saaha-Möller, C.R.; Schreier, P. J. Org. Chem. 1998,
63, 6123. c) Adam, W.; Saha-Möller, C.R.; Schmid, K.S. J.
Org. Chem. 2000, 65, 1431.
(7) Lipoxygenase (Type-I, activity = 127,000 units/mg of
protien) and peroxidase (from horseradish, Type VI-A, 1100
units/mg solid) were obtained from SIGMA and used as
obtained.
It is important to note that regioselective formation of diol
6, which corresponds to oxygenation at the appropriate
olefinic site, decreased on changing the distal groups. For
instance, increasing the distal group from n-C5H11 to
n-C8H17 resulted in a substantial decrease in selectivity
from 98:2 to 1:1 ratio of 6/7. Further change in the distal
unit (n-C9H19) led to reversal of selectivity (Table 2). It
should be noted that hydrophobicity constant for n-alkyl
groups increase uniformly as alkyl chains lengthen steadi-
ly. Thus it is clear that the regiospecificity is largely influ-
enced by the difference in hydrophobic content between
the proximal and the distal unit (the hydrocarbon units,
flanking the cis-cis pentadienyl moiety).
(8) Funk, M.O.; Andre, J.C.; Otsuki, T. Biochemistry 1987, 26,
6880.
(9) Selected spectral data - 4a: IR (TF) 3410, 1740 cm-1; 1H
NMR (200 MHz, CDCl3) 0.9 (t, J = 7Hz, 3H), 1.3 (br s, 6H),
1.5-1.8 (m, 8H), 2.3 (t, J = 7Hz, 2H), 2.4 (t, J = 7Hz, 2H), 2.5
(br s, 2H, - OH), 3.6 (t, J = 7Hz, 2H), 4.1 (t, J = 7Hz, 2H), 4.15
(q, J = 7Hz, 1H), 5.46 (dd, J = 10, 15 Hz, 1H), 5.7 (dd, J = 7,
15Hz, 1H), 6.15 (dd, J = 10, 10.4Hz, 1H), 6.53 (dd, J = 10,
15Hz, 1H); 13C NMR (50 MHz, CDCl3) 14, 20.9, 22.5, 26,
26.9, 27.1, 29.4, 31.6, 34, 62.1, 64, 72, 124, 127.4, 128.5,
130.2, 173; [ ]D25 = +11 (c = 0.75, CHCl3). 6a: IR (TF) 3410,
1085 cm-1; 1H NMR (200 MHz, CDCl3) 0.85 (t, J = 7Hz,
3H), 1.26 (m, 6H), 1.44 (m, 6H), 2.2(br s, 2H, -OH), 2.4 (q,
J = 7Hz, 2H), 3.7 (t, J = 7Hz, 2H), 4.1 (q, J = 7Hz, 1H), 5.45
(dd, J = 7, 10.4 Hz, 1H), 5.72 (dd, J = 7, 15 Hz, 1H), 6.14 (dd,
J = 10, 10.4 Hz, 1H), 6.55 (dd, J = 10, 15 Hz, 1H); 13C NMR
(50 MHz, CDCl3): 13.9, 22.5, 26.0, 27.0, 29.5, 32.0, 62.5,
65.0, 124.3, 127.5, 128.6, 130.3; HRMS calcd for C12H22O2
198.1619, Obsd. m/e 198.1592 (M+).
Though HRP reduces the hydroperoxides to correspond-
ing alcohols in a stereoselective way, the points regarding
stereoselection or asymmetric amplification by HRP has
little relevance in this context. In all the cases the hydrop-
eroxides obtained from SBLO catalysed reactions are op-
tically rich with only one isomer (Table 1). So the
enantioselection is fully governed by SBLO only.
Our present Study demonstrates a synthetically useful en-
zymatic method for asymmetric oxidation-reduction reac-
tion sequence of unnatural alkenes. These results lead us
directly to the design of new unnatural substrates and
studies their activities with soybean lipoxygenase and
horseradish peroxidase are currently under investigation.
Acknowledgement
Article Identifier:
1437-2096,E;2001,0,06,0787,0788,ftx,en;D00901ST.pdf
One of the authors (SN) is grateful to CSIR, New Delhi for awar-
ding research fellowship.
Synlett 2001, No. 6, 787–788 ISSN 0936-5214 © Thieme Stuttgart · New York