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Isolation Synthesis and Absolute Configuration of Filbertone Ц the Principal Flavor Component of the Hazelnut.

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Isolation, Synthesis, and Absolute Configuration
of Filbertone - the Principal Flavor Component
of the Hazelnut **
(heptafluorobutanoy1)-( 1R)-l O-methylcamphorate]
in
8 min at 50 "C [phase I] (Fig. 1) and by chiral inclusion gas
chromatography on heptakis(2,6-di-O-methyl-3-O-trifluo-
By Johann Jauch, Dieter Schmalzing, Volker Schurig,*
Roland Emberger, Rudolf Hopp, Manfred Kopsel,
Wilhelm Silberzahn, and Peter Werkhoff
Chiral, acyclic, aliphatic (conjugated) enones occur in nature as pheromones and flavors. For example, (E,6S')-4,6dimethyloct-4-en-3-one, manicone, has been identified in the
mandibular gland of the ant Manica mutica"] and (E)-5methylhept-2-en-4-one, filbertone 5, has been isolated 1'1
from hazelnut extracts and identified as the principal flavor
component of filbertsL3](name synonymous with hazelnut;
derived from St. Philibert).
The determination of the enantiomeric composition, the
assignment of the absolute configuration, and the establishment of a relationship between chirality and olfaction necessitated both the enantioselective synthesis and the quantitative enantiomer separation of 5. (E,SS')-Methylhept-2-en4-one ( 3 - 5 was prepared according to Scheme 1 utilizing the
(S)-sec-butyl chirality pool.
0
5
10
f [minl-
Fig. 1. Enantiomer separation of racemic (@-5-methylhept-2-en-4-one 5 (filbertone) by chiral complexatlon gas chromatography on nickel(11)bis[3-(heptafluorobutanoy1)-(1R)-10-methylcamphorate]in OV-1 (ca. 0.1 m) at 5O'C
[phase 11 [I I ] . Column: 10 m x 0.25 mm (I.D.) fused silica capillary. Carrier:
0.5 bar N,.
roacetyl)-P-cyclodextrinI'2' in 12 min at 110 "C [phase 21
(Fig. 2), thus permitting the simple complementary enantiomer analysis of synthetic ( 8 - 5 and trace amounts of natural 5.
The enantiomeric excess of synthetic ( 8 - 5 was determined
on phase 1 and 2 to be ee = 92 i 1 % (Fig. 2c). On both
2
1
3
(9-5
4
Scheme 1. a) K,Cr,O,/H,SO,/HIO;
70"C/140 torr. b) LIC-CCHJTHF;
- 8O'C, 2 h; allowed to warm in ca. 12 h to
25 ' C . c) LiAIH,/THF, 4 h
reflux. d) MnO,/n-pentane, 40 h.
+
Commercially available (9-(
-)-2-methylbutan-l-ol 1
was oxidized to (9-(
+)-2-methylbutanal 2,r4,'1 which was
then coupled with propynyllithium[6] to give (4R/4S,5S)-5methylhept-2-yn-4-01 3. Stereoselective LiAIH, reduction
afforded exclusively (E,4R/4S,5S)-5-methylhept-2-en-4-014,
which was oxidized with MnO, to (E,SS)-5-methylhept-2en-4-one (S)-5 (purity (by GLC) > 98 % ;overall yield, 20 %,
with steps a and d not yet optimized).[']
Whereas racemic and natural 5 had previously been separated by GLCf9]after conversion to the oxime on a diamide
phase,"'] the underivatized enone can be resolved directly by
chiral complexation gas chromatography on nickel(I1) bis[3-
['I
I**]
Prof. Dr. V. Schurig, Dipl.-Chem. J. Jaucb, DipLChem. D. Schmalzing
Institut fur Organische Chemie der Universitat
Auf der Morgenstelle 18, D-7400 Tubingen (FRG)
Dr. R. Emberger, Dr. R. Hopp, Dr. M . Kopsel, Dr. W. Silberzahn.
Dr. P. Werkhoff
Haarmann & Reimer GmbH, Research Department
D-3450 Holzminden (FRG)
This work was supported by the Fonds der Chemischen Industrie. The
identification and isolation offilbertone was performed in Holzminden, its
enantiomer separation and the synthesis of the (S)enantiomer was carried
out in Tubingen. The supply of filberts by H . Teckenburg, Schwartau
GmbH, Bad Schwartau (FRG), is gratefully acknowledged.
1022
0 VCH
Verlagsgesellschafi mhH, 0-6940 Weinheim, 1989
0
5
10
t [minl-
15
0
5
10
t[minl-
15
Fig. 2. Enantiomer separation of (@-5-methylhept-2-en-4-one5 by chiral inclusion gas chromatography on beptakis(2,6-di-O-methyl-3-O-trifluoroacetyl)P-cyclodextrin in OV-1701 (0.05 m) at 110°C [phase 21 [12]. Column:
40 m xO.25 mm (I.D.) glass capillary. Carrier: 1 bar N,. a) Natural 5 (filhertone), isolated from filberts 121; b) racemic 5, synthesized according to
Scheme 1 ; c) (9-5, synthesized according to Scheme 1.
phases 1 and 2, the (S)enantiomer is eluted as the second
peak. Surprisingly, natural 5 exhibits only a rather low enantiomeric excess, varying with the origin of the hazelnuts:
ee = 54.4% [phase I], ee = 56.2% [phase 21 (Fig. 2a) (filberts from Turkey) and ee = 62.5 % [phase l ], ee = 62.6 %
[phase 21 (filberts from Italy). Comparison of the elution
order of synthetic (S)-5 and natural filbertone established (S')
configuration for the enantiomer in excess (Fig. 2 a and 2c).
Thus, it has been shown that 5 isolated from filberts is enriched with the (S)enantiomer of varying degree of enantiomeric purity.['l The observation of differing enantiomeric
compositions of natural compounds has a precedent in chiral
pheromones (e.g., for endo-brevicomin [I3]).
0570-0833j89j0808-l022$02.50/0
Angen. Chem. Inr. Ed. Engl. 28 (1989) No. 8
Aided by the efficient analytical enantiomer separation
(cf. Fig. 2c), racemization studies on ( 8 - 5 were also performed. No racemization, albeit appreciable thermal decomposition of 5 was observed in the gas phase at 120 "C over 21
days. In the presence of silica no racemization occurred at
22 "C over 21 h, but the ee dropped from 92 % to 74 % in the
presence of basic alumina at 22 "C over 21 h. These results
imply that the low enantiomeric purity of natural filbertone
is an inherent property of the natural compound and is unlikely to have been caused by racemization during isolation.[21
The sensory evaluation of R- and S-filbertone, separated
on phase 2 (Fig. 2 b), established a marked olfactory difference between the enantiomers in respect to odor intensity
and quality. Also the sensory evaluation of synthetic racemic
5 and enantiomeric ( 8 - 5 , prepared according to Scheme 1,
revealed striking olfactory differences. Thus, filbertone represents a new example of the importance of chirality in olfaction, thereby stressing the need to develop efficient chromatographic procedures for stereochemical analyses.['41
Received: April 3,1989 [Z 3269 IE]
German version: Angew. Chem. 101 (1989) 1039
[I] H. J. Bestmann, A. B. Attygalle, J. Glasbrenner, R. Riemer, 0. Vostrowsky. Angew. Chem. 99 (1987) 784; Angew Chem. Inr. Ed. Engl. 26 (1987)
784.
121 Good quality raw hazelnuts (filberts) (600 g) of a Turkish variety (1988
crop) were ground to a fine slurry in an Ultra-Turrax blender with 1.5 L of
distilled water and subsequently transferred into a 10-L round-bottom
flask. 4.5 L of distilled water was added and the finely chopped nuts were
subjected to a simultaneous steam distillationlextraction procedure with
150 mL of n-pentaneldiethyl ether (2: 1, vlv) for 2.5 h at atmospheric pressure as described by Likens-Nickerson (S. T. Likens, G. B. Nickerson,
Proc. Am. Sac. Brew. Chem. 1964, 5.). The extract was dried over anhydrous sodium sulfate and the organic solvent was removed on a 25-cm x 1cm Vigreux distillation column. The process was repeated several times.
and about 50 mL of an aroma concentrate was obtained from a total of
about 5 kg of raw filberts. The aroma concentrates had the characteristic
and intense odor of raw filberts. A separation according to the polarity of
the volatile components was carried out by adsorption Chromatography
The total flavor extract was preseparated on silica gel 60 (70-230 mesh.
Merck, Darmstadt) by using a stepwise gradient of increasing amounts of
diethyl ether in n-pentane. Filbertone was isolated from a medium-polar
silica fraction by means of preparative capillary gas chromatography em-ploying a wide-bore fused silica open-tubular column (30-m x 0.53-mm
1.D.. DB-l/film thickness 1 pm).
[3] R. Emberger, M. Kopsel, J. Briining, R. Hopp, T. Sand, DOS 3 525604 Al
(22. Jan. 1987), Chem. Abstr. 106 (1987) 155899f; DOS 3345784 (27 June
1985), Chem. Abstr. 103 (1985) 140657q; R. Emberger, 43. Dbkussionstagung des Forschungskreises der Ernahrungsinduslrie, 25/26 March 1985,
Bremen.
141 D. Seebach, V. Ehrig, M. Teschner, Justus Liebigs Ann. Chem. 1976,1357.
[S] W. Kirmse, H. Arold, Chem. Ber. 104 (1971) 1800.
[6] Very expensive propyne has been replaced by the cheap welding gas mixture MAPP (Methyl-Acetylene, Propadiene, Propene) (Messer-Griesheim,
4000 Dusseldorf), which contains up to 13.5% propyne.
[7] M. L. Midland, A. Tramontano, A. Kazubski, R. S. Graham, D. J. S . Tsai,
D. B. Cardin, Tetrahedron 40 (1984) 1371.
[S] " C NMR (62.9 MHz, CDCI,): 6 = 18.1 (Cl), 142.2 (C2). 130.4 (C3),
203.8 (C4), 45.1 (CS), 26.0 (C6). 11.5 (C7). 16.0(C8). 'H NMR(250 MHz,
CDCI,): 6 = 1.81 (dd, Hl), 6.82 (dquart. H2), 6.15 (dquart. H3), 2.66
(sext, H5), 1.70 (m, H6), 1.40 (m. H6), 0.80 (t, H7), 1.07 (d, H8).
191 W. Silberzahn, Disserlution, Technische Universitat Berlin, Berlin 1988.
[lo] W. A. Konig, I. Benecke, K. Ernst, J. Chromatogr. 253 (1982) 267.
1111 V. Schurig, R. Link in D. Stevenson, I. D. Wilson (Eds.): Chiral Separalions. Plenum. London, 12 (1989) 383.
[12l H.-P. Nowotny, D. Schmalzing, D. Wistuba, V. Schurig, H R C & C C , J.
High Resolut. Chromatogr. Chromatogr. Commun., 12 (1989) 383.
[131 R. Weber, V. Schurig, Nuturwissenschaften 71 (1984) 408.
I141 V. Schurig in P. Schreier (Ed.): Bioflavour 8 7 , de Gruyter, Berlin 1988,
p. 35
Angew Chem. Int. Ed. Engl. 28 (1989) No. 8
0
The Active Site of Glutathione Reductase:
An Example of Near Transition-State Structures**
By Reiner Sustmann,* Willi Sicking, and Ceorg E. Schulz *
Dedicated to Professor Christoph Ruchardt on the occasion of
his 60th birthday
The ubiquitous flavoenzyme glutathione reductase serves
an important function in intracellular redox processes by
making available free thiols in the form of reduced glutathione."' The catalysis proceeds in two separable steps. In the
first step investigated here, two electrons are transferred
from NADPH to the enzyme, leading to the reduced enzyme
as a stable intermediate.
He
+ NADPH + E , , e
E,,,H,
+ NADPO
In the second step, glutathione disulfide is reduced.
+ G S S G e E , , + 2 GSH
E,,,H,
The active center consists of isoalloxazine (from FAD)
and the adjacent disulfide bridge Cys58: Cys63, both of
which are buried in the protein. The reducing NADPH binds
to the isoalloxazine side of this arrangement and the glutathione being reduced binds to the disulfide side.['' The geometries have been determined by X-ray structure analysis at
resolutions between 2.0 8, and 1.54
Since the active center is located in a very densely packed,
internal region of the protein, which is far apart from all
intermolecular contacts with the enzyme crystal lattice[31
and since the enzyme remains active in the crystal, it is safe
to assume that the geometry established for the average positions of the non-hydrogen atoms agrees with the geometry
of the cellular enzyme to within 0.1 -0.2 A. The mechanism
of the reduction is still not known in detail: the important
question of whether a hydride transfer or successive oneelectron transfers are involved remains unanswered.
Using calculations at the MNDO level that incorporate
the most recent PM3 p a r a m e t r i z a t i ~ n we
, ~ ~have
~ begun to
analyze the interactions in the active center of the enzyme.
These calculations are supplemented using perturbation calculations with the PERVAL"' program, also subjected to
PM3 parametrization. The latter allows the direct determination of the interactions of two molecules and the interpretation of the results on the basis of covalent stabilization as
well as of polar and noncovalent contributions (closed shell
repulsion). The analysis is based on the coordinates of the
non-hydrogen atoms obtained from the X-ray structure
analysis, complemented with hydrogen atoms introduced
with standard bond lengths and bond angles. Since even
semiempirical calculations are too unwieldy for FAD and
NADPH, only the partial structures important for the reaction, that is, the isoalloxazine ring with and without ribitol
and dihydronicotinamide with ribose, were included in the
calculations. Hydrogen atoms were placed at the sites of the
omitted moieties. The disulfide bridge between Cys58 and
Cys63 was treated similarly; the bonds to the remaining
protein were replaced by hydrogen atoms. The calculations
indeed show that the frontier orbitals of the reactants impor~
[*I Prof. Dr. R. Sustmann, DipLIng. W. Sicking
Institut fur Organische Chemie der Universitat
Postfach 103764, D-4300 Essen 1 (FRG)
Prof. Dr. G. E. Schulz
Institut fur Organische Chemie und Biochemie der Universitat
Albertstrasse 21, D-7800 Freiburg (FRG)
["I This work was supported by the Deutsche Forschungsgemeinschaft
.CH Verlug.~~esell,schafi
mhH, 0-6940 Weinheim. 1989
0570-0833/89/0X0X-10233 02.5010
f 023
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principaux, synthesis, flavor, filbertone, isolation, absolute, components, configuration, hazelnuts
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