Simple and efficient preparation of [10,20-13C2]- and [10-CH3,13-13C2]-10-methylretinal: Introduction of substituents at the 2-position of 2,3-unsaturated nitriles

Article abstract of DOI:10.1021/jo0009595

In this paper, we present the synthesis of [10,20-¹³C₂]-10-methylretinal and [10-CH₃,13-¹³C₂]-10-methylretinal, two doubly ¹³C-labeled chemically modified retinals that have been recently used to study the structural and functional details behind the photocascade of bovine rhodopsin (Verdegem et al. Biochemistry 1999, 38, 11316; de Lange et al. Biochemistry 1998, 37, 1411). To obtain both doubly ¹³C-labeled compounds, we developed a novel synthetic method to directly and regiospecifically introduce a methyl substituent on the 2-position of 3-methyl-5-(2′,6′,6′-trimethyl-1′ -cyclohexen-1′-yl)-2,4-pentadienenitrile. Encouraged by these results, we investigated the scope of this novel reaction by developing a general method for the introduction of a variety of substituents to the 2-position of 3-methyl-2,3-unsaturated nitriles, paving the way for simple and efficient synthesis of a wide variety of 10-, 14-, and 10,14-substituted chemically modified retinals, and other biologically important compounds.

Full text of DOI:10.1021/jo0009595

J . Org. Chem. 2001, 66, 1269-1282
1269
Sim p le a n d Efficien t P r ep a r a tion of [10,20-13C2]- a n d
[10-CH3,13-13C2]-10-Meth ylr etin a l: In tr od u ction of Su bstitu en ts a t
th e 2-P osition of 2,3-Un sa tu r a ted Nitr iles
Peter J . E. Verdegem,†,‡ Menno C. F. Monnee,†,§ and J ohan Lugtenburg*,†
Leiden Institute of Chemistry, Leiden University, Gorlaeus Laboratoria, P.O. Box 9502,
2300 RA Leiden, The Netherlands
lugtenbu@chem.leidenuniv.nl
Received September 22, 2000
In this paper, we present the synthesis of [10,20-13C2]-10-methylretinal and [10-CH3,13-13C2]-10-
methylretinal, two doubly 13C-labeled chemically modified retinals that have been recently used to
study the structural and functional details behind the photocascade of bovine rhodopsin (Verdegem
et al. Biochemistry 1999, 38, 11316; de Lange et al. Biochemistry 1998, 37, 1411). To obtain both
doubly 13C-labeled compounds, we developed a novel synthetic method to directly and regiospe-
cifically introduce a methyl substituent on the 2-position of 3-methyl-5-(2′,6′,6′-trimethyl-1′-
cyclohexen-1′-yl)-2,4-pentadienenitrile. Encouraged by these results, we investigated the scope of
this novel reaction by developing a general method for the introduction of a variety of substituents
to the 2-position of 3-methyl-2,3-unsaturated nitriles, paving the way for simple and efficient
synthesis of a wide variety of 10-, 14-, and 10,14-substituted chemically modified retinals, and
other biologically important compounds.
In tr od u ction
Retinoids form an important class of biologically active
compounds. Retinal (1), a C20 conjugated aldehyde, the
structure of which is depicted in Figure 1, is the most
prominent species of this class and has several distinct
functions in nature. For vertebrates, mollusks, and
anthropods, the 11-Z isomer serves as the chromophore
of rhodopsin, the visual signal transduction membrane
protein.1 The absorption of a photon by this colored
cofactor, results in the highly efficient triggering of a
biochemical photocascade, eventually leading to excita-
tion of the optic nerve and visual awareness.2,3 For other
organisms, like bacteria, the light-absorbing character-
istics of this molecule are put to use for entirely different
processes. Bacteriorhodopsin, the light-driven proton
pump in Halobacterium salinarium, uses the all-E form
of retinal as its chromophore. Activation of this protein
by light results in the generation of a net proton gradient
across the cell membrane, which can be exploited to
generate energy.4,5 Other intrinsic membrane proteins
with retinylidene chromophores include halorhodopsin,6
a chloride ion pump, and sensory rhodopsin,7 a phototaxis
F igu r e 1. Structure of all-E-retinal (1) and all-E-10-meth-
ylretinal (2) including the IUPAC-approved numbering.50
system for bacteria. Retinoids are also known to play an
important role in embryo development8 and are used for
specific forms of cancer treatment.9,10
The details behind the functioning of retinoids as
chromophores of intrinsic membrane proteins have been
a topic of research since its discovery. A variety of
biophysical techniques has been applied to tackle this
question.
A well-established approach in retinal proteins func-
tion research is the use of chemically modified retinoids.11
It is widely accepted that the intrinsic structural and
electronic characteristics of retinal are essential for the
highly efficient processes it drives in nature. Hence,
* To whom correspondence should be addressed.
† Leiden Institute of Chemistry, Leiden University, P.O. Box 9502,
2300 RA Leiden, The Netherlands.
‡ Present address: Numico Research B.V., P.O. Box 7005, 6700 CA
Wageningen, The Netherlands.
§ Present address: Department of Medicinal Chemistry, Utrecht
Institute for Pharmaceutical Sciences, Utrecht University, P.O. Box
80082, 3508 TB Utrecht, The Netherlands.
(1) Wald, G. Nature 1968, 219, 800.
(7) Spudich, J . L.; Zacks, D. N.; Bogomolni, R. A. Isr. J . Chem. 1995,
35, 495.
(8) Pijnappel, W. W. M.; Folkers, G. E.; de J onge, W. J .; Verdegem,
P. J . E.; de Laat, S. W.; Lugtenburg, J .; Hendriks, H. F. J .; van der
Saag, P. T.; Durston, A. J . Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 15424.
(9) The retinoids; Sporn, M. B.; Roberts, A. B.; Goodman, D. S., Eds.;
Academic Press Inc.: Orlando, 1984.
(2) Peteanu, L. A.; Schoenlein, R. W.; Wang, Q.; Mathies, R. A.;
Shank, C. V. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 11762.
(3) Hofmann, K. P.; J ager, S.; Ernst, O. P. Isr. J . Chem. 1995, 35,
339.
(4) Mathies, R. A.; Lin, S. W.; Ames, J . B.; Pollard, W. T. Annu.
Rev. Biophys. Biophys. Chem. 1991, 20, 491.
(5) Schulten, K.; Humphrey, W.; Logunov, I.; Sheves, M.; Xu, D. Isr.
J . Chem. 1995, 35, 447.
(6) Oesterhelt, D. Isr. J . Chem. 1995, 35, 475.
(10) Retinoids, Differentiation and disease; CIBA Foundation, Sym-
posium 113, Pithon: London, 1985.
(11) Nakanishi, K.; Crouch, R. Isr. J . Chem. 1995, 35, 253.
10.1021/jo0009595 CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/30/2001

1270 J . Org. Chem., Vol. 66, No. 4, 2001
Verdegem et al.
replacement of particular atoms or groups in the retinal
skeleton by means of organic synthesis, followed by
incorporation studies and functional studies in the
protein, can deliver a wealth of information on the
intriguing structure-activity relationships.
One member of the class of chemically modified reti-
nals, 10-methylretinal (2) (See Figure 1), has recently
been studied by de Lange et al.12 In this study, it was
anticipated that the presence of an extra methyl moiety
on the 10-position of retinal would lead to an increased
out-of-plane distortion of the chromophore of bovine
rhodopsin. It is widely accepted that this out-of-plane
distortion is essential for the fast and efficient kinetics
of the rhodopsin to bathorhodopsin transformation in the
visual phototransduction cascade. To set out a profound
basis for such studies, a detailed study of the geometric
structure of the 10-methylretinilydene chromophore is a
prerequisite. Recently, some of us developed a novel MAS
NMR method that enables the measurement of intramo-
lecular distances in ligands, such as the retinylidene
chromophore of rhodopsin, between pairs of 13C atoms.13
For the determination of the out-of-plane distortion of
10-methylrhodopsin, we focused on the measurement of
the C10‚‚‚C20 and 10-CH3‚‚‚C13 distances. For this
study, the availability of [10,20-13C2]-10-methylretinal
(2a ) and [10-CH3,13]-13C2-10-methylretinal (2b) was a
prerequisite.
Two different syntheses of 10-methylretinal (2) have
been reported in the literature. These include a procedure
by Tanis et al.14 and a procedure developed by Liu et al.,15
involving Peterson olefination reactions. In these ap-
proaches the modification of the retinal structure is
effected by linear synthesis using C1 to C3 building blocks.
The published synthetic sequences by Tanis and Liu,
however, do not allow the incorporation of 13C labels at
predetermined positions in 10-methylretinal, because the
starting synthons are not available in 13C-labeled form.
We decided to investigate the possibility of introducing
the 10-methyl directly at the conjugated chain of retinal,
with 13C-iodomethane, that is a relatively cheap 13C-
labeled synthon.
Our research was boosted by the discovery of a base-
induced self-condensation of retinal, that was recently
described by us in the literature.16 In this reaction, the
C20 methyl moiety of retinal (see Figure 1) is deproto-
nated using a strong nonnucleophilic base and subse-
quently the anion reacts with a neutral retinal molecule
at C13, forming a C40 condensation compound. On the
basis of this observation, we started to investigate the
synthetic possibilities of the anion of the corresponding
nitrile compound, 3-methyl-5-(2′,6′,6′-trimethyl-1′-cyclo-
hexen-1′-yl)-2,4-pentadienal, for the synthesis of the two
13C-labeled 10-methylretinals. The anion itself only yielded
a self-condensation product, when treated with methyl
iodide under various experimental conditions. Therefore,
we focused on the corresponding nitrile compound 3-meth-
F igu r e 2. The four 3-methyl-2-butenenitriles that where
tested in the 2-functionalization reaction. 3-Methyl-5-(2′,6′,6′-
trimethyl-1′-cyclohexen-1′-yl)-2,4-pentadienenitrile (3); 3,7-di-
methyl-9-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4,6,8-nona-
tetraenenitrile (4); 3-methyl-2-butenenitrile (5); 2-butene-
nitrile (6).
yl-5-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4-pentadi-
enenitrile (3). It is known that the anions of such nitriles
are less prone to self-condensation. Moreover, the nitrile
moiety can be readily converted to the aldehyde func-
tionality after the substitution reaction. The aldehyde
moiety can subsequently be extended to the complete
retinal skeleton via known synthetic methods.11
We realized that the development of such a synthetic
procedure for 10-methylretinals does not limit itself to
these compounds, but could serve to develop novel ways
of introducing substituents at the 10 position of retinal.
Moreover, novel 10-substituted retinals could become
available. The 2,3-unsaturated nitrile moiety, that is
present in 3, is also present in 3,7-dimethyl-9-(2′,6′,6′-
trimethyl-1′-cyclohexen-1′-yl)-2,4,6,8-nonatetraeneni-
trile (4), another well-known intermediate for retinal
syntheses. Hence, novel 14-substituted retinals could also
become available. Moreover, by combining the two ap-
proaches, 10,14-disubstituted retinals could be synthe-
sized. A complete investigation to all possibilities de-
scribed above is beyond the scope of our research, but
we focused on the two nitriles described above: 3-methyl-
5-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4-pentadieneni-
trile (3) and 3,7-dimethyl-9-(2′,6′,6′-trimethyl-1′-cyclohexen-
1′-yl)-2,4,6,8-nonatetraenenitrile (4). In addition, we
studied 3-methyl-2-butenenitrile (5) and 2-butenenitrile
(6) (see Figure 2). Compounds 5 and 6 represent the most
simple members of the 3-methyl-2-butenenitrile family.
With the successful functionalization of these smaller
compounds, it may be expected that a large number of
compounds with the same functionality will react in a
similar way.
To explore the scope of the synthetic applications of
these anions, we applied three distinct electrophiles.
From the wide variety of electrophiles available, io-
domethane, methyl thiocyanate, and elemental iodine
were chosen. This restrictive choice is an effort to cover
the characteristics of the electrophile variety. Iodomethane
represents a member of the alkyl halogenide family and
serves as a paradigm for the introduction of alkyl
substituents at the 2-position. Methyl thiocyanate is a
sulfur electrophile and stands for the family of O, S,and
Se electrophiles, that can introduce heteroatoms in the
molecular structure. Iodine is a paradigm for the elemen-
tal halogenides, such as, for example, bromine.
(12) De Lange, F.; Bovee-Geurts, P. H. M.; Van Oostrum, J .; Portier,
M. D.; Verdegem, P. J . E.; Lugtenburg, J .; de Grip, W. J . Biochemistry
1998, 37, 1411.
(13) Verdegem, P. J . E.; Bovee-Geurts, P. H. M.; de Grip, W. J .;
Lugtenburg, J .; de Groot, H. J . M. Biochemistry 1999, 38, 11316.
(14) Tanis, S. P.; Brown, R. H.; Nakanishi, K. Tetrahedron Lett.
1978, 869.
(15) Liu, R. S. H.; Crescitelli, F.; Denny, M.; Matsumoto, H.; Asato,
A. E. Biochem. 1986, 25, 7026.
(16) Verdegem, P. J . E.; Monnee, M. C. F.; Lugtenburg, J . Tetrahe-
dron Lett. 1997, 38, 5355.
All synthesized end products were purified using HPLC

[10,20-13C2]- and [10-CH3,13-13C2]-10-Methylretinal
J . Org. Chem., Vol. 66, No. 4, 2001 1271
Sch em e 1. Syn th etic Sch em e of 10-Meth ylr etin a l (2) (a , b ) 12C), [10,20-13C2]-10-Meth ylr etin a l (2a ) (a ) 13C),
a n d [10-CH3,13-13C2]-10-Meth ylr etin a l (2b) (b). Th e Solid Ba lls In d ica te th e P osition of th e 13C La bels
and subsequently characterized using mass spectrometry
starting nitrile compound. The reaction mixture was
quenched with water at -80 °C and subsequently ex-
tracted with diethyl ether. After silica gel column chro-
matography, the 2,3-dimethyl-5-(2′,6′,6′-trimethyl-1′-
cyclohexen-1′-yl)-2,4-pentadienenitrile (8) was isolated as
an E/Z mixture in 96% yield. This nitrile was subse-
quently reduced using DIBALH following known proce-
dures.17 In this way, 2,3-dimethyl-5-(2′,6′,6′-trimethyl-1′-
cyclohexen-1′-yl)-2,4-pentadienal (9) was obtained in 92%
yield as an E/Z-mixture. The C16 aldehyde 9 was subse-
quently reacted with the anion of triethylphosphono-
acetate in a Horner-Emmonds reaction to form 4,5-
dimethyl-7-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4,6-
heptatrienoate (10) in 72% yield, again as a mixture of
E/Z-isomers. The ester 10 can be reacted with methyl-
lithium to form the corresponding methyl ketone (11).
In this reaction procedure overalkylation is a particular
problem. Gebhard et al.18,19 have developed a procedure
that prevents this problem by performing the reaction
at strictly -100 °C in dry THF containing 5 equiv of
freshly distilled chlorotrimethylsilane. After the addition
1
and high-resolution H and 13C NMR.
Resu lts
Syn th esis
10-Meth ylr etin a l a n d Its Isotop om er s. The syn-
thetic sequence for the isotopomers of 10-methylretinal
was optimized using nonlabeled compounds. â-Ionone (7)
is the synthon of choice for the synthesis of 10-methyl-
retinal (see Scheme 1). In a Horner-Emmonds reaction
sequence, 7 is reacted with the anion of diethyl cyano-
methylphosphonate in dry THF at -80 °C. In this way,
3-methyl-5-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4-pen-
tadienenitrile (3) is formed in 79% yield as a mixture of
E and Z isomers. To introduce the methyl substituent at
the 2-position, this nitrile synthon is deprotonated using
a strong nonnucleophilic base in an anhydrous environ-
ment at low temperature. We applied lithium diisopro-
pylamide (LDA) in dry tetrahydrofuran at low temper-
ature (-70 °C to -90 °C). At these conditions the nitrile
was smoothly deprotonated. At the same low tempera-
ture, the resulting anion was reacted with a solution of
iodomethane in THF. After about 2 h of stirring at low
temperature, TLC analysis showed no presence of the
(17) Groesbeek, M.; Kirillova, Y. G.; Boeff, R.; Lugtenburg, J . Recl.
Trav. Chim. Pays-Bas 1994, 113, 45.
(18) Gebhard, R. Thesis, Leiden University, 1991.
(19) Cooke, M. P., J r. J . Org. Chem. 1986, 51, 951.

1272 J . Org. Chem., Vol. 66, No. 4, 2001
Verdegem et al.
Sch em e 2. Syn th etic Sch em e for th e P r ep a r a tion of 10-Th iom eth ylr etin a l (19) a n d 10-Iod or etin a l (20)
of the methyllithium, the formed alcoholate is trapped
by the chlorotrimethylsilane, to form the respective
trimethylsilyl alcohol, which is stable under these condi-
tions. Subsequent hydrolysis yields the desired 5,6-
dimethyl-8-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-3,5,7-
octatrien-2-one (11) in 80% yield. To finish the retinal
skeleton, 11 is reacted with commercially available
cyanomethyl diethylphosphonate to form the C21 nitrile
2,3,7-trimethyl-9-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-
2,4,6,8-nonatetraenenitrile (12) (95% yield), that is sub-
sequently reduced using DIBALH to form 10-methylret-
inal (2) in 83% yield. In this way, 10-methylretinal can
be synthesized starting from â-ionone (7) in 31% overall
yield. The product is isolated from the reaction mixture
by column chromatography as an E/Z-mixture. The all-
E-isomer was isolated from this mixture with straight-
phase HPLC and subsequently characterized.
able in 13C-labeled form, the introduction of the 20 label
cannot be effected by the methylation of ester 10a as for
the synthesis of natural abundance 10-methylretinal. A
good alternative has been found in the Grignard reagent
methylmagnesium iodide, which can be obtained in 13C-
labeled form starting from 13C-iodomethane. When the
Grignard reagent is reacted with the ester 10a directly,
the addition of chlorotrimethylsilane cannot prevent
overalkylation of the ester moiety. A successful approach
has been found in the transformation of the ester 10a to
the N-methyl-N-methoxy amide 33a by reaction of the
ester with the anion of N-methyl-N-methoxyamine (yield
74%).21,22 During the subsequent reaction of this amide
with 13C-methylmagnesium iodide, the intermediately
formed alcoholate is stabilized by the ligating effect of
the magnesium between the alcohol anion and the amide
nitrogen. After hydrolysis of this intermediate during
workup, the methyl ketone (11a ) is isolated in 17% yield.
Subsequently, the synthetic sequence is followed as for
the natural abundance 10-methylretinal to complete the
retinal skeleton of 11a .
For the synthesis of both doubly 13C-labeled 10-
methylretinals, some of the reagents in Scheme 1
were substituted for their 13C-labeled isotopomer. For
[10-CH3,13-13C2]-10-methylretinal (2b), the labels are
introduced by 13C-iodomethane, that is commercially
available, and [1-13C]-triethylphosphonoacetate. This
labeled synthon is synthesized starting from [1-13C]-acetic
acid according to literature procedures.19,20 In short,
[1-13C]-acetic acid is reacted with bromine and phospho-
rus tribromide in a Hell-Vollhardt-Zelinsky reaction to
form [1-13C]-bromoacetyl bromide, which is subsequently
quenched with ethanol to form [1-13C]-ethyl bromoacetate
in 86% overall yield, after distillation of the product
under reduced pressure. [1-13C]-triethylphosphonoacetate
is formed by an Arbusov reaction of [1-13C]-ethyl bro-
moacetate and triethyl phosphite in 90% yield.
Oth er Ch em ica lly Mod ified Retin oid s. Encouraged
by the success obtained with the synthesis of 10-meth-
ylretinal, we continued to investigate the synthetic scope
of the 2-functionalization reaction for 2,3-unsaturated-
3-methylnitriles. We decided to react the anion of 3 with
methyl thiocyanate and iodine, leading to 10-thiometh-
ylretinal (19) and 10-iodoretinal (20) as depicted in
Scheme 2. The anion of 3 was reacted with 1.5 equiv of
methyl thiocyanate or iodine under identical experimen-
tal conditions as for the synthesis of 10-methylretinal.
Typically, 3 h of stirring at the low temperature was
needed to completely react the electrophile according to
thin-layer chromatography. Concomitantly, 3-methyl-5-
(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2-thiomethyl-2,4-
pentadienenitrile (13) and 2-iodo-3-methyl-5-(2′,6′,6′-
trimethyl-1′-cyclohexen-1′-yl)-2,4-pentadienenitrile (14)
were prepared in 97% and 90% yield, respectively. The
nitrile moieties of 13 and 14 are subsequently reduced
using DIBALH to yield the respective aldehydes 15 and
16. In this case the completion of the retinal C20 skeleton
can be effected by a shorter synthetic route, by reacting
the aldehydes with the anion of 4-(diethylphosphono)-3-
methyl-2-butenenitrile. The resulting nitriles are again
reduced using DIBALH to yield 10-thiomethylretinal (19)
and 10-iodoretinal (20) as a mixture of isomers in the
For [10,20-13C2]-10-methylretinal (2a ), the labels are
introduced by ([2-13C]-cyanomethyl)-diethylphosphonate
and by 13C-iodomethane in a Grignard reaction. ([2-13C]-
Cyanomethyl)diethylphosphonate is prepared starting
from commercially available [2-13C]-acetonitrile. In a one-
pot reaction, [2-13C]-acetonitrile is reacted with 2 equiv
of LDA in THF at -80 °C. The first equivalent of LDA is
used to deprotonate [2-13C]-acetonitrile. Subsequently 1
equiv of diethyl chlorophosphate is added to the reaction
mixture to form ([2-13C]-cyanomethyl)diethylphospho-
nate, which is deprotonated by the remaining second
equivalent of LDA. Then â-ionone (7) is added, so that
the Horner-Emmonds reaction commences. The intro-
duction of the C13 methyl in 10-methylretinal is effected
by methyllithium, but since methyllithium is not avail-
(21) Groesbeek, M.; Lugtenburg, J . Photochem. Photobiol. 1992, 56,
903.
(20) Harwood, H. J . Chem. Rev. 1962, 62, 99.
(22) Nahm, S.; Weinreb, S. M. Tetrahedron Lett. 1981, 22, 3815.

[10,20-13C2]- and [10-CH3,13-13C2]-10-Methylretinal
J . Org. Chem., Vol. 66, No. 4, 2001 1273
Sch em e 3. Syn th etic Sch em e for th e P r ep a r a tion of 14-Meth ylr etin a l (24), 14-Th iom eth ylr etin a l (25), a n d
14-Iod or etin a l (26)
respective overall isolated yields of 11% and 23%. The
low overall yield for the synthesis of 10-iodoretinal is
mainly due to the low reaction yield for the DIBALH
reduction of 18. After the reaction, a considerable amount
of 3 was found, indicating that dehalogenation by DIBALH
is an important side reaction, which is in agreement with
literature reports.23
Sch em e 4. Syn th etic Sch em e for th e P r ep a r a tion
of 2,3-Dim eth yl-2-bu ten en itr ile (27),
3-Meth yl-2-th iom eth yl-2-bu ten en itr ile (28),
3-Meth yl-2-iod o-2-bu ten en itr ile (29),
2-Meth yl-2-bu ten en itr ile (30),
2-Th iom eth yl-2-bu ten en itr ile (31), a n d
2-Iod o-2-bu ten en itr ile (32)
For the synthesis of 14-substituted retinals, depicted
in Scheme 3, 3,7-dimethyl-9-(2′,6′,6′-trimethyl-1′-cyclo-
hexen-1′-yl)-2,4,6,8-nonatetraenenitrile (4) (obtained by
literature procedure.21 The synthesis and characteriza-
tion of this compound has been previously described in
the literature24) was deprotonated using LDA at -85 °C.
Subsequently one of the three electrophiles, methyl
iodide, methyl thiocyanate, or iodine, was added (1.5
equiv). Again, about 3 h of stirring at the low temperature
was needed to completely react the electrophile according
to thin-layer chromatography. In this way 2,3,7-tri-
methyl-9-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4,6,8-nona-
tetraenenitrile (21), 3,7-dimethyl-2-thiomethyl-9-(2′,6′,6′-
trimethyl-1′-cyclohexen-1′-yl)-2,4,6,8-nonatetraeneni-
trile (22), and 3,7-dimethyl-2-iodo-9-(2′,6′,6′-trimethyl-1′-
cyclohexen-1′-yl)-2,4,6,8-nonatetraenenitrile (23) were
prepared in the respective yields of 80%, 96%, and 70%.
Subsequent DIBALH reduction of the nitrile moieties of
21, 22, and 23 results in the formation of 14-methylreti-
nal (24), 14-thiomethylretinal (25), and 14-iodoretinal
(26) in the respective overall yields of 61%, 66%, and 11%.
Again, the reductive removal of the substituent by
DIBALH is responsible for the lower overall yield of 26.23
Sm a ller F u n ction a lized Nitr iles. For the synthesis
of the 2-substituted 3-methyl-2-butenenitriles and butene-
nitriles depicted in Scheme 4, compounds 5 and 6 were
deprotonated using the same reaction conditions and
were reacted with the same electrophiles, again adding
1.5 equiv. 2,3-Dimethylbutenenitrile (27), 3-methyl-2-
thiomethylbutenenitrile (28), and 2-iodo-3-methylbutene-
nitrile (29) were formed in 53%, 80%, and 89% yield. The
volatility of 27 presumably explains the lower isolated
yield of this compound. This effect is even stronger for
the 2-substituted butenenitrile 30. This compound, of
which its presence in the reaction mixture was inferred
from the proton NMR spectrum, could not be isolated
after standard reaction workup. Compounds 31 and 32,
that have a higher molecular weight than 30, were
isolated in 28% and 14%, respectively.
It can be concluded that for all nitriles, the 2-function-
alization reaction gives exclusively 2-reaction in very good
yields. Moreover, no remaining starting material can be
observed in the reaction mixtures.
Ch a r a ct er iza t ion of t h e Novel R et in a l Com -
p ou n d s. We limit the discussion of the characterization
of the compounds to the novel retinal compounds. All
spectroscopic data of the other compounds can be found
in the Experimental Section. All retinal compounds were
isolated as mixtures of geometric isomers. Preparative
HPLC (see Experimental Section for procedures) allowed
us to purify the all-E isomers. After separation of the
isomers, the fraction containing the all-E-isomer was run
again, resulting in a single-peak HPLC trace, proving
that the isolated all-E-isomers of all compounds were
obtained with purity close to unity. The all-E-isomers
were subsequently characterized using mass spectroscopy
and high-resolution NMR.
Ma ss Sp ectr om etr y. The single focus electron impact
mass spectra of 10-methylretinal (2) appears as a
typical retinoid mass spectrum with the base peak at m/z
) 298, the molecular ion peak. The spectra of the
isotopomers of 2, [10,20-13C2]-10-methylretinal (2a ) and
[(10-CH3),13-13C2]-10-methylretinal (2b) reveal their base
(23) March, J . Advanced Organic Chemistry; J ohn Wiley & Sons:
New York, 1992; p 438.
(24) Pardoen, J . A.; Winkel, C.; Mulder, P. P. J .; Lugtenburg, J . Rec.
Trav. Chim. Pays-Bas 1984, 103, 135.

1274 J . Org. Chem., Vol. 66, No. 4, 2001
Verdegem et al.
Ta ble 1. Exp er im en ta lly Deter m in ed a n d Ca lcu la ted
Molecu la r Ma sses of 10-Meth ylr etin a l (2),
[10,20-13C2]-10-Meth ylr etin a l (2a ),
a label incorporation of g99%, indicating that no loss of
label has occurred during the synthetic steps.
NMR. The NMR spectra which are discussed in this
section are enclosed in the Supporting Information.
The proton spectra of the all-E isomers of 10-
methylretinal (2), [10,20-13C2]-10-methylretinal (2a ) and
[(10-CH3),13-13C2]-10-methylretinal (2b), were recorded
in chloroform at a proton resonance frequency of 300
MHz. The spectra can be assigned by comparison to the
1H spectrum of retinal, that has been known in the
literature for a long time.9
[10-CH3,13-13C2]-10-Meth ylr etin a l (2b),
10-Th iom eth ylr etin a l (19), 10-Iod or etin a l (20),
14-Meth ylr etin a l (24), 14-Th iom eth ylr etin a l (25),
14-Iod or etin a l (26), 2,3-Dim eth yl-2-bu ten en itr ile (27),
3-Meth yl-2-th iom eth yl-2-bu ten en itr ile (28),
2-Iod o-3-m eth yl-2-bu ten en itr ile (29),
2-Meth yl-2-bu ten en itr ile (30),
2-Th iom eth yl-2-bu ten en itr ile (31), 2-Iod o-2-bu ten en itr ile
(32). Th e Da ta Ar e Obta in ed Usin g Electr on Im p a ct (EI,
70 eV) Dou ble F ocu s Ma ss Sp ectr om etr y
The substitution at the C10 position of retinal by a
methyl moiety as for 10-methylretinal can be inferred
from the proton spectrum. The H10 response, with δ )
6.18 ppm for retinal, is not present in the spectrum of 2.
Moreover, the AMX pattern including the H11 response
in the retinal spectrum has changed to an AX pattern
comprising the resonance of H11 at δ ) 7.42 ppm and
H12 at δ ) 6.44 ppm. In the downfield region of the
spectrum of natural abundance all-E-10-methylretinal,
an aldehyde and five other resonances can be observed,
corresponding to the five inequivalent vinylic protons,
present in the molecular structure.
calcd mass experimental mass
compd molecular formula
(a.m.u.)
(a.m.u.)
2
C
C
C
C
C
C
C
C
1H3016O1
298.22967
300.23638
300.23638
330.20174
410.11054
299.23749c
330.20174
410.11054
298.23106
300.23682
300.23855
330.20029
410.11400
299.23618c
330.20015
410.11180
12
21
20
20
21
20
21
21
20
13C1 H3016O1
12
12
12
12
12
12
12
1
2a
2b
19
20
24b
25
26
1
13C1 H3016O1
1H3016O132S1
1H27127I116O1
1H3016O1
1H3016O132S1
1H27127I116O1
a
b
No data available. Measurement performed using chemical
ionization. c Mass of [M + H]+•
.
The incorporation of the 13C labels can be inferred from
the proton spectra by comparing them to the spectrum
of the natural abundance 10-methylretinal. The incor-
poration of the 13C label at the C10 position can be clearly
observed in the resonance of H8 at δ ) 6.68 ppm: an
extra splitting of the doublet due to the J coupling is
observed. The resonance of H12 at δ ) 6.44 ppm has
changed from a doublet in natural abundance 10-meth-
ylretinal to a double doublet, due to the extra three-bond
couplings with both 13C10 and 13C20. The presence of
13C20 is also inferred from the coupling of 13C20 with the
H14 resonance. In the aliphatic region of the spectrum
of all-E-[10,20-13C2]-10-methylretinal, the H20 singlet is
now split into a doublet, confirming the position of the
13C methyl label. Within experimental error, no signal
in the middle of the doublet can be observed, proving that
the label incorporation is very high. In a similar fashion,
the spectrum of all-E-[10-CH3,13-13C2]-10-methylretinal
can be explained. The presence of both 13C labels is
reflected in the H11 resonance, that is split into a double
double doublet, due the combined three-bond couplings
with the 13C at the C10 methyl moiety and the 13C13.
The remaining vinylic resonances are unaffected by the
13C incorporation.
peak at m/z ) 300, providing evidence that the two 13C
labels have been incorporated into the molecular skeleton
of the compounds. The single focus electron impact mass
spectra of the other analogues of retinal, 10-thiomethyl-
retinal (19), 10-iodoretinal (20), 14-methylretinal (24) (CI
method used), 14-thiomethylretinal (25), and 14-iodoreti-
nal (26) appear also as typical retinoid mass spectra, with
their respective base peaks at m/z ) 330, 410, 330, 299,
and 410, the molecular ion peaks. All spectra of the
retinal analogues showed a peak due to the expulsion of
a CH3 unit and a strong peak at m/z ) 149, assigned to
the â-cyclocitral ethene cation, as for retinal.9 The
spectrum of 20 reveals the presence of an iodine sub-
stituent in the molecular structure by a peak at m/z )
283, assigned to the fragment that is formed by expulsion
of the iodine atom. The molecular ion peak in the
spectrum of 24 is assigned to the [M + H]+•, caused by
the use of the chemical ionization technique. The use of
the electron impact as ionization technique resulted in
the absence of the M+• peak in the mass spectrum,
probably due to complete fragmentation of the molecular
ion before it is detected.
Double focus mass spectrometry is used to determine
the exact mass of the synthesized compounds. Table 1
lists the experimentally determined molecular masses of
all synthesized retinal compounds. The experimentally
found exact masses of the synthesized compounds are all
according to expectations. The difference between the
calculated and experimentally determined exact masses
is never greater than 0.0008%. The degree of incorpora-
tion of the 13C labels in the isotopomers of 10-methyl-
retinal are inferred from the double-focus mass spectra.
This is performed by comparing the exact mass of the
M+• peak with the [M - 1]+• and [M - 2]+• peak, after
correction for the natural abundance contributions to the
intensity of the peaks. Both doubly 13C-labeled isoto-
pomers have a label incorporation of g98%. This implies
that the label incorporation at the individual positions
in the isotopomers amounts to g99%. This is also shown
The proton spectra of all-E-10-methylretinal clearly
prove that the 10 position is substituted with a methyl
group. Moreover, the chemical shifts and J coupling
constants are in agreement with an all-E configuration.
The spectra of all-E-[10,20-13C2]-10-methylretinal and all-
E-[10-CH3,13-13C2]-10-methylretinal indicate that the 13
C
labels are at their predetermined positions and, within
experimental error, incorporated for 99%.
1
The 300.1 MHz H NMR spectra of the all-E isomers
of the other retinal analogues 10-thiomethylretinal (19),
10-iodoretinal (20), 14-methylretinal (24), 14-thiometh-
ylretinal (25), and 14-iodoretinal (26) have been recorded
in deuterated chloroform.
The substitution at the C10 position of retinal by a
thiomethyl moiety and an iodine atom, as for 19 and 20,
respectively, can be inferred from the proton spectrum.
For 10-thiomethylretinal no H10 resonance is present.
Instead, the resonance of H11 appears in an AB pattern,
due to the J coupling with H12 at δ ) 7.26. The presence
1
by the H NMR spectra of the labeled compounds (see
below). The commercially available 13C-labeled starting
compounds we used for the synthesis of the retinals have

[10,20-13C2]- and [10-CH3,13-13C2]-10-Methylretinal
J . Org. Chem., Vol. 66, No. 4, 2001 1275
Sch em e 5. P r op osed Rea ction Mech a n ism for th e 2-F u n ction a liza tion of 3-Meth yl-2-bu ten en itr iles
of the 10-thiomethyl moiety is reflected in the spectrum
by the strong additional singlet at δ ) 2.38 ppm. The
strong magnetic anisotropy effect of the iodine atom is
reflected in the upfield shift for the H11 resonance25
compared to the chemical shifts for the H11 proton in
all-E-retinal at δ ) 7.14 ppm.
atom and the γ position. Hence, for a deprotonated
3-methyl-2-butenenitrile, a nucleophilic attack can either
occur from the 4- or the 2-position. The nitriles applied
in this research, however, exclusively give 2-substitution
products, the expected product of a kinetic reaction
between the nitrile anion and electrophiles. The condi-
tions are such that no unreacted material is present nor
further reaction to disubstitution takes place. This
substantially increases the synthetic versatility of this
reaction. After nucleophilic coupling, the structure of the
product must be a â,γ-unsaturated nitrile. With our
reaction conditions, however, the 2-substituted products
all give immediate isomerization to the “reconjugated”
compound, presumably promoted by the basic conditions
in the reaction mixture.
Substitution at the C14 position of the retinal chain,
as for 14-methylretinal (21), 14-thiomethylretinal (22),
and 14-iodoretinal (23), results in the disappearance of
the H14 signal. For all three compounds, the spectra lack
the H14 signal, and contain the 15 aldehyde resonance
appearing as a singlet, since the three-bond J coupling
with H14 is no longer present. The introduction of the
iodine atom at C14 in 23 is reflected in the upfield shift
of the aldehyde resonance to δ ) 9.26 ppm, as compared
to the H15 shift for all-E-retinal. The overall structure
of all three 14-substituted retinals is supported by the
1H NMR data.
It is particularly important to maintain the reaction
temperature at values close to -80 °C throughout the
whole reaction. If the reaction temperature is allowed to
rise to room temperature after the addition of the
electrophile, a complex mixture of products is observed
that might be explained by competition by a Thorpe type
reaction,26 resulting in nitrile adduct products. If the
temperature is raised to room temperature before the
addition of the electrophiles, no substitution products can
be observed at all, but only unidentified products.
This novel reaction allowed us to introduce the 13C
methyl moiety in a one-step procedure. This reduces the
costs for the preparation of the labeled compound sub-
stantially. In the recent past, our group has developed
efficient routes for the introduction of 13C labels at any
position or combination of positions in the skeleton of
retinal.21 These methods can easily be adapted to the
synthesis of labeled 10-methylretinals. Thus, 13C-labeled
10-methylretinals can be synthesized with labels at any
position or combination of positions.
1
The H-noise-decoupled 75.4 MHz 13C NMR spectra of
all-E-10-methylretinal (2), all-E-[10,20-13C2]-10-methyl-
retinal (2a ), and all-E-[10-CH3,13-13C2]-10-methylretinal
(2b) were also recorded in chloroform. The spectrum of
natural abundance all-E-10-methylretinal shows 20 car-
bon resonances, in agreement with the structure of all-
E-10-methylretinal, that comprises 21 carbon atoms, of
which two (C16 and C17) are equivalent. The resonance
at δ ) 190.99 originates from the C15 aldehyde carbon.
The vinylic region of the spectrum contains five reso-
nances with a large amplitude, representing the vinylic
carbons possessing a proton and five smaller amplitude
resonances, from the quaternary carbons. The aliphatic
region of the spectrum shows the remaining nine carbon
resonances, representing the ionone ring and the methyl
substituent carbons, confirming the structure of all-E-
10-methylretinal.
13C NMR is used to prove that the incorporation of the
13C isotopes in all-E-10-methylretinal at their predeter-
mined positions has been successful. Both spectra of the
labeled compounds show two strong resonances, flanked
by weak signals from the natural abundance background
13C. Comparison of the position of the label signals in the
spectrum with the spectrum of natural abundance all-
E-10-methylretinal, indicates the position of the label.
From the NMR experiments we can conclude that the
all all-E-isomers of the synthesized compounds were
obtained without presence of other isomers or compounds.
Concomitantly, the purity of the samples is close to 100%.
It is anticipated that the 2-functionalization reaction
for 3-methyl-2-butenenitriles as presented in this paper
will have a wide applicability in the synthesis of chemi-
cally modified retinals. Retinal analogues have played
an important role in retinal protein structure and func-
tion research during the past decades.11 A large variety
of 10- and 14-substituted retinals has been synthe-
sized. Among them, 10-chlororetinal,27 10-fluororeti-
nal,28,29 10-methylretinal,28,30 10-ethylretinal,28 14-meth-
ylretinal, and 14-fluororetinal.31,32 A second set of retinal
analogues contains a ring system, attached to the C10
position in retinal, among them, 10,20-methanoreti-
Discu ssion
The mechanism for the 2-functionalization reaction for
3-methyl-2-butenenitriles presented in this paper is
depicted in Scheme 5. One of the γ hydrogens at the
3-methyl moiety, that are relatively acidic because of the
conjugation effect of the corresponding anion with the
nitrile group, is abstracted by the LDA as a strong base.
Because of the conjugation effect, negative charge density
will be spread out in the region between the nitrogen
(26) March, J . Advanced Organic Chemistry; J ohn Wiley & Sons:
New York, 1992, p 963.
(27) Ko¨brich, G.; Breckoff, W. E.; Drischel, W. J ust. Lieb. Ann. Chem.
1967, 704, 51.
(28) Asato, A. E.; Denny, M.; Matsumoto, H.; Mirzadegan, T.; Ripka,
W. C.; Crescitelli, F.; Liu, R. S. H. Biochemistry 1986, 25, 7021.
(29) Asato, A. E.; Denny, M.; Liu, R. S. H. J . Am. Chem. Soc. 1978,
100, 5957.
(30) Shichida, Y.; Ono, T.; Yoshizawa, T.; Matsumoto, H.; Asato, A.
E.; Zingoni, J .; Liu, R. S. H. Biochemistry 1987, 26, 4422.
(31) Tanis, S. P.; Brown, R. H.; Nakanishi, K. Tetrahedron Lett.
1978, 869.
(25) The assignment of the H11 proton in 23 is ambiguous: the H11
and H12 resonances could be reversed.
(32) Steinberg, G.; Ottolenghi, M.; Sheves, M. Biophys. J . 1993, 64,
1499.

1276 J . Org. Chem., Vol. 66, No. 4, 2001
Verdegem et al.
nal,33,34 10,20-ethanoretinal,35,36 10,20-propanoretinal,37,38
10,12-ethanoretinal,39 and 10,12-propanoretinal.44 A large
variety of synthetic steps has been applied to introduce
the substituent at the C10 or C14 position. The reaction
presented in this paper appears to be a versatile method
to introduce a variety of substituents on the 10 and 14
positions of retinal in excellent yields. Moreover, the
scheme can be adapted to introduce substituents at both
the 10- and the 14-position of retinal. To demonstrate
the scope of the reaction we reacted 3-methyl-5-(2′,6′,6′-
trimethyl-1′-cyclohexen-1′-yl)-2,4-pentadienenitrile (3) and
3,7-dimethyl-9-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-
2,4,6,8-nonatetraenenitrile (4) with a selection of elec-
trophiles. From the wide variety of electrophiles avail-
able, we chose iodomethane, methyl thiocyanate, and
elemental iodine. This choice is not trivial. We tried to
cover the characteristics of different electrophiles. Io-
domethane is a soft electrophile that represents the
family of alkyl halogenides. Since the introduction of the
methyl moiety in 10-methylretinal is so effective, one may
anticipate that other alkyl halogenides, such as iodo-
ethane, will react in a similar fashion. In this way the
sequence will allow for the easy synthesis of, for example,
10-ethylretinal or 14-ethylretinal, two retinal analogues
that are known in the literature.45
Another class of well-studied chemically modified re-
tinoids include ring structures.11 The newly developed
2-functionalization reaction for 3-methyl-2-butenenitriles
will facilitate the synthesis of these kind of ring-contain-
ing retinal analogues via coupling with bifunctional
electrophiles, that allow ring closure of the system.
Methyl thiocyanate is an electrophile with a cyanide
moiety as leaving group. With this electrophile, 10-
thiomethylretinal (19) and 14-thiomethylretinal (25) have
been synthesized. In a similar fashion, different hetero-
atom-containing retinal analogues can be synthesized by
applying different electrophiles.
electrophile will allow for the synthesis of 10-bromoreti-
nal. The presence of a iodo substituent in retinal may be
very important, if good three-dimensional crystals of
rhodopsin will be available in the future. These crystals
will be investigated using X-ray crystallography. This
technique, probing the overall structure of the protein,
is only successful if the so-called phase problem can be
solved. This requires the presence of a heavy atom at a
known position in the crystal. The iodine atom, with a
nuclear mass of 127, can serve to solve the phase problem
when the 10-iodoretinal analogue is bound in the binding
pocket of the rhodopsin crystals.
The novel 2-functionalization reaction for 3-methyl-2-
butenenitriles does not limit itself to the synthesis of
retinals. Other biomolecules of which the molecular
skeleton comprises isoprene units, such as carotenoids,
possess the same structural units as for retinals. The
function of proteins containing these carotenoids as
chromophores are also investigated using chemically
modified skeletons.46-48 The new procedure can be ex-
ploited for efficient synthesis of known and novel chemi-
cally modified carotenoids.
As shown, the reaction is also applicable to the smaller
nitriles 5 and 6, in the same high yields. Since the nitrile
moiety of these substituted unsaturated compounds can
be functionalized to different groups, e.g., carboxylic
acids, ketones, or aldehydes, this synthetic approach
opens the way to efficient introduction of substituents
on vinylic carbons.
Con clu sion
In this paper we presented an efficient method for the
introduction of substituents at the 2-position of 3-methyl-
2-butenenitriles. The procedure is simple, can be applied
with very different electrophiles, and yields exclusively
the desired 2-functionalization in high yields. We dem-
onstrated the versatility of the reaction with the prepara-
tion of two doubly 13C-labeled 10-methylretinals and five
10- and 14-substituted retinals, four of which are novel
compounds which have a wide application in retinal
proteins function research. In this way, starting from
3-methyl-5-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4-pen-
tadienenitrile and 3,7-dimethyl-9-(2′,6′,6′-trimethyl-1′-
cyclohexen-1′-yl)-2,4,6,8-nonatetraenenitrile, 10-methyl-
retinal (31% overall yield), [10,20-13C2]-10-methylretinal
(3% overall yield), [10-CH3,13-13C2]-10-methylretinal (5%
overall yield), 14-methylretinal (61% overall yield), 14-
thiomethylretinal (66% overall yield), 14-iodoretinal (11%
overall yield), 10-thiomethylretinal (11% overall yield),
and 10-iodoretinal (23% overall yield) were synthesized.
[10,20-13C2]-10-Methylretinal, [10-CH3,13-13C2]-10-meth-
ylretinal, 10-thiomethyl-, 14-thiomethyl, 10-iodo-, and 14-
iodoretinal are novel retinal compounds. 10-Methylreti-
nal and 14-methylretinal are known modified retinals
that have been presented in the literature, albeit syn-
thesized via different approaches.
10-Iodoretinal is synthesized by using iodine as elec-
trophile. This novel analogue adds to the list of 10-
halogenated retinal analogues. 10-Fluororetinal29,30 and
10-chlororetinal27 are already described in the literature.
Applying the same reaction procedure with bromine as
(33) Liang, J .; Steinberg, G.; Livnah, N.; Sheves, M.; Ebrey, T.;
Tsuda, M. Biophys. J . 1994, 67, 848.
(34) Groesbeek, M. Thesis, Leiden University, 1993.
(35) van der Steen, R.; Groesbeek, M.; van Amsterdam, L. J . P.;
Lugtenburg, J .; van Oostrum, J .; de Grip, W. J . Recl. Trav. Chim. Pays
Bas 1989, 108, 20.
(36) de Grip, W. J .; van Oostrum, J .; Bovee-Geurts, P. H. M.; van
der Steen, R.; van Amsterdam, L. J . P.; Groesbeek, M.; Lugtenburg,
J . Eur. J . Biochem. 1990, 191, 211.
(37) Bhattacharya, S.; Ridge, K. D.; Knox, B. E.; Khorana, H. G. J .
Biol. Chem. 1992, 267, 6763.
(38) Akita, H.; Tanis, S.; Adams, M.; Balogh-Nair, V.; Nakanishi,
K. J . Am. Chem. Soc. 1980, 102, 6370.
(39) Buchert, J .; Stefancic, V.; Doukas, A. G.; Alfano, R. R.; Cal-
lender, R. H.; Pande, J .; Akita, H.; Balogh-Nair, V.; Nakanishi, K.
Biophys. J . 1983, 43, 279.
(40) Birge, R. R.; Murray, L. P.; Pierce, B. M.; Akita, H.; Balogh-
Nair, V.; Findsen, L. A.; Nakanishi, K. Proc. Natl. Acad. Sci. U.S.A.
1985, 82, 4117.
(41) Hu, S.; Franklin, P. J .; Wang, J .; Ruiz Silva, B. E.; Derguini,
F.; Nakanishi, K. Biochemistry 1994, 33, 408.
Starting from 3-methyl-2-butenenitrile and 2-butene-
nitrile, we synthesized 2,3-dimethyl-2-butenenitrile (53%
yield), 3-methyl-2-thiomethyl-2-butenenitrile (80% yield),
(42) Mizukami, T.; Kandori, H.; Shichida, Y.; Chen, A.-H.; Derguini,
F.; Caldwell, C.; Bigge, C.; Nakanishi, K.; Yoshizawa, T. Proc. Natl.
Acad. Sci. U.S.A. 1993, 90, 4072.
(43) Nakanishi, K.; Chen, A.-H.; Derguini, F.; Franklink, F.; Hu,
S.; Wang, J . Pure Appl. Chem. 1994, 66, 981.
(46) Frank, H. A.; Desamero, R. Z. B.; Chynwat, V.; Gebhard, R.;
van der Hoef, I.; J ansen, F. J .; Lugtenburg, J .; Gosztola, J .; Wasielew-
ski, M. R. J . Phys. Chem. 1997, 101, 149.
(44) Groesbeek, M.; Kirillova, Y. G.; Boeff, R.; Lugtenburg, J . Recl.
Trav. Chim. Pays-Bas 1994, 113, 45.
(45) Ga¨rtner, W.; Hopf, H.; Hull, W. E.; Oesterhelt, D.; Scheutzow,
D.; Towner, P. Tetrahedron Lett. 1980, 347.
(47) Farhoosh, R.; Chynwat, V.; Gebhard, R.; Lugtenburg, J .; Frank,
H. A. Photochem. Photobiol. 1997, 66, 97.
(48) Farhoosh, R.; Chynwat, V.; Gebhard, R.; Lugtenburg, J .; Frank,
H. A. Photosynth. Res. 1994, 42, 157.

[10,20-13C2]- and [10-CH3,13-13C2]-10-Methylretinal
J . Org. Chem., Vol. 66, No. 4, 2001 1277
layers were separated in a separation funnel. The aqueous
layer was extracted three times with diethyl ether and
subsequently the organic layers were combined and washed
with a saturated sodium chloride solution. The organic layer
was dried over magnesium sulfate and after filtration of the
solids, the crude product was concentrated in vacuo. The
product was purified using silica gel column chromatography,
using diethyl ether/petroleum ether as eluent.
2-iodo-3-methyl-2-butenenitrile (89% yield), 2-methyl-2-
butenenitrile no pure compound was isolated), 2-thio-
methyl-2-butenenitrile (28% yield), and 2-iodo-2-butene-
nitrile (14% yield). To our knowledge, 3-methyl-2-thio-
methyl-2-butenenitrile, 2-iodo-3-methyl-2-butenenitrile,
2-thiomethyl-2-butenenitrile, and 2-iodo-2-butenenitrile
are novel compounds.
Gen er a l P r oced u r e for th e P h osp h on a te Cou p lin g of
a n Ald eh yd e (fr om n ow on r efer r ed to a s p r oced u r e 2).
A three-necked round-bottomed flask of suitable size was
equipped with a Teflon magnetic stirrer, a pentane thermom-
eter, and a dropping funnel. The complete setup was flame-
dried using a Bunsen burner, while continuously flushed with
dry nitrogen, using two needles through septa as inlet and
outlet for the gas stream. The nitrogen flow was continued
during the complete reaction until aqueous workup. The flask
was loaded with a mixture of 1.5 equiv of the phosphono
compound and approximately 100 mL of dry THF. The solution
was cooled to -80 °C, and via a syringe, 1.55 equiv of 1.6 M
butyllithium in hexanes was slowly added. The mixture was
stirred for 20 min at -80 °C, and via the dropping funnel, 1.0
equivalent of aldehyde in 15 mL of dry THF was added. The
mixture was allowed to warm to room temperature and stirred
for 2 h. To quench anions, a saturated ammonium chloride
solution was added, and the aqueous and organic layers were
separated in a separation funnel. The aqueous layer was
extracted three times with diethyl ether, and subsequently the
organic layers were combined and washed with a saturated
sodium chloride solution. The organic layer was dried over
magnesium sulfate, and after filtration of the solids, the crude
product was concentrated in vacuo. Removal of excess phospho-
no compound was effected with a silica gel flash column using
diethyl ether/petroleum ether (1:1 v/v) as eluent. The eluate
was subsequently concentrated in vacuo.
Gen er a l P r oced u r e for th e DIBALH Red u ction of a
Nitr ile (fr om n ow on r efer r ed to a s p r oced u r e 3). A three-
necked round-bottomed flask of suitable size was equipped
with a Teflon magnetic stirrer and a pentane thermometer.
The complete setup was flame-dried using a Bunsen burner,
while continuously flushing with dry nitrogen, using two
needles through septa as inlet and outlet for the gas stream.
The nitrogen flow was continued during the complete reaction
until aqueous workup. The flask was loaded with a mixture
of 1.0 equiv of nitrile and approximately 100 mL of dry
petroleum ether. The solution was cooled to -50 °C. Via a
syringe, 2.5 equiv of 1.0 M DIBALH in hexanes was slowly
added. The mixture was stirred for 20 min at -50 °C.
Subsequently 1.75 g of silica gel slurry (100 g of SiO2 and 37.5
mL of distilled water), for each milliliter of DIBALH used, was
added. The thick slurry was stirred at room temperature for
2 h. To absorb the water content, magnesium sulfate was
added. The solids were filtered off and thoroughly rinsed with
diethyl ether. The filtrate was concentrated in vacuo, and the
product was purified using silica gel column chromatography,
using diethyl ether/petroleum ether (1:4 v/v) as eluent.
Gen er a l P r oced u r e for th e Sep a r a tion of a Isom er
Mixtu r e of Retin oid s Usin g HP LC. Typically, 1-2 mL of
the stock solution of the mixture of isomers in n-pentane (∼10
mg/mL) was injected on the silica gel column and separated
using diethyl ether/petroleum ether (1:4 v/v) as mobile phase.
The working pressure was approximately 60 bar and the flow
rate 15 mL/min. The eluted isomers were detected using a
UV-vis detector operating at 360 nm. The all-E isomer is
always the fraction with the longest retention time. The pure
isomers were collected in a round-bottomed flask, that was
packed in aluminum foil to prevent isomerization. Subse-
quently, the isomers were spectroscopically characterized vide
infra. The isomers were concentrated in vacuo in the dark and
stored in n-pentane at -80 °C.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es. All commercially
available chemicals were purchased at either Acros, Aldrich,
Fluka, or Merck and were used without further purification.
The 13C-labeled compounds were purchased at Cambridge
Isotope Laboratories (MA). In all cases, the chemically pure
or higher quality of the chemicals was used. Petroleum ether
refers to the distillate with boiling range 40-60 °C. Dry
solvents and reagents were stored under a dry argon atmos-
phere. Dry argon and nitrogen was prepared by leading the
gas through
a column of sodium hydroxide pellets. Dry
tetrahydrofuran (THF) was prepared by storing THF one week
on 4 Å molecular sieves. Dry petroleum ether was prepared
by distilling from phosphorus pentoxide and was stored on
sodium wire. Dry diisopropylamine, dichloromethane, and
chlorotrimethylsilane were prepared by distilling from freshly
grinded calcium hydride and stored on 4 Å molecular sieves.
Reactions were followed with thin-layer chromatography using
Merck silica gel (60 F254). Spots were detected with UV-light
(254 nm) or by spraying the chromatogram with a coloring
spray (2 g of potassium permanganate and 4 g of sodium
hydrogen carbonate in 100 mL of water). Silica gel column
chromatography was performed using Merck silica gel 60
(230-400 Mesh). HPLC purifications were performed using a
preparative Zorbax silica gel column 21.2 mm × 25 cm (Du
Pont, Delaware). 1H NMR spectra were recorded at 199.50 and
300.13 MHz, respectively, and were internally referenced to
the protons of tetramethylsilane that resonate at δ ) 0 ppm.
1
13C NMR spectra were recorded using H decoupling at 50.10
and 75.48 MHz, respectively, and were internally referenced
to the carbon of deuteriochloroform which resonates at δ )
77.00 ppm. Mass spectra were recorded on a Finnigan MAT
900, equipped with a programmable direct insertion probe
(DIP). Temperature was raised from 30 to 300 °C in 5 min.
Exact mass determination was performed using the peak
matching procedure with perfluorokerosine as internal cali-
bration compound. UV-vis spectra were recorded on a Varian
DMS 200 spectrophotometer. All manipulations with isomeri-
cally pure retinoids were performed in dim red light (λ > 620
nm) or in the dark. All compounds are named according to
the IUPAC convention on organic nomenclature. Compounds
comprising the retinoid structure are named according to the
IUPAC convention on carotenoid nomenclature.
Gen er a l P r oced u r e for Con ver sion of 2-Bu ten en itr iles
in to 2-Su bstitu ted Der iva tives via An ion F or m a tion a n d
Tr ea tm en t w ith Electr op h iles (fr om n ow on r efer r ed to
a s p r oced u r e 1). A three-necked round-bottomed flask of
suitable size, was equipped with a Teflon magnetic stirrer, a
pentane thermometer, and a dropping funnel. The complete
setup was flame-dried using a Bunsen burner, while continu-
ously flushed with dry nitrogen, using two needles through
septa as inlet and outlet for the gas stream. The nitrogen flow
was continued during the complete reaction until aqueous
workup. The flask was loaded with a mixture of 1.55 equiv of
dry diisopropylamine and dry THF. The solution was cooled
to -85 °C, and via a syringe, 1.5 equiv of 1.6 M butyllithium
in hexanes were added and the mixture was stirred for 15 min.
At this temperature, 1.0 equiv of the 3-methyl-2-butenenitrile
in 20 mL of dry THF was added via the dropping funnel. The
mixture was stirred for 20 min and while still at -85 °C 1.5
equiv of electrophile was added. The temperature was main-
tained at -85 °C until the starting compound had disappeared
according to TLC. Subsequently, a saturated solution of
ammonium chloride was added. The mixture was allowed to
warm to room temperature, and the aqueous and organic
10-Meth ylr etin a l (2). 1.41 g (4.77 mmol) of 3,6,7-trimethyl-
9-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4,6,8-nonatetraeneni-
trile (12) was reacted with DIBALH following procedure 3. The
product was purified using silica gel column chromatography
with diethyl ether/petroleum ether (1:9 v/v) as eluent. Yield

1278 J . Org. Chem., Vol. 66, No. 4, 2001
Verdegem et al.
1.12 g (79%) as a yellow oil. all-E-2:1H NMR (300.1 MHz,
CDCl3): δ 10.13 (d, 1H, J ) 8.2 Hz); 7.42 (d, 1H, J ) 15.5 Hz);
6.68 (d, 1H, J ) 16.0 Hz); 6.44 (d, 1H, J ) 15.5 Hz); 6.39 (d,
1H, J ) 16.0 Hz); 6.03 (d, 1H, J ) 8.2 Hz); 2.36 (s, 3H); 2.09
(s, 3H); 2.04 (t, 2H, J ) 6.0 Hz); 1.99 (s, 3H); 1.75 (s, 3H); 1.63
(m, 2H); 1.49 (m, 2H); 1.05 (s, 6H) ppm. 13C NMR (75.5 MHz,
1H-noise-decoupled, CDCl3): δ 190.99; 155.37; 138.24; 137.10;
135.43; 132.64; 130.55; 130.44; 130.12; 129.48; 129.02; 39.49;
34.16; 33.07; 28.94; 21.81; 19.12; 14.16; 13.83; 13.17 ppm. UV-
vis (n-hexane) λmax 371 nm.
times with diethyl ether, and subsequently the organic layers
were combined and washed with a saturated sodium chloride
solution. The organic layer was dried over magnesium sulfate,
and after filtration of the solids, the crude product was
concentrated in vacuo. Removal of excess phosphono compound
was effected with a silica gel flash column using diethyl ether/
petroleum ether (1:1 v/v) as eluent. The eluate was subse-
quently concentrated in vacuo. Yield 2.48 g (96%) as a light
yellow oil. all-E-3a : 1H NMR (199.5 MHz, CDCl3): δ 6.57 (d,
1H, J ) 16.1 Hz); 6.15 (dd, 1H, J ) 16.1 Hz, J ) 5.5 Hz); 5.16
(d, 1H, J ) 172.0 Hz); 2.20 (d, 3H, J ) 5.8 Hz); 2.05 (t, 2H, J
) 6.1 Hz); 1.76 (s, 3H); 1.63 (m, 2H); 1.47 (m, 2H); 1.03 (s,
6H) ppm. 13C NMR (50.0 MHz, 1H-noise-decoupled, CDCl3): δ
95.94 ppm.
2,3-Dim et h yl-5-(2′,6′,6′-t r im et h yl-1′-cycloh exen -1′-yl)-
2,4-p en ta d ien en itr ile (8). 0.70 g (3.2 mmol) of all-E-3-
methyl-5-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4-pentadi-
enenitrile (3) was reacted with iodomethane as electrophile
following procedure 1. The product was purified using silica
gel column chromatography with diethyl ether/petroleum ether
(1:4 v/v) as eluent. Yield 0.72 g (96%) as a light yellow oil. all-
E-8: 1H NMR (300.1 MHz, CDCl3): δ 6.52 (d, 1H, J ) 16.0
Hz); 6.42 (d, 1H, J ) 16.0 Hz); 2.20 (s, 3H); 2.04 (t, 2H, J )
6.0 Hz); 1.98 (s, 3H); 1.72 (s, 3H); 1.63 (m, 2H); 1.47 (m, 2H);
1.03 (s, 6H) ppm. 13C NMR (75.5 MHz, 1H-noise-decoupled,
CDCl3): δ 148.21; 137.17; 134.51; 131.64; 128.27; 120.65;
103.92; 39.30; 33.94; 32.93; 28.71; 21.55; 18.88; 18.07; 15.46
ppm.
[10,20-13C2]-10-Meth ylr etin a l (2a ). 130 mg (0.44 mmol)
of [(3-CH3),6-13C2]-3,6,7-trimethyl-9-(2′,6′,6′-trimethyl-1′-cyclo-
hexen-1′-yl)-2,4,6,8-nonatetraenenitrile (12a ) was reacted with
DIBALH following procedure 2. The product was purified using
silica gel column chromatography with diethyl ether/petroleum
ether (1:9 v/v) as eluent. Yield 115 mg (88%) as a yellow oil.
all-E-2a : 1H NMR (300.1 MHz, CDCl3): δ 10.13 (d, 1H, J )
8.2 Hz); 7.42 (d, 1H, J ) 15.5 Hz); 6.68 (dd, 1H, J ) 16.0 Hz,
J ) 3.6 Hz); 6.44 (ddd, 1H, J ) 15.5 Hz, J ) 5.1 Hz, J ) 5.1
Hz); 6.39 (d, 1H, J ) 16.0 Hz); 6.03 (dd, 1H, J ) 8.2 Hz, J )
7.8 Hz); 2.36 (d, 3H, J ) 127.7 Hz); 2.09 (d, 3H, J ) 6.1 Hz);
2.04 (t, 2H, J ) 6.0 Hz); 1.99 (d, 3H, J ) 4.9 Hz, 10-CH3); 1.75
(s, 3H); 1.63 (m, 2H); 1.49 (m, 2H); 1.05 (s, 6H) ppm. 13C NMR
(75.5 MHz, 1H-noise-decoupled, CDCl3): δ 129.53; 13.23 ppm.
12
1
MS (EI, 70 eV):
C
13C2 H3016O1; experimental mass 300.23682;
19
theoretical mass 300.23638; m/z 300 (M+•).
[(10-CH3),13-13C2]-10-Met h ylr et in a l (2b ). 260 mg (0.88
mmol) of [3,(6-CH3)-13C2]-3,6,7-trimethyl-9-(2′,6′,6′-trimethyl-
1′-cyclohexen-1′-yl)-2,4,6,8-nonatetraenenitrile (12b) was re-
acted with DIBALH following procedure 2. The product was
purified using silica gel column chromatography with diethyl
ether/petroleum ether (1:9 v/v) as eluent. Yield 230 mg (88%)
as a yellow oil. all-E-2b: 1H NMR (300.1 MHz, CDCl3): δ 10.13
(d, 1H, J ) 8.2 Hz); 7.42 (ddd, 1H, J ) 15.5 Hz, J ) 5.6 Hz, J
) 5.6 Hz); 6.68 (d, 1H, J ) 16.0 Hz); 6.44 (dd, 1H, J ) 15.5
Hz, J ) 2.3 Hz); 6.39 (d, 1H, J ) 16.0 Hz); 6.03 (d, 1H, J ) 8.2
Hz); 2.36 (d, 3H, J ) 6.0 Hz); 2.09 (s, 3H); 2.04 (t, 2H, J ) 6.0
Hz); 1.99 (d, 3H, J ) 126.0 Hz, 10-CH3); 1.75 (s, 3H); 1.63 (m,
[2-13C]-2,3-Dim eth yl-5-(2′,6′,6′-tr im eth yl-1′-cycloh exen -
1′-yl)-2,4-p en ta d ien en itr ile (8a ). 2.95 g (13.6 mmol) of
[2-13C]-2E,4E-3-methyl-5-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-
2,4-pentadienenitrile (3a ) was reacted with iodomethane as
electrophile following procedure 1. The product was purified
using silica gel column chromatography with diethyl ether/
petroleum ether (1:9 v/v) as eluent. Yield 3.08 g (98%) as a
light yellow oil. all-E-8a : 1H NMR (199.5 MHz, CDCl3): δ 6.52
(d, 1H, J ) 16.0 Hz); 6.42 (dd, 1H, J ) 16.0 Hz, J ) 3.1 Hz);
2.20 (d, 3H, J ) 6.5 Hz); 2.04 (t, 2H, J ) 6.0 Hz); 1.98 (d, 3H,
J ) 2.5 Hz); 1.72 (s, 3H); 1.63 (m, 2H); 1.47 (m, 2H); 1.03 (s,
6H) ppm. 13C NMR (50.0 MHz, 1H-noise-decoupled, CDCl3): δ
103.42 ppm.
1
2H); 1.49 (m, 2H); 1.05 (s, 6H) ppm. 13C NMR (75.5 MHz, H-
noise-decoupled, CDCl3): δ 155.47; 13.90 (10-CH3) ppm. MS
12
1
(EI, 70 eV):
C
13C2 H3016O1; experimental mass 300.23835;
19
[(2-CH3)-13C]-2,3-Dim eth yl-5-(2′,6′,6′-tr im eth yl-1′-cyclo-
h exen -1′-yl)-2,4-p en ta d ien en itr ile (8b). 3.54 g (16.4 mmol)
of 3-methyl-5-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4-penta-
dienenitrile (3) was reacted with [13C]-iodomethane as elec-
trophile following procedure 1. The product was purified using
silica gel column chromatography with diethyl ether/petroleum
ether (1:9 v/v) as eluent. Yield 3.60 g (95%) as a light yellow
oil. all-E-8b: 1H NMR (300.1 MHz, CDCl3): δ 6.52 (d, 1H, J
) 16.0 Hz); 6.42 (d, 1H, J ) 16.0 Hz); 2.20 (s, 3H); 2.04 (t, 2H,
J ) 6.0 Hz); 1.98 (d, 3H, J ) 133.4 Hz); 1.72 (s, 3H); 1.63 (m,
theoretical mass 300.23638; m/z 300 (M+•).
3-Met h yl-5-(2′,6′,6′-t r im et h yl-1′-cycloh exen -1′-yl)-2,4-
p en ta d ien en itr ile (3). 17.0 g (89 mmol) of â-ionone (7) was
reacted with 2-(diethylphosphono)acetonitrile as phosphonate
following procedure 2. Yield 15.0 g (79%) as a light yellow oil.
all-E-3: 1H NMR (199.5 MHz, CDCl3): δ 6.57 (d, 1H, J ) 16.1
Hz); 6.15 (d, 1H, J ) 16.1 Hz); 5.16 (s, 1H); 2.20 (s, 3H); 2.05
(t, 2H, J ) 6.1 Hz); 1.76 (s, 3H); 1.63 (m, 2H); 1.47 (m, 2H);
1.03 (s, 6H) ppm. The synthesis and characterization of this
compound has been previously described in the literature.49
[2-13C]-3-Met h yl-5-(2′,6′,6′-t r im et h yl-1′-cycloh exen -1′-
yl)-2,4-p en ta d ien en itr ile (3a ). A dry reaction setup was
loaded with a mixture of 3.50 mL (25.0 mmol) of dry lithium
diisopropylamide and 100 mL of dry THF. The solution was
cooled to -80 °C using a cooling bath with a mixture of ethanol
and liquid nitrogen. Via a syringe, 15.2 mL (24.3 mmol) of 1.6
M butyllithium in hexanes was slowly added. The mixture was
stirred for 20 min at -80 °C, and via the dropping funnel, 0.50
g of (11.9 mmol) [2-13C]-acetonitrile in 15 mL dry THF was
added. The mixture was stirred for 20 min at -80 °C, and
subsequently 1.80 mL (12.5 mmol) diethyl chlorophosphate in
15 mL of dry THF was added via the dropping funnel. The
mixture was allowed to warm to room temperature and stirred
for 1 h. Via the dropping funnel, 2.51 g (13.7 mmol) of â-ionone
(7) in 25 mL of dry THF was added. After 2 h stirring at room
temperature, a saturated ammonium chloride solution was
added, and the aqueous and organic layers were separated in
a separation funnel. The aqueous layer was extracted three
1
2H); 1.47 (m, 2H); 1.03 (s, 6H) ppm. 13C NMR (75.4 MHz, H-
noise-decoupled, CDCl3): δ 15.47 ppm.
2,3-Dim et h yl-5-(2′,6′,6′-t r im et h yl-1′-cycloh exen -1′-yl)-
2,4-p en ta d ien a l (9). 6.0 g (26 mmol) of all-E-2,3-dimethyl-
5-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4-pentadienenitrile
(8) was reacted with DIBALH following procedure 3. The
product was purified using silica gel column chromatography
with diethyl ether/petroleum ether (1:4 v/v) as eluent. Yield
5.6 g (92%) as a light yellow oil. all-E-9: 1H NMR (300.1 MHz,
CDCl3): δ 10.27 (s, 1H); 6.76 (d, 1H, J ) 16.1 Hz); 6.68 (d,
1H, J ) 16.1 Hz); 2.33 (s, 3H); 2.06 (t, 2H, J ) 6.0 Hz); 1.88
(s, 3H); 1.77 (s, 3H); 1.63 (m, 2H); 1.49 (m, 2H); 1.07 (s, 6H)
ppm. 13C NMR (75.5 MHz, 1H-noise-decoupled, CDCl3):
δ
191.57;0.149.43; 137.49; 134.55; 132.11; 132.00; 131.97; 39.34;
33.96; 33.00; 28.73; 21.58; 18.85; 12.38; 10.39 ppm.
[2-13C]-2,3-Dim eth yl-5-(2′,6′,6′-tr im eth yl-1′-cycloh exen -
1′-yl)-2,4-p en ta d ien a l (9a ). 3.60 g (15.6 mmol) of 2E/Z,4E-
[2-13C]-2,3-dimethyl-5-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-
2,4-pentadienenitrile (8a ) was reacted with DIBALH following
procedure 3. The product was purified using silica gel column
chromatography with diethyl ether/petroleum ether (1:4 v/v)
as eluent. Yield 2.00 g (55%) as a light yellow oil. all-E-9a :
1H NMR (199.5 MHz, CDCl3): δ 10.27 (d, 1H, J ) 23.0 Hz);
6.76 (d, 1H, J ) 16.1 Hz); 6.68 (dd, 1H, J ) 16.1 Hz, J ) 2.4
(49) Pardoen, J . A.; Neijenhesch, H. N.; Mulder, P. P. J .; Lugtenburg,
J . Recl. Trav. Chim. Pays-Bas 1983, 102, 341.
(50) IUPAC Commission on Nomenclature of Organic Chemistry and
IUPAC IUB Commission on Biochemical Nomenclature. Pure Appl.
Chem. 1975, 41, 407.

[10,20-13C2]- and [10-CH3,13-13C2]-10-Methylretinal
J . Org. Chem., Vol. 66, No. 4, 2001 1279
Hz); 2.33 (d, 3H, J ) 4.1 Hz); 2.06 (t, 2H, J ) 6.0 Hz); 1.88 (d,
3H, J ) 6.2 Hz); 1.77 (s, 3H); 1.63 (m, 2H); 1.49 (m, 2H); 1.07
(s, 6H) ppm. 13C NMR (50.0 MHz, 1H-noise-decoupled,
CDCl3): δ 132.14 ppm.
with diethyl ether/petroleum ether (1:4 v/v) as eluent. The
eluate was subsequently concentrated in vacuo. Yield 1.95 g
(74%) as a yellow oil. all-E-33a : 1H NMR (199.5 MHz,
CDCl3): δ 7.80 (dd, 1H, J ) 14.6 Hz, J ) 2.8 Hz); 6.37 (d, 1H,
J ) 16.1 Hz); 6.15 (d, 1H, J ) 16.1 Hz); 6.07 (d, 1H, J ) 14.6
Hz); 3.70 (s, 3H); 3.26 (s, 3H); 2.05 (s, 3H); 2.05 (s, 3H); 2.03
(t, 2H, J ) 6.1 Hz); 1.71 (s, 3H); 1.61 (m, 2H); 1.62 (m, 2H);
1.03 (s, 6H) ppm. 13C NMR (50.0 MHz, 1H-noise-decoupled,
CDCl3): δ 117.69 ppm.
[(2-CH3)-13C]-2,3-Dim eth yl-5-(2′,6′,6′-tr im eth yl-1′-cyclo-
h exen -1′-yl)-2,4-p en ta d ien a l (9b). 3.08 g (13.4 mmol) of [(2-
CH3)-13C]-2,3-dimethyl-5-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-
2,4-pentadienenitrile (8a ) was reacted with DIBALH following
procedure 3. The product was purified using silica gel column
chromatography with diethyl ether/petroleum ether (1:4 v/v)
as eluent. Yield 2.10 g (67%) as a light yellow oil. all-E-9b:
1H NMR (199.5 MHz, CDCl3): δ 10.27 (d, 1H, J ) 2.7 Hz);
6.76 (d, 1H, J ) 16.1 Hz); 6.68 (d, 1H, J ) 16.1 Hz); 2.33 (s,
3H); 2.06 (t, 2H, J ) 6.0 Hz); 1.88 (d, 3H, J ) 127.7 Hz); 1.77
(s, 3H); 1.63 (m, 2H); 1.49 (m, 2H); 1.07 (s, 6H) ppm. 13C NMR
5,6-Dim et h yl-8-(2′,6′,6′-t r im et h yl-1′-cycloh exen -1′-yl)-
3,5,7-octa tr ien -2-on e (11). A dry reaction setup was loaded
with a mixture of 4.99 g (17.9 mmol) of ethyl 4,5-dimethyl-7-
(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4,6-heptatrienoate (10)
and 30 mL of dry THF. The reaction mixture was cooled to
-100 °C, and via a syringe, 10.3 mL (82.1 mmol) freshly
distilled chlorotrimethylsilane was slowly added. 10.31 mL of
1.0 M methyllithium was added in aliquots of 0.25 equiv. The
mixture was stirred for 2 h. To quench anions, a saturated
ammonium chloride solution was added, and the aqueous and
organic layers were separated in a separation funnel. The
aqueous layer was extracted three times with diethyl ether,
and subsequently the organic layers were combined and
washed with a saturated sodium chloride solution. The organic
layer was dried over magnesium sulfate, and after filtration
of the solids, the crude product was concentrated in vacuo. The
product was purified using silica gel column chromatography
with diethyl ether/petroleum ether (1:4 v/v) as eluent. The
eluate was subsequently concentrated in vacuo. Yield 3.69 g
(80%) as a yellow oil. all-E-11: 1H NMR (199.5 MHz, CDCl3):
δ 7.80 (d, 1H, J ) 15.6 Hz); 6.68 (d, 1H, J ) 16.1 Hz); 6.48 (d,
1H, J ) 16.1 Hz); 6.26 (d, 1H, J ) 15.6 Hz); 2.32 (s, 3H); 2.13
(s, 3H); 2.05 (s, 3H); 2.03 (t, 2H, J ) 6.1 Hz); 1.95 (s, 3H); 1.75
(s, 3H); 1.61 (m, 2H); 1.52 (m, 2H); 1.05 (s, 6H) ppm.
1
(50.0 MHz, H-noise-decoupled, CDCl3): δ 10.48 ppm.
Eth yl 4,5-Dim eth yl-7-(2′,6′,6′-tr im eth yl-1′-cycloh exen -
1′-yl)-2,4,6-h ep ta tr ien oa te (10). 5.60 g (24.8 mmol) of 2,3-
dimethyl-5-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4-pentadi-
enal (9) was reacted with ethyl 2-(diethylphosphono)acetate
as phosphonate following procedure 2. Yield 4.99 g (70%) as a
yellow oil. all-E-10: 1H NMR (199.5 MHz, CDCl3): δ 8.02 (d,
1H, J ) 15.5 Hz); 6.66 (d, 1H, J ) 15.8 Hz); 6.42 (d, 1H, J )
15.8 Hz); 5.93 (d, 1H, J ) 15.8 Hz); 4.23 (q, 2H, J ) 7.2 Hz);
2.11 (s, 3H); 2.04 (t, 2H, J ) 6.1 Hz); 1.94 (s, 3H); 1.74 (s, 3H);
1.61 (m, 2H); 1.49 (m, 2H); 1.31 (t, 3H, J ) 7.2 Hz); 1.04 (s,
6H) ppm.
Eth yl [4-13C]-4,5-Dim eth yl-7-(2′,6′,6′-tr im eth yl-1′-cyclo-
h exen -1′-yl)-2,4,6-h ep ta tr ien oa te (10a ). 2.00 g (8.60 mmol)
of [2-13C]-2,3-dimethyl-5-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-
2,4-pentadienal (9a ) was reacted with ethyl 2-(diethylphospho-
no)acetate as phosphonate following procedure 2. Yield 1.95 g
(74%) as a yellow oil. all-E-10a : 1H NMR (199.5 MHz,
CDCl3): δ 8.02 (dd, 1H, J ) 15.5 Hz, J ) 16.1 Hz); 6.66 (dd,
1H, J ) 15.8 Hz, J ) 3.4 Hz); 6.42 (d, 1H, J ) 15.8 Hz); 5.93
(dd, 1H, J ) 15.8 Hz, J ) 5.1 Hz); 4.23 (q, 2H, J ) 7.2 Hz);
2.11 (d, 3H, J ) 5.8 Hz); 2.04 (t, 2H, J ) 6.1 Hz); 1.94 (d, 3H,
J ) 6.2 Hz); 1.74 (s, 3H); 1.61 (m, 2H); 1.49 (m, 2H); 1.31 (t,
[1,5-13C2]-5,6-Dim eth yl-8-(2′,6′,6′-tr im eth yl-1′-cycloh exen -
1′-yl)-3,5,7-octa tr ien -2-on e (11a ). A dry reaction setup was
loaded with a mixture of 73 mg (3.0 mmol) of magnesium
pallets 30 mL of dry diethyl ether. Via a syringe, 0.51 mL (3.5
mmol) of [13C]-iodomethane was slowly added. During the
addition of the iodomethane, the nitrogen flow was stopped to
prevent evaporation of reactant. The mixture was slowly
stirred while the diethyl ether started boiling, indicating the
formation of the Grignard reagent. After 20 min, when all solid
magnesium had reacted, 0.90 g (2.8 mmol) of [4-13C]-N-
methoxy-N,4,5-trimethyl-7-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-
yl)-2,4,6-heptatrienamide (33a ) in 15 mL of dry THF was
added via the dropping funnel. The mixture was stirred for 2
h. To quench anions, a saturated ammonium chloride solution
was added, and the aqueous and organic layers were separated
in a separation funnel. The aqueous layer was extracted three
times with diethyl ether, and subsequently the organic layers
were combined and washed with a saturated sodium chloride
solution. The organic layer was dried over magnesium sulfate,
and after filtration of the solids, the crude product was
concentrated in vacuo. The product was purified using silica
gel column chromatography with diethyl ether/petroleum ether
(1:4 v/v) as eluent. The eluate was subsequently concentrated
in vacuo. Yield 130 mg (17%) as a yellow oil. all-E-11a : 1H
NMR (199.5 MHz, CDCl3): δ 7.80 (dd, 1H, J ) 15.5 Hz, J )
1.8 Hz); 6.68 (dd, 1H, J ) 16.1 Hz, J ) 3.5 Hz); 6.48 (d, 1H, J
) 16.1 Hz); 6.26 (dd, 1H, J ) 15.5 Hz, J ) 4.9 Hz); 2.32 (d,
3H, J ) 127.0 Hz); 2.13 (d, 3H, J ) 4.8 Hz); 2.05 (s, 3H); 2.03
(t, 2H, J ) 6.1 Hz); 1.95 (d, 3H, J ) 5.9 Hz); 1.75 (s, 3H); 1.61
(m, 2H); 1.52 (m, 2H); 1.05 (s, 6H) ppm. 13C NMR (50.0 MHz,
1H-noise-decoupled, CDCl3): δ 128.47; 27.82 ppm.
1
3H, J ) 7.2 Hz); 1.04 (s, 6H) ppm. 13C NMR (50.0 MHz, H-
noise-decoupled, CDCl3): δ 128.17 ppm.
Eth yl [1,4-CH3-13C2]-4,5-Dim eth yl-7-(2′,6′,6′-tr im eth yl-
1′-cycloh exen -1′-yl)-2,4,6-h ep ta tr ien oa te (10b). 2.10 g (9.0
mmol) of [(2-CH3)-13C]-2,3-dimethyl-5-(2′,6′,6′-trimethyl-1′-cy-
clohexen-1′-yl)-2,4-pentadienal (9b) was reacted with ethyl
[1-13C]-2-(diethylphosphono)acetate as phosphonate following
procedure 2. To prevent loss of 13C label, only 1.1 equiv of
phosphonate and 1.15 equiv of butyllithium were used. Yield
1.27 g (46%) as a yellow oil. all-E-10b: 1H NMR (199.5 MHz,
CDCl3): δ 8.02 (ddd, 1H, J ) 15.5 Hz, J ) 5.7 Hz, J ) 5.7
Hz); 6.66 (d, 1H, J ) 15.8 Hz); 6.42 (d, 1H, J ) 15.8 Hz); 5.93
(dd, 1H, J ) 15.5 Hz, J ) 2.4 Hz); 4.23 (q, 2H, J ) 7.2 Hz);
2.11 (s, 3H); 2.04 (t, 2H, J ) 6.1 Hz); 1.94 (d, 3H, J ) 126.7
Hz); 1.74 (s, 3H); 1.61 (m, 2H); 1.49 (m, 2H); 1.31 (t, 3H, J )
7.2 Hz); 1.04 (s, 6H) ppm. 13C NMR (50.0 MHz, 1H-noise-
decoupled, CDCl3): δ 167.63; 13.72 ppm.
[4-13C]-N-Meth oxy-N,4,5-tr im eth yl-7-(2′,6′,6′-tr im eth yl-
1′-cycloh exen -1′-yl)-2,4,6-h ep ta tr ien a m id e (33a ). A dry
reaction setup was loaded with a mixture of 0.51 g (8.4 mmol)
of N-methoxymethylamine and 100 mL of dry THF. The
solution was cooled to -15 °C using an ice/salt cooling bath.
Via a syringe, 5.22 mL (8.4 mmol) of 1.6 M butyllithium in
hexanes was slowly added. The mixture was stirred for 20 min
at -15 °C, and via the dropping funnel, 1.95 g (6.4 mmol) of
ethyl [4-13C]-4,5-dimethyl-7-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-
yl)-2,4,6-heptatrienoate (10a ) in 15 mL of dry THF was added.
The mixture was allowed to warm to room temperature and
stirred for 2 h. To quench anions, a saturated ammonium
chloride solution was added, and the aqueous and organic
layers were separated in a separation funnel. The aqueous
layer was extracted three times with diethyl ether, and
subsequently the organic layers were combined and washed
with a saturated sodium chloride solution. The organic layer
was dried over magnesium sulfate, and after filtration of the
solids, the crude product was concentrated in vacuo. The
product was purified using silica gel column chromatography
[2,(5-CH3)-13C2]-5,6-Dim eth yl-8-(2′,6′,6′-tr im eth yl-1′-cy-
cloh exen -1′-yl)-3,5,7-octatr ien -2-on e (11b). 1.27 g (4.2 mmol)
ethyl of [1,(4-CH3)-13C2]-4,5-dimethyl-7-(2′,6′,6′-trimethyl-1′-
cyclohexen-1′-yl)-2,4,6-heptatrienoate (10b), 2.63 mL (20.9
mmol) of freshly distilled chlorotrimethylsilane, and 2.61 mL
of 1.0 M methyllithium were reacted to yield 11b. Yield 260
mg (23%) as a yellow oil. all-E-11b : 1H NMR (199.5 MHz,
CDCl3): δ 7.80 (ddd, 1H, J ) 15.6 Hz, J ) 6.2 Hz, J ) 6.2 Hz);
6.68 (d, 1H, J ) 16.1 Hz); 6.48 (d, 1H, J ) 16.1 Hz); 6.26 (dd,
1H, J ) 15.6 Hz, J ) 2.8 Hz); 2.32 (d, 3H, J ) 5.8 Hz); 2.13 (s,
3H); 2.05 (s, 3H); 2.03 (t, 2H, J ) 6.1 Hz); 1.95 (d, 3H, J )

1280 J . Org. Chem., Vol. 66, No. 4, 2001
Verdegem et al.
126.7 Hz); 1.75 (s, 3H); 1.61 (m, 2H); 1.52 (m, 2H); 1.05 (s,
6H) ppm. 13C NMR (50.0 MHz, 1H-noise-decoupled, CDCl3): δ
198.57; 13.83 ppm.
3. The product was purified using silica gel column chroma-
tography with diethyl ether/petroleum ether (1:9 v/v) as eluent.
Yield 0.36 g (70%) as a light yellow solid. all-E-16: 1H NMR
(300.1 MHz, CDCl3): δ 9.27 (s, 1H); 6.79 (s, higher order AB,
1H); 6.79 (s, higher order AB, 1H); 2.54 (s, 3H); 2.11 (t, 2H, J
) 6.2 Hz); 1.83 (s, 3H); 1.63 (m, 2H); 1.50 (m, 2H); 1.12 (s,
6H) ppm. 13C NMR (75.5 MHz, 1H-noise-decoupled, CDCl3): δ
184.97;0.156.84; 139.27; 138.53; 136.98; 135.64; 108.61; 39.73;
34.02; 33.62; 28.84; 21.95; 18.77; 16.74 ppm.
3,7-Dim eth yl-6-th iom eth yl-9-(2′,6′,6′-tr im eth yl-1′-cyclo-
h exen -1′-yl)-2,4,6,8-n on a tetr a en en itr ile (17). 0.19 g (0.72
mmol) of 3-methyl-2-thiomethyl-5-(2′,6′,6′-trimethyl-1′-cyclo-
hexen-1′-yl)-2,4-pentadienal (15) was reacted with 4-(dieth-
ylphosphono)-3-methyl-2-butenenitrile as phosphonate follow-
ing procedure 2. Yield 0.12 g (51%) as a yellow oil. all-E-17:
1H NMR (300.1 MHz, CDCl3): δ 7.39 (d, 1H, J ) 16.2 Hz);
7.08 (d, 1H, J ) 15.0 Hz); 7.00 (d, 1H, J ) 15.0 Hz); 6.53 (d,
1H, J ) 16.2 Hz); 5.32 (s, 1H); 2.26 (s, 3H); 2.13 (s, 3H); 2.10
(s, 3H); 2.06 (t, 2H, J ) 6.0 Hz); 1.77 (s, 3H); 1.64 (m, 2H);
1.49 (m, 2H); 1.07 (s, 6H) ppm. 13C NMR (75.5 MHz, 1H-noise-
decoupled, CDCl3): δ 156.91; 144.73; 137.80; 133.80; 132.96;
132.21; 131.49; 131.36; 130.80; 118.08; 97.46; 39.65; 34.18;
33.27; 29.83; 21.81; 19.06; 18.48; 16.93; 15.38 ppm.
3,7-Dim eth yl-6-iod o-9-(2′,6′,6′-tr im eth yl-1′-cycloh exen -
1′-yl)-2,4,6,8-n on a tetr a en en itr ile (18). 0.35 g (1.0 mmol) of
2-iodo-3-methyl-5-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4-
pentadienal (16) was reacted with 4-(diethylphosphono)-3-
methyl-2-butenenitrile as phosphonate following procedure 2.
Yield 0.29 g (70%) as a yellow oil. all-E-18: 1H NMR (300.1
MHz, CDCl3): δ 6.86 (d, 1H, J ) 16.0 Hz); 6.72 (s, higher order
AB, 1H); 6.72 (s, higher order AB, 1H); 6.55 (d, 1H, J ) 16.0
Hz); 5.33 (s, 1H); 2.32 (s, 3H); 2.26 (s, 3H); 2.07 (t, 2H, J ) 5.8
Hz); 1.80 (s, 3H); 1.63 (m, 2H); 1.49 (m, 2H); 1.09 (s, 6H) ppm.
13C NMR (75.5 MHz, 1H-noise-decoupled, CDCl3): δ 155.93;
143.68; 140.15; 137.27; 137.18; 133.91; 133.81; 132.83; 117.69;
102.24; 98.38; 39.67; 34.04; 33.39; 28.90; 21.96; 18.94; 17.34;
16.55 ppm.
10-Th iom et h ylr et in a l (19). 100 mg (0.31 mmol) of 3,7-
dimethyl-6-thiomethyl-9-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-
2,4,6,8-nonatetraenenitrile (17) was reacted with DIBALH
following procedure 3. The product was purified using silica
gel column chromatography with diethyl ether/petroleum ether
(1:9 v/v) as eluent. Yield 66 mg (65%) as a yellow oil. The
individual regioisomers were separated using HPLC (see
procedure 4). all-E-19: 1H NMR (300.1 MHz, CDCl3): δ 10.14
(d, 1H, J ) 8.1 Hz); 7.41 (d, 1H, J ) 16.3 Hz); 7.26 (d, 1H, J
) 15.1 Hz); 7.06 (d, 1H, J ) 15.1 Hz); 6.54 (d, 1H, J ) 16.3
Hz); 6.10 (d, 1H, J ) 8.1 Hz); 2.38 (2, 3H); 2.15 (s, 2H); 2.12
(s, 3H); 2.07 (t, 2H, J ) 6.2 Hz); 1.78 (s, 3H); 1.64 (m, 2H);
1.49 (m, 2H); 1.08 (s, 6H) ppm. 13C NMR (75.5 MHz, 1H-noise-
decoupled, CDCl3): δ 191.15; 154.72; 144.62; 137.90; 134.59;
133.95; 132.81; 132.26; 131.59; 131.44; 129.79; 39.71; 34.25;
33.34; 29.00; 21.88; 19.12; 18.53; 15.49; 13.52 ppm.
3,6,7-Tr im et h yl-9-(2′,6′,6′-t r im et h yl-1′-cycloh exen -1′-
yl)-2,4,6,8-n on a tetr a en en itr ile (12). 1.0 g (4.3 mmol) of 5,6-
dimethyl-8-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-3,5,7-octatrien-
2-one (11) was reacted with diethyl cyanomethylphosphonate
as phosphonate following procedure 2. Yield 1.2 g (95%) as a
light yellow oil. all-E-12: 1H NMR (199.5 MHz, CDCl3): δ 7.22
(d, 1H, J ) 15.8 Hz); 6.67 (d, 1H, J ) 16.1 Hz); 6.37 (d, 1H, J
) 15.8 Hz); 6.30 (d, 1H, J ) 16.1 Hz); 5.22 (s, 1H); 2.23 (s,
3H); 2.06 (s, 3H); 1.95 (s, 3H); 2.02 (t, 2H, J ) 6.0 Hz); 1.75 (s,
3H); 1.62 (m, 2H); 1.48 (m, 2H, H′5); 1.06 (s, 6H) ppm. 13C
NMR (75.5 MHz, 1H-noise-decoupled, CDCl3): δ 157.04; 138.33;
137.10; 135.20; 132.29; 130.09; 130.01; 129.07; 127.21; 118.21;
96.48; 39.65; 34.27; 33.19; 29.05; 21.92; 19.29; 16.72; 14.24;
13.89 ppm.
[(3-CH3),6-13C2]-3,6,7-Tr im eth yl-9-(2′,6′,6′-tr im eth yl-1′-
cycloh exen -1′-yl)-2,4,6,8-n on a tetr a en en itr ile (12a ). 130
mg (0.47 mmol) of [1,5-13C2]-5,6-dimethyl-8-(2′,6′,6′-trimethyl-
1′-cyclohexen-1′-yl)-3,5,7-octatrien-2-one (11a ) was reacted
with 2-(diethylphosphono)acetonitrile as phosphonate follow-
ing procedure 1. Yield 100 mg (72%) as a yellow oil.
[(3,(6-CH3)-13C2]-3,6,7-Tr im eth yl-9-(2′,6′,6′-tr im eth yl-1′-
cycloh exen -1′-yl)-2,4,6,8-n on a tetr a en en itr ile (12b). 260
mg (0.95 mmol) of [2,(5-CH3)-13C2]-5,6-dimethyl-8-(2′,6′,6′-
trimethyl-1′-cyclohexen-1′-yl)-3,5,7-octatrien-2-one (11b) was
reacted with 2-(diethylphosphono)acetonitrile as phosphonate
following procedure 1. Yield 250 mg (89%) as a yellow oil.
3-Meth yl-2-th iom eth yl-5-(2′,6′,6′-tr im eth yl-1′-cycloh exen -
1′-yl)-2,4-pen tadien en itr ile (13). 0.70 g (3.3 mmol) of 3-meth-
yl-5-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4-pentadieneni-
trile (3) was reacted with thiocyanomethane as electrophile
following procedure 1. The product was purified using silica
gel column chromatography with diethyl ether/petroleum ether
(1:9 v/v) as eluent. Yield 0.83 g (97%) as a light yellow oil. all-
E-13: 1H NMR (300.1 MHz, CDCl3): δ 6.90 (d, 1H, J ) 16.1
Hz); 6.63 (d, 1H, J ) 16.1 Hz); 2.41 (s, 3H); 2.27 (s, 3H); 2.04
(t, 2H, J ) 6.0 Hz); 1.76 (s, 3H); 1.63 (m, 2H); 1.48 (m, 2H);
1.06 (s, 6H) ppm. 13C NMR (75.5 MHz, 1H-noise-decoupled,
CDCl3): δ 151.32; 136.93; 136.48; 133.35; 128.68; 116.17;
104.88; 39.54; 33.97; 33.27; 28.76; 21.64; 18.85; 18.85; 16.96
ppm.
2-Iod o-3-m et h yl-5-(2′,6′,6′-t r im et h yl-1′-cycloh exen -1′-
yl)-2,4-p en ta d ien en itr ile (14). 2.50 g (11.6 mmol) of 3-meth-
yl-5-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4-pentadieneni-
trile (3) was reacted with a solution of iodine in THF as
electrophile following procedure 1. The product was purified
using silica gel column chromatography with diethyl ether/
petroleum ether (1:19 v/v) as eluent. Yield 3.58 g (90%) as a
light yellow solid. all-E-14: 1H NMR (300.1 MHz, CDCl3): δ
6.80 (d, 1H, J ) 15.8 Hz); 6.70 (d, 1H, J ) 15.8 Hz); 2.19 (s,
3H); 2.03 (t, 2H, J ) 6.2 Hz); 1.74 (s, 3H); 1.62 (m, 2H); 1.48
(m, 2H); 1.04 (s, 6H) ppm. 13C NMR (75.5 MHz, 1H-noise-
decoupled, CDCl3): δ 159.34; 136.60; 136.60; 133.57; 129.26;
117.87; 56.61; 39.39; 34.00; 33.25; 28.85; 22.65; 21.84; 18.88
ppm.
10-Iod or etin a l (20). 233 mg (0.57 mmol) of 3,7-dimethyl-
6-iodo-9-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4,6,8-nonatet-
raenenitrile (18) was reacted with DIBALH following proce-
dure 3. The product was purified using silica gel column
chromatography with diethyl ether/petroleum ether (1:9 v/v)
as eluent. Yield 124 mg (53%) as a yellow oil. The individual
regioisomers were separated using HPLC (see procedure 4).
all-E-20: 1H NMR (300.1 MHz, CDCl3): δ 10.14 (d, 1H, J )
8.0 Hz); 6.88 (d, 1H, J ) 16.0 Hz); 6.79 (d, 1H, J ) 14.5 Hz);
6.56 (d, 1H, J ) 16.0 Hz); 6.19 (d, 1H, J ) 14.5 Hz); 6.10 (d,
1H, J ) 8.0 Hz); 2.38 (s, 3H); 2.25 (s, 3H); 2.08 (t, 2H, J ) 6.0
Hz); 1.81 (s, 3H); 1.64 (m, 2H); 1.49 (m, 2H); 1.09 (s, 6H) ppm.
13C NMR (75.5 MHz, 1H-noise-decoupled, CDCl3): δ 191.00;
153.63; 143.59; 140.47; 140.41; 137.43; 134.01; 133.62; 132.78;
130.32; 102.98; 39.78; 34.17; 33.50; 29.01; 22.07; 19.04; 16.70;
14.02 ppm.
3-Meth yl-2-th iom eth yl-5-(2′,6′,6′-tr im eth yl-1′-cycloh exen -
1′-yl)-2,4-p en ta d ien a l (15). 0.50 g (1.9 mmol) of 3-methyl-
2-thiomethyl-5-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4-pen-
tadienenitrile (13) was reacted with DIBALH following
procedure 3. The product was purified using silica gel column
chromatography with diethyl ether/petroleum ether (1:4 v/v)
as eluent. Yield 0.17 g (34%) as a light yellow oil. all-E-15:
1H NMR (300.1 MHz, CDCl3): δ 10.15 (s, 1H); 7.40 (d, 1H, J
) 16.0 Hz); 6.89 (d, 1H, J ) 16.0 Hz); 2.41 (s, 3H); 2.21 (s,
3H); 2.09 (t, 2H, J ) 6.2 Hz); 1.80 (s, 3H); 1.65 (m, 2H); 1.50
(m, 2H); 1.10 (s, 6H) ppm. 13C NMR (75.5 MHz, 1H-noise-
decoupled, CDCl3): δ 188.56;0.156.11; 137.37; 136.63; 133.98;
133.08; 133.08; 39.64; 34.13; 33.43; 28.87; 21.80; 18.89; 17.65;
14.62 ppm.
2,3,7-Tr im et h yl-9-(2′,6′,6′-t r im et h yl-1′-cycloh exen -1′-
yl)-2,4,6,8-n on a t et r a en en it r ile (21). 0.70 g (2.5 mmol) of
3,7-dimethyl-9-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4,6,8-no-
natetraenenitrile (4) was reacted with iodomethane as electro-
phile following procedure 1. The product was purified using
silica gel column chromatography with diethyl ether/petroleum
2-Iod o-3-m et h yl-5-(2′,6′,6′-t r im et h yl-1′-cycloh exen -1′-
yl)-2,4-p en ta d ien a l (16). 0.52 g (1.5 mmol) of all-E-2-iodo-
3-methyl-5-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4-pentadi-
enenitrile (14) was reacted with DIBALH following procedure

[10,20-13C2]- and [10-CH3,13-13C2]-10-Methylretinal
J . Org. Chem., Vol. 66, No. 4, 2001 1281
ether (1:19 v/v) as eluent. Yield 0.58 g (80%) as a yellow oil.
all-E-21: 1H NMR (300.1 MHz, CDCl3): δ 6.93 (dd, 1H, J )
12.0 Hz, J ) 15.0 Hz); 6.55 (d, 1H, J ) 15.0 Hz); 6.31 (d, 1H,
J ) 15.8 Hz); 6.18 (d, 1H, J ) 12.0 Hz); 6.15 (d, 1H, J ) 15.8
Hz); 2.21 (s, 3H); 2.02 (t, 2H, J ) 6.0 Hz); 2.00 (s, 3H); 1.99 (s,
3H); 1.71 (s, 3H); 1.62 (m, 2H); 1.48 (m, 2H); 1.03 (s, 6H) ppm.
13C NMR (75.5 MHz, 1H-noise-decoupled, CDCl3): δ 147.99;
140.27; 137.33; 136.83; 131.57; 129.73; 129.35; 128.92; 126.74;
104.04; 39.30; 33.96; 32.85; 28.70; 21.50; 18.94; 18.07; 15.46;
12.68 ppm.
raenenitrile (23) was reacted with DIBALH following proce-
dure 3. The product was purified using silica gel column
chromatography with diethyl ether/petroleum ether (1:9 v/v)
as eluent. Yield 63 mg (16%) as a yellow oil. The reaction
mixture contained substantional amounts of retinal, that
originated from dehalogenation reaction (see text for discus-
sion). Major isomer 26: 1H NMR (300.1 MHz, CDCl3): δ 9.26
(s, 1H); 7.00 (d, 1H, J ) 15.0 Hz); 7.35 (dd, 1H, J ) 11.5 Hz,
J ) 15.0 Hz); 6.43 (d, 1H, J ) 16.1 Hz); 6.34 (d, 1H, J ) 11.5
Hz); 6.22 (d, 1H, J ) 16.1 Hz); 2.56 (s, 3H); 2.06 (s, 3H); 2.04
(t, 2H, J ) 6.6 Hz); 1.74 (s, 3H); 1.63 (m, 2H); 1.47 (m, 2H);
1.05 (s, 6H) ppm.
3,7-Dim eth yl-2-th iom eth yl-9-(2′,6′,6′-tr im eth yl-1′-cyclo-
h exen -1′-yl)-2,4,6,8-n on a t et r a en en it r ile (22). 0.65 g (2.3
mmol) of 3,7-dimethyl-9-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-
2,4,6,8-nonatetraenenitrile (4) was reacted with thiocya-
nomethane as electrophile following procedure 1. The product
was purified using silica gel column chromatography with
diethyl ether/petroleum ether (1:19 v/v) as eluent. Yield 0.73
g (96%) as a yellow oil. all-E-22: 1H NMR (300.1 MHz,
CDCl3): δ 7.09 (dd, 1H, J ) 12.1 Hz, J ) 16.9 Hz); 6.90 (d,
1H, J ) 16.9 Hz); 6.34 (d, 1H, J ) 16.0 Hz); 6.20 (d, 1H, J )
12.1 Hz); 6.16 (d, 1H, J ) 16.0 Hz); 2.41 (s, 3H); 2.28 (s, 3H);
2.02 (t, 2H, J ) 6.0 Hz); 2.00 (s, 3H); 1.72 (s, 3H); 1.62 (m,
2,3-Dim eth yl-2-bu ten en itr ile (27). 0.50 g (6.2 mmol) of
3-methyl-2-butenenitrile (5) was reacted with iodomethane as
electrophile following procedure 1. The product was purified
using silica gel column chromatography with diethyl ether/
petroleum ether (1:9 v/v) as eluent. Yield 0.31 g (53%) as a
1
4
yellow oil. H NMR (300.1 MHz, CDCl3): δ 2.06 (q, 3H, J )
1.4 Hz); 1.87 (q, 3H, J ) 1.4 Hz); 1.84 (s, 3H) ppm. 13C NMR
(75.5 MHz, 1H-noise-decoupled, CDCl3): δ 152.02; 119.72;
103.18; 24.19; 19.83; 15.81 ppm. MS (EI, 70 eV): m/z 95 (M+•).
1
2H); 1.48 (m, 2H); 1.03 (s, 6H) ppm. 13C NMR (75.5 MHz, H-
3-Meth yl-2-th iom eth yl-2-bu ten en itr ile (28). 0.45 g (5.6
mmol) of 3-methyl-2-butenenitrile (5) was reacted with me-
thylthiocyanate as electrophile following procedure 1. The
product was purified using silica gel column chromatography
with diethyl ether/petroleum ether (1:9 v/v) as eluent. Yield
noise-decoupled, CDCl3): δ 150.68; 141.17; 137.36; 136.85;
132.64; 130.03; 129.46; 129.41; 127.46; 116.33; 105.09; 39.37;
34.02; 32.94; 28.77; 21.58; 18.98; 18.74; 16.95; 12.79 ppm.
3,7-Dim eth yl-2-iod o-9-(2′,6′,6′-tr im eth yl-1′-cycloh exen -
1′-yl)-2,4,6,8-n on a tetr a en en itr ile (23). 1.17 g (4.2 mmol) of
3,7-dimethyl-9-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4,6,8-
nonatetraenenitrile (4) was reacted with a solution of iodine
in THF as electrophile following procedure 1. The product was
purified using silica gel column chromatography with diethyl
ether/petroleum ether (1:19 v/v) as eluent. Yield 1.19 g (70%)
as a yellow solid. all-E-23: 1H NMR (300.1 MHz, CDCl3): δ
7.11 (dd, 1H, J ) 12.9 Hz, J ) 14.8 Hz); 6.55 (d, 1H, J ) 14.8
Hz); 6.41 (d, 1H, J ) 16.0 Hz); 6.23 (d, 1H, J ) 12.9 Hz); 6.18
(d, 1H, J ) 16.0 Hz); 2.30 (s, 3H); 2.02 (t, 2H, J ) 5.9 Hz);
2.02 (s, 3H); 1.73 (s, 3H); 1.63 (m, 2H); 1.48 (m, 2H); 1.04 (s,
6H) ppm. 13C NMR (75.5 MHz, 1H-noise-decoupled, CDCl3): δ
155.62; 143.02; 137.31; 136.64; 135.97; 132.55; 130.72; 130.44;
1
0.56 g (80%) as a yellow oil. H NMR (300.1 MHz, CDCl3): δ
2.38 (s, 3H); 2.16 (q, 3H, 4J ) 0.5 Hz); 2.04 (q, 3H, J ) 0.5 Hz)
ppm. 13C NMR (75.5 MHz, 1H-noise-decoupled, CDCl3):
δ
156.41; 115.25; 104.18; 24.67; 21.53; 19.57 ppm. MS (EI, 70
eV): m/z 127 (M+•).
2-Iod o-3-m eth yl-2-bu ten en itr ile (29). 0.50 g (6.2 mmol)
of 3-methyl-2-butenenitrile (5) was reacted with a solution of
iodine in THF as electrophile following procedure 1. The
product was purified using silica gel column chromatography
with diethyl ether/petroleum ether (1:9 v/v) as eluent. Yield
1
1.14 g (89%) as a yellow solid. H NMR (300.1 MHz, CDCl3):
δ 2.24 (s, 3H); 2.07 (s, 3H) ppm. 13C NMR (75.5 MHz, 1H-noise-
decoupled, CDCl3): δ 163.25; 117.22; 53.25; 28.60; 24.28 ppm.
MS (EI, 70 eV): m/z 207 (M+•).
128.92; 118.84; 54.89; 39.38; 34.00; 33.01; 28.80; 21.65; 18.96;
12
18.85; 12.97 ppm. MS:
C
1H2614N1127I1 + 1H1; experimental
20
2-Met h yl-2-b u t en en it r ile (30). 0.50 g (7.2 mmol) of
2-butenenitrile (6) was reacted with iodomethane as electro-
phile following procedure 1. The product was purified using
silica gel column chromatography with diethyl ether/petroleum
ether (1:9 v/v) as eluent. Due to the low-boiling point no pure
30 could be isolated.
mass 407.99659; theoretical mass 408.11870; m/z 282 ([[M -
I] + H]+); 408 ([M + H]+).
14-Met h ylr et in a l (24). 310 mg (1.05 mmol) of 2,3,7-tri-
methyl-9-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4,6,8-nona-
tetraenenitrile (21) was reacted with DIBALH following
procedure 3. The product was purified using silica gel column
chromatography with diethyl ether/petroleum ether (1:9 v/v)
as eluent. Yield 239 mg (76%) as a yellow oil. The individual
regioisomers were separated using HPLC (see procedure 4).
all-E-24: 1H NMR (300.1 MHz, CDCl3): δ 10.26 (s, 1H); 7.17
(dd, 1H, J ) 11.6 Hz, J ) 15.0 Hz); 6.81 (d, 1H, J ) 15.0 Hz);
6.34 (d, 1H, J ) 16.2 Hz); 6.26 (d, 1H, J ) 11.6 Hz); 6.18 (d,
1H, J ) 16.2 Hz); 2.34 (s, 3H); 2.04 (s, 3H); 2.01 (t, 2H, J )
6.0 Hz); 1.91 (s, 3H); 1.73 (s, 3H); 1.63 (m, 2H); 1.47 (m, 2H);
1.04 (s, 6H) ppm.
14-Th iom et h ylr et in a l (25). 400 mg (1.22 mmol) of 3,7-
dimethyl-2-thiomethyl-9-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-
2,4,6,8-nonatetraenenitrile (22) was reacted with DIBALH
following procedure 3. The product was purified using silica
gel column chromatography with diethyl ether/petroleum ether
(1:9 v/v) as eluent. Yield 278 mg (69%) as a yellow oil. The
individual regioisomers were separated using HPLC (see
procedure 4). all-E-25: 1H NMR (300.1 MHz, CDCl3): δ 10.13
(s, 1H); 7.50 (d, 1H, J ) 15.2 Hz); 7.26 (dd, 1H, J ) 12.0 Hz,
J ) 15.2 Hz); 6.38 (d, 1H, J ) 16.0 Hz); 6.33 (d, 1H, J ) 12.0
Hz); 6.20 (d, 1H, J ) 16.0 Hz); 2.42 (s, 3H); 2.23 (s, 3H); 2.05
(s, 3H); 2.04 (t, 2H, J ) 6.6 Hz); 1.73 (s, 3H); 1.63 (m, 2H);
1.47 (m, 2H); 1.04 (s, 6H) ppm. 13C NMR (75.5 MHz, 1H-noise-
decoupled, CDCl3): δ 188.39; 155.72; 141.95; 137.59; 137.08;
133.98; 133.56; 132.31; 130.76; 130.24; 130.00; 39.56; 33.17;
28.96; 21.79; 19.14; 17.71; 14.86; 13.07 ppm.
2-Th iom eth yl-2-bu ten en itr ile (31). 0.50 g (7.2 mmol) of
2-butenenitrile (6) was reacted with methyl thiocyanate as
electrophile following procedure 1. The product was purified
using silica gel column chromatography with diethyl ether/
petroleum ether (1:9 v/v) as eluent. Yield 0.23 g (28%) as a
yellow oil. Major isomer: 1H NMR (300.1 MHz, CDCl3): δ 6.63
(q, 1H, J ) 7.1 Hz); 2.44 (s, 3H); 1.93 (d, 3H, J ) 7.1 Hz)
ppm.13C NMR (75.5 MHz, 1H-noise-decoupled, CDCl3):
δ
143.88; 115.49; 111.49; 16.45; 15.86 ppm. Minor isomer: 1H
NMR (300.1 MHz, CDCl3): δ 6.58 (q, 1H, J ) 7.1 Hz); 2.38 (s,
3H); 2.06 (d, 3H, J ) 7.1 Hz) ppm.13C NMR (75.5 MHz, 1H-
noise-decoupled, CDCl3): δ 145.32; 112.22; 110.03; 17.89; 15.63
ppm. MS (EI, 70 eV): m/z 113 (M+•).
2-Iod o-2-bu ten en itr ile (32). 0.50 g (7.2 mmol) of 2-butene-
nitrile (6) was reacted with a solution of iodine in THF as
electrophile following procedure 1. The product was purified
using silica gel column chromatography with diethyl ether/
petroleum ether (1:9 v/v) as eluent. Yield 0.19 g (14%) as a
yellow solid. Major isomer: 1H NMR (300.1 MHz, CD3-
COCD3): δ 7.14 (q, 1H, J ) 7.1 Hz); 1.93 (d, 3H, J ) 7.1 Hz)
ppm.13C NMR (75.5 MHz, 1H-noise-decoupled, CD3COCD3): δ
155.57; 118.84; 62.07; 22.79 ppm. Minor isomer: 1H NMR
(300.1 MHz, CD3COCD3): δ 7.31 (q, 1H, J ) 7.1 Hz); 1.99 (d,
3H, J ) 7.1 Hz) ppm.13C NMR (75.5 MHz, 1H-noise-decoupled,
CD3COCD3): δ 159.18; 116.84; 52.25; 21.08 ppm. MS (EI, 70
eV): m/z 193 (M+•).
14-Iod or etin a l (26). 400 mg (0.98 mmol) of 3,7-dimethyl-
2-iodo-9-(2′,6′,6′-trimethyl-1′-cyclohexen-1′-yl)-2,4,6,8-nonatet-

1282 J . Org. Chem., Vol. 66, No. 4, 2001
Verdegem et al.
couplings of the synthesized retinal compounds. NMR spectra,
as well as the mass spectra of all synthesized 10- and
14-substituted retinals are included. This material is available
free of charge via the Internet at http://pubs.acs.org.
Ack n ow led gm en t. This research was financed in
part by Biotechnology grant Bio2 CT-930467 of the
European Union.
Su p p or tin g In for m a tion Ava ila ble: The Supporting
Information contains 1H and 13C NMR assignments and J
J O0009595