The preparation of all-trans uniformly 13C-labeled retinal via a modular total organic synthetic strategy. Emerging central contribution of organic synthesis toward the structure and function study with atomic resolution in protein research

Article abstract of DOI:10.1021/ja012368h

Uniformly [¹³C₂₀]-labeled all-trans-retinal (1) has been prepared via a convergent modular total organic strategy with high isotope incorporation (>99%) and without isotope dilution starting from commercially available 99% enriched <

Full text of DOI:10.1021/ja012368h

Published on Web 05/10/2002
The Preparation of All-Trans Uniformly 13C-Labeled Retinal via
a Modular Total Organic Synthetic Strategy. Emerging Central
Contribution of Organic Synthesis toward the Structure and
Function Study with Atomic Resolution in Protein Research
Alain F. L. Creemers and Johan Lugtenburg*
Contribution from the Leiden Institute of Chemistry, Leiden UniVersity, P.O. Box 9502,
2300 RA, Leiden, The Netherlands
Received October 15, 2001
Abstract: Uniformly [13C20]-labeled all-trans-retinal (1) has been prepared via a convergent modular total
organic strategy with high isotope incorporation (>99%) and without isotope dilution starting from
commercially available 99% enriched 13C-labeled starting materials. For this purpose we have developed
a strategy that is based on four different modules: [1,2,3,4,(3-CH3)-13C5]-4-(diethylphosphono)-3-methyl-
2-butenenitrile (3), [1,2,3,4-13C4]-ethyl acetoacetate (7), [U-13C5]-4-bromo-2-methyl-2-butene (13), and
[U-13C10]-2,6,6-trimethylcyclohex-2-ene-1-ylcarbonitrile (16). This scheme permits the synthesis of the full
cassette of all isotopomers with 13C-labels at any position or combination of positions by using different
13C-labeled starting materials. In addition, modifications of the synthesized modules will give access to a
broad range of chemically modified 13C-labeled retinoids and carotenoids. This modular strategy enables
the synthesis of multifold and uniformly stable isotopically labeled (bio)macromolecules that can be used
for studying proteins with atomic resolution, providing detailed functional information of the studied biological
system.
Introduction
labeled molecule. Another spectroscopic method is solid-state
magic angle spinning (MAS) NMR spectroscopy, which probes
the electron density at the atoms. This technique allows the
establishment of electronic charges in the atoms, protonation
states, and configurations and conformations around bonds of
the stable isotopically labeled molecule.8,9
Comparison of the structural parameters obtained via these
techniques for intermediate I and intermediate I + 1 in the
biochemical process of the studied system provides functional
information, that is, changes in protonation states, bond lengths,
configuration, and conformation around bonds on the time scale
involved.7,8 When sufficient structural and functional informa-
tion at the atomic level of the native form has been obtained, a
whole new dimension can be attained by studying in a similar
fashion systems with mutations in the protein chain and systems
with rationally designed chemical changes in the cofactors.9,10
These studies will lead to an even deeper understanding of the
biochemical process.
The primary structures of all human proteins are now
available with the completion of the human genome project.1
In the post-genomic era in a very rapid process, the total
genomes of a plethora of other organisms are also becoming
available in addition to mutants that lead to malfunctioning or
nonfunctioning proteins leading to genetic diseases. Furthermore,
efficient procedures are available via biotechnology to obtain
the proteins using these genetic codes.2,3 The fundamental
challenge now is to study the chemical processes of these
proteins involving (bio)macromolecules without perturbation in
the native states at the atomic level in time scales ranging from
femtoseconds up to days.
Nature provides us with the ultimate probe via stable isotopes.
Isotopes combine the same chemistry with different physical
properties.4 Study of a system with site-directed isotope labeling
with a high incorporation allows the determination of the whole
force field via vibrational techniques such as FT infrared
spectroscopy and (Resonance) Raman spectroscopy based on
the difference in isotopic mass.5-7 These techniques probe, for
instance, the electron density in chemical bonds of the isotope-
The implementation of the above-mentioned program has now
utmost urgency. Without this program the now increasingly
(7) Mathies, R. A.; Lugtenburg, J. Molecular Mechanisms in Visual Trans-
duction; Stavenga, D. G., DeGrip, W. J., Pugh, E. N., Jr., Eds; Elsevier:
Amsterdam, 2000.
(8) Verdegem, P. J. E.; Bovee-Geurts, P. H. M.; DeGrip, W. J.; Lugtenburg,
J.; De Groot, H. J. M. Biochemistry 1999, 38, 11316-11324.
(9) Verhoeven, M. A.; Creemers, A. F. L.; Bovee-Geurts, P. H. M.; DeGrip,
W. J.; Lugtenburg, J.; De Groot, H. J. M. Biochemistry 2001, 40, 3282-
3288.
* To whom correspondence should be addressed. E-mail: Lugtenbu@
chem.leidenuniv.nl. Fax: +31-715274488. Telephone: +31-715274504.
(1) Venter, J. C.; et al. Science 2001, 291, 1304-1351.
(2) Murhammer, D. W. Appl. Biochem. Biotechnol. 1991, 31, 283-310.
(3) Vissers, P. M. A. M.; DeGrip, W. J. FEBS Lett. 1996, 396, 26-30.
(4) Lockhart, I. M. Isotopes: Essential Chemistry & Applications; Elvidge, J.
A., Jones, J. R., Eds; The Chemical Society: London, 1979.
(5) Torres J.; Adams, P. D.; Arkin, I. T. J. Mol. Biol. 2000, 300, 677-685.
(6) Eyring, G.; Curry, B.; Broek, A.; Lugtenburg, J.; Mathies, R. A.
Biochemistry 1982, 21, 384-393.
(10) DeLange, F.; Bovee-Geurts, P. H. M.; VanOostrum, J.; Portier, M. D.;
Verdegem, P. J. E.; Lugtenburg, J.; DeGrip, W. J. Biochemistry 1998, 37,
1411-1420.
9
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J. AM. CHEM. SOC. 2002, 124, 6324-6334
10.1021/ja012368h CCC: $22.00 © 2002 American Chemical Society

[U-13C ]-all-trans-Retinal
A R T I C L E S
20
available genetic information in the post-genomic era cannot
be translated into the required structural and functional informa-
tion that will lead to the expected quantum jump in the
understanding of the various processes in human (animal) health
and diseases and the expected rational approach to treat these
diseases.
The access to a full number of possible site-directed stable
isotopically enriched building blocks (amino acids and cofactors)
up to the uniformly labeled systems is a “condition sine qua
non” for the proposed structural and functional investigations.
All uniformly isotopically labeled amino acids and several
cofactors are available via photosynthetic organisms that are
grown in media containing 13CO2 and 15NH3.11
To start the above-mentioned program, access to building
blocks with isotope labels at each defined atomic position and
combination of positions up to the uniformly labeled form is
required. The only way to obtain access to the whole cassette
of desired isotopomers is a modular synthetic approach such
that one synthetic scheme can give in a rational way the required
building block as a cassette of all isotopomers. This approach
may seem Herculean; however, only 20 different amino acids
and a limited number of cofactors are required. The synthesis
of these cassettes has to be based on a limited number of
commercially available highly enriched stable isotopically
labeled starting materials.
In this paper we describe the method to prepare the full
cassette of all site-directed 13C-labeled isotopomers of all-trans-
retinal up to the uniformly 13C-labeled form. The selection of
[U-13C20]-labeled all-trans-retinal and all its isotopomers in the
new modular approach is based on the fact that retinal and
retinoids play a very important role in many life processes.12-14
In addition, structural and functional studies with isotope-
sensitive techniques have already been initiated in the field of
rhodopsin.8,9,15,16
Rhodopsin serves as the paradigm for the superfamily of
seven transmembrane helix G-protein coupled receptors
(GPCRs).17 The GPCRs mediate a broad array of important
physiologically and pharmacologically signal transduction pro-
cesses. GPCRs trigger a wide variety of physiological processes
that involve signaling by neurotransmitters, hormones, and
neuropeptides.18 Consequently, GPCRs are the major pharma-
ceutical targets for pharmacological intervention in human (and
veterinary) pathology. In rhodopsin the photoreactive ligand is
11-cis-retinal that is covalently bound in the interior of the
protein via a protonated Schiff base linkage with lysine residue
296 to form the retinylidene chromophore.12 Absorption of a
photon leads to the ultrafast isomerization of the C11dC12 bond
of the retinylidene ligand from the 11-cis to the all-trans
configuration in only ∼200 fs, the fastest photochemical reaction
on earth to date.12,19
The full vibration analysis of the chromophore of rhodopsin
and its photoproduct, bathorhodopsin, has been reported via
about 70 isotopomers.6,20 Furthermore, the chemical shift values
of the sp2 carbons in the tail end of the chromophore have been
reported.9,15 The distances between C10-C20 and C11-C20 could
be determined with very high precision (0.1 Å) via the 1-D
rotational resonance solid-state 13C NMR technique.8 From these
distance measurements the precise chromophore structure could
be derived. The molecular torsional angle around a bond in the
chromophore labeled with two 13C isotopes could be directly
established via this method.21 Furthermore, we recently pub-
lished the ultrahigh-field solid-state MAS NMR study on the
[8,9,10,11,12,13,14,15,19,20-13C10]-11-cis-retinylidene chromo-
phore in its natural lipid membrane environment.9 This study
showed that the use of multispin labeling in combination with
2-D solid-state MAS NMR correlation spectroscopy improves
the relative accuracy of the shift measurements in solids. This
allows the electronic structure of the retinylidene chromophore
to be analyzed at high levels of understanding: (1) by specifying
the interactions between the 13C-labeled ligand and the G-protein
coupled receptor target and (2) by making an assessment of
the various factors contributing to the charge distribution in the
chromophore. In one short experimental session, information
of much higher quality about 10 carbon atoms at the same time
was obtained that thus far has taken a decade to collect.
Nevertheless, these results were obtained via studies of the
almost unlimited supply of bovine rhodopsin from cattle eyes.
However, cone pigments of man and other animals and the
various opsins with site-directed mutations have to be obtained
via biotechnological expression systems, which implies that now
the availability is the limiting factor.3,22 Recently we have
published a solid-state 15N MAS NMR study of rod visual
pigment rhodopsin in which 99% 15N enriched [R,ꢀ-15N2]-L-
lysine was incorporated by using the baculovirus/Sf9 cell
expression system.16
It is clear that the demand for [U-13C20]-labeled all-trans-
retinal, synthesized via a modular synthetic scheme, is urgently
needed. In addition, this scheme must also allow an easy access
to the complete cassette of all desired isotopomers. In this paper
we describe a modular synthetic scheme, which enables us to
prepare [U-13C20]-labeled all-trans-retinal and all isotopomers
via an efficient and economic way.
Results and Discussion
Synthesis. For the synthesis of [U-13C20]-labeled all-trans-
retinal (1) and all its isotopomers with 13C-labels at any carbon
position or combination of positions, we have developed a
modular convergent synthetic scheme. This synthetic scheme
has to meet several important requirements, which allows the
introduction of the stable isotopes: (1) the synthesis should be
based on starting compounds of low molecular weight (e.g.
acetic acid, acetonitrile, acetone) that are commercially available
(11) Winkler, F. J.; Ku¨hnl, K.; Schwarz-Kaske, R.; Schmidt, H.-L. Isot. EnViron.
Health Stud. 1995, 31, 161-190.
(12) Wald, G. Nature 1968, 219, 800-807.
(13) Durston, A. J.; Van der Wees, J.; Pijnappel, W. W. M.; Schilthuis, J. G.;
Godsave, S. V. Cell Mol. Life Sci. 1997, 53, 339-349.
(14) Pijnappel, W. W. M.; Folkers, G. E.; De Jonge, W. J.; Verdegem, P. J. M.;
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, 15424-15429.
(15) Smith, S. O.; Palings, I.; Miley, M. E.; Courtin, J.; De Groot, H. J. M.;
Lugtenburg, J.; Mathies, R. A.; Griffin, R. G. Biochemistry 1990, 29, 8158-
8164.
(19) Hubbart, R.; Croft, A. Proc. Natl. Acad. Sci. U.S.A. 1958, 44, 130-139.
(20) Palings, I.; Pardoen, J. A.; Van den Berg, E.; Winkel, C.; Lugtenburg, J.;
Mathies, R. A. Biochemistry 1987, 26, 2544-2556.
(16) Creemers, A. F. L.; Klaassen, C. H. W.; Bovee-Geurts, P. H. M.; Kelle,
R.; Kragl, U.; Raap, J.; DeGrip, W. J.; Lugtenburg, J.; De Groot, H. J. M.
Biochemistry 1999, 38, 7195-7199.
(21) Feng, X.; Verdegem, P. J. E.; Lee, Y. K.; Sandstro¨m, D.; Ede´n, M.; Bovee-
Geurts, P.; DeGrip, W. J.; Lugtenburg, J.; De Groot, H. J. M.; Levitt, M.
H. J. Am. Chem. Soc. 1997, 119, 6853-6857.
(17) Baldwin, J. M.; Schertler, G. F. X.; Unger, V. M. J. Mol. Biol. 1997, 272,
144-164.
(22) Vissers, P. M. A. M.; Bovee-Geurts, P. H. M.; Portier, M. D.; Klaassen,
C. H. W.; DeGrip, W. J. Biochem. J. 1998, 330, 1201-1208.
(18) Ji, T. H.; Grossmann, M.; Ji, I. J. Biol. Chem. 1998, 273, 17299-17302.
9
J. AM. CHEM. SOC. VOL. 124, NO. 22, 2002 6325

A R T I C L E S
Creemers and Lugtenburg
Scheme 1. Synthesis of [U-13C20]-all-trans-Retinal (1) via a Horner-Emmons Reaction Sequence Using the 5-Fold 13C-Labeled
C5-Phosphonate Module, [1,2,3,4,(3-CH3)-13C5]-4-(Diethylphosphono)-3-methyl-2-butene-nitrile (3)a
a The asterisks (*) indicate the positions of the 13C-labels.
Scheme 2. Synthetic Scheme for the Preparation of [1,2,3,4,(3-CH3)-13C5]-4-(Diethylphosphono)-3-methyl-2-butenenitrile (3) (upper part)
and [U-13C10]-2,6,6-Trimethylcyclohex-1-enylcarbaldehyde (2) (lower part)a
a Both compounds were prepared from the commercially available module [1,2,3,4-13C4]-ethyl acetoacetate (7). An important intermediate that is synthesized
via this method is [U-13C]-chloroacetone (9), which is not commercially available.
in specifically and high enriched (>99%) form; (2) since these
starting materials are quite expensive, an efficient, convergent,
and thoroughly optimized synthetic scheme is required; and (3)
care should be taken that no scrambling or dilution of the 13C-
label can occur at any stage in the course of the synthesis. To
meet these requirements, we have developed an economic and
convergent scheme for the preparation of all-trans-retinal (1)
that is based on four different modules: [1,2,3,4,(3-CH3)-13C5]-
4-(diethylphosphono)-3-methyl-2-butenenitrile (3), [1,2,3,4-
13C4]-ethyl acetoacetate (7), [U-13C5]-4-bromo-2-methyl-2-
butene (13), and [U-13C10]-2,6,6-trimethylcyclohex-2-ene-1-
ylcarbonitrile (16). With the exception of â-keto ester 7, which
is commercially available, these modules are prepared in a small
number of steps from commercially available 99% enriched 13C-
labeled starting materials of low molecular weight. Via these
modules 1 can be obtained with 13C-labels introduced at any
carbon position or combination of positions in a highly enriched
form. In addition, modification of the modules makes it possible
to prepare a range of chemically modified retinoids and
carotenoids. Furthermore, two modules in this scheme are used
twice in the synthesis of 1, leading to an efficient use of the
relatively expensive isotopically enriched starting materials.
Subsequently, nitrile 4 was reduced with DIBAL-H to give the
corresponding aldehyde [U-13C15]-3-methyl-5-(2,6,6-trimethyl-
cyclohex-1-enyl)penta-2,4-dienal (5) in 79% yield. The same
procedure was used to convert C15-aldehyde 5 into [U-13C20]-
3,7-dimethyl-9-(2,6,6-trimethyl-cyclohex-1-enyl)nona-2,4,6,8-
tetraenal (1). Coupling of 5-fold labeled phosphonate 3 to
aldehyde 5 via a second Horner-Emmons reaction gave
[U-13C20]-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-
2,4,6,8-tetraenenitrile (6) in 97% yield, which was reduced with
DIBAL-H to obtain the final product, retinal 1, as a mixture of
isomers. This reaction sequence directly shows the potency of
the use of modules for the preparation of multifold labeled
molecules. In addition, chemically modified retinoids with
various kinds of substituents introduced at positions C-10 and
C-14 can be synthesized based on the methods described by
Verdegem et al.25 The aforementioned â-cyclocitral 2 and C5-
phosphonate 3 are both prepared from another important and
commercially available module, [1,2,3,4-13C4]-ethyl acetoacetate
(7) (Scheme 2).
The 5-fold 13C-labeled C5-phosphonate 3 was prepared in four
steps from the commercially available â-keto ester module 7.
First, chlorination of 7 gives [1,2,3,4-13C4]-ethyl 2-chloro-
acetoacetate (8) in a quantitative yield.26 Subsequently, acid-
catalyzed hydrolysis of the ester and decarboxylation of the acid
group gave [U-13C3]-chloroacetone (9). This hydrolysis of ester
8 is the crucial step in the synthesis of the 5-fold 13C-labeled
phosphonate 3. Generally, the hydrolysis of esters is carried
Via a Horner-Emmons reaction sequence using the 5-fold
13C-labeled C5-phosphonate module, [1,2,3,4,(3-CH3)-13C5]-4-
(diethylphosphono)-3-methyl-2-butenenitrile (3), we elongate in
just four consecutive steps the polyene chain that is attached to
the ring moiety, starting from [U-13C10]-2,6,6-trimethylcyclohex-
1-enylcarbaldehyde (2) (Scheme 1). The unlabeled phosphonate
has already been applied many times for the synthesis of several
carotenoids and retinoids.23,24 In the first chain elongation, the
anion of phosphonate 3 was coupled with â-cyclocitral 2
affording the nitrile [U-13C15]-3-methyl-5-(2,6,6-trimethyl-cy-
clohex-1-enyl)penta-2,4-dienenitrile (4) in a yield of 81%.
(23) Gebhard, R.; Van Dijk, J. T. M.; Van Ouwerkerk, E.; Boza, M. V. T. J.;
Lugtenburg, J. Recl. TraV. Chim. Pays-Bas 1991, 110, 459-469.
(24) Lugtenburg, J.; Creemers, A. F. L.; Verhoeven, M. A.; Van Wijk, A. A.
C.; Verdegem, P. J. E.; Monnee, M. C. F.; Jansen, F. J. H. M. Pure Appl.
Chem. 1999, 71, 2245-2251.
(25) Verdegem, P. J. E.; Monnee, M. C. F.; Lugtenburg, J. J. Org. Chem. 2001,
66, 1269-1282.
(26) De Kimpe, N.; De Cock, W.; Schamp, N. Synthesis 1987, 188-190.
9
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[U-13C ]-all-trans-Retinal
A R T I C L E S
20
Scheme 3. (A) Synthetic Scheme for the Preparation of [U-13C5]-4-Bromo-2-methyl-2-butene (13) from the Commercially Available
[
13C2]-Acetic Acid (18a) and [13C3]-Acetone (22) and (B) Synthesis of [2,3,4-13C3]-Ethyl Acetoacetate (7)a
a The key step is a Blaise reaction where [13C2]-acetonitrile (10) is coupled to ester 25 to give in two steps â-keto ester 7. The three 13C-labeles are
indicated with 4, O, and 0 visualizing the three different positions of 13C-labeling when different starting materials are used.
out with a basic solution. However, the hydrolysis of â-keto
ester 8 cannot be carried out in an alkaline solution since under
these conditions a retro-Claisen condensation occurs. Therefore,
an acidic solution was used to hydrolyze the ester bond.
Subsequently, the decarboxylation of the carboxyl group gave
the uniformly 13C-labeled ketone 9. In addition, even though
one 13C-label is lost via this way because of the decarboxylation
of the ester 8, this method was used for the preparation of the
phosphonate due to the fact that the 4-fold 13C-labeled ethyl
acetoacetate is commercial available.
Coupling of ketone 9 with the anion of the in situ prepared
phosphonate 11 via a Horner-Emmons reaction gave [U-13C5]-
4-chloro-3-methyl-2-butenenitrile (12) in a yield of 70% (two
steps). This coupling is based on a reaction that has been used
many times in our group.24,27 After the deprotonation of [13C2]-
acetonitrile (10) with 1 equiv of LDA, the formed anion reacted
with diethyl chlorophosphate to give [13C2]-diethyl cyanometh-
ylphosphonate (11), which was immediately deprotonated by
the second equivalent of LDA. [13C2]-Acetonitrile (10) was in
this way converted in situ into the reactive anion of the [13C2]-
diethyl cyanomethylphosphonate (11), which reacted with ketone
9 to give nitrile 12. The chlorine atom of nitrile 12 was
substituted via an Arbuzov reaction with triethyl phosphite to
afford the first module, [1,2,3,4,(3-CH3)-13C5]-4-(diethylphospho-
no)-3-methyl-2-butenenitrile (3) in 95% yield.
The first step in the synthesis of â-cyclocitral 2 is an
alkylation at the R-position by adding the third module,
[U-13C5]-4-bromo-2-methyl-2-butene (13), to the anion of â-keto
ester 7, which was also used for the preparation of phosphonate
3. Subsequently, saponification and acid-catalyzed decarboxy-
lation gave [U-13C8]-6-methyl-5-hepten-2-one (14) in a yield
of 75% based on bromide 13. The Horner-Emmons reaction
with the anion of the in situ prepared [13C2]-diethyl cyanom-
ethylphosphonate (11) gave [U-13C10]-3,7-dimethylocta-2,6-
dienenitrile (15) in 72% yield. Next, according to the method
described by Jansen et al., the open-chain nitrile 15 was cyclized
to give the cyclic compound [U-13C10]-2,6,6-trimethylcyclohex-
2-ene-1-ylcarbonitrile (16).28 In the workup procedure of this
reaction it proved to be important to keep the conditions acidic
to avoid a double bond shift to the â-position. Nitrile 16 might
be seen as the fourth module, due to the fact that this
intermediate can also be used for the synthesis of various
carotenoids and retinoids containing chemical modifications in
the ring moiety.28,29 Reduction of nitrile 16 with DIBAL-H gave
[U-13C10]-2,6,6-trimethylcyclohex-2-ene-1-ylcarbaldehyde (17)
in a yield of 68%. Subsequently, isomerization of the double
bond from the R-position to the â-position yielded â-cyclocitral
2 in a yield of 80%.
Bromide 13, which was coupled to the â-keto ester 7, was
synthesized in 6 steps from the commercially available 13C-
labeled starting materials [13C2]-acetic acid (18a) and [13C3]-
acetone (22) (Scheme 3A). Bromination of [13C2]-acetic acid
(18a) with bromine and trifluoroacetic anhydride (TFAA) gave
[13C2]-bromoacetic acid (19a) in an excellent yield of 93%.30,31
Subsequently, acid 19a was esterificated to give [13C2]-ethyl
bromoacetate (20a) in 93% yield with oxalyl chloride and
ethanol. Next, substitution of the bromine atom of ester 20a
via an Arbuzov reaction with triethyl phosphite gave [1,2-13C2]-
ethyl-2-(diethylphosphono) acetate (21) in a quantitative yield.
Coupling of phosphonate 21 with [13C3]-acetone (22) via a
Horner-Emmons reaction gave [1,2,3,4,(3-CH3)-13C5]-ethyl-
3-methyl-2-butenoate (23) in a yield of 91%, based on acetone.
The coupling was done by using an excess of 1.5 equiv of the
phosphonate. Using an equimolar amount of 21 resulted in a
much lower yield (∼30%) that stands for the loss of a large
quantity of the expensive [13C3]-acetone. Ester 23 was reduced
with DIBAL-H to give the corresponding alcohol [U-13C5]-3-
methyl-2-buten-1-ol (24). Even though TLC analysis and change
of color showed an instantaneous conversion of the ester group,
the optimized yield of the reaction was only 55%. However, in
the meantime a method is described by Vassilikogiannakis et
al. using LiAlH4 as reductor leading to an increase of the yield
up to 90%.32 Subsequently, alcohol 24 was converted into the
bromide by using 47% hydrobromic acid to obtain the fourth
module, bromide 13.
(29) Kutney, J. P.; Gunning, P. J.; Clewley, R. G.; Sommerville, J.; Rettig, S.
J. Can. J. Chem. 1992, 70, 2094-2114.
(30) Roberts, J. L.; Poulter, C. D. J. Org. Chem. 1978, 43, 1547-1550.
(31) Ouwerkerk, N.; Van Boom, J. H.; Lugtenburg, J.; Raap, J. Eur. J. Org.
Chem. 2000, 861-866.
(27) Raap, J.; Nieuwenhuis, S. A. M.; Creemers, A.; Hexspoor, S.; Kragl, U.;
Lugtenburg, J. Eur. J. Org. Chem. 1999, 2609, 9-2621.
(28) Jansen, F. J. H. M.; Lugtenburg, J. Eur. J. Org. Chem. 2000, 829-836.
(32) Vassilikogiannakis, G.; Chronakis, N.; Orfanopoulos, M. J. Am. Chem. Soc.
1998, 120, 9911-9920.
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A R T I C L E S
Creemers and Lugtenburg
We have prepared [U-13C20]-retinal as a mixture of isomers
via the above-described method. Through column chromatog-
raphy the all-trans-retinal was isolated from the cis isomers.
Subsequently, cis-to-trans isomerization was accomplished via
irradiation of a solution of the cis isomers under a tungsten lamp
in the presence of iodine crystals. Finally, a total amount of
125 mg of [U-13C20]-all-trans-retinal (1), which has identical
chemical properties (λmax, Rf value) as the natural abundance
all-trans-retinal, was obtained via this modular synthetic scheme.
In just two months time the [U-13C20]-all-trans-retinal (1) was
synthesized from 2.0 g of [13C2]-acetic acid, 4.4 g of [1,2,3,4-
13C4]-ethyl acetoacetate, 1.0 g of [13C3]-acetone, and 1.2 g of
[13C2]-acetonitrile. These starting materials were a kind gift from
Cambridge Isotope Laboratories.
To also have the ability to synthesize the complete cassette
of isotopomers, a synthetic method has been developed for the
preparation of ethyl acetoacetate (7), which is not commercially
available with a 13C-label at all position combinations. The
â-keto ester 7 can be obtained via a Blaise reaction.33,34 In this
reaction a zinc ester enolate is coupled to a nitrile to give an
enamino ester that is hydrolyzed to obtain the corresponding
â-keto ester. Although the synthesis of [2,3,4-13C3]-ethyl
acetoactetate is discussed in this paper, all combinations of the
13C-labeled â-keto ester can be prepared via this method
(Scheme 3B). For the synthesis of the 3-fold 13C-labeled ester
7, the zinc enolate was prepared from [2-13C]-ethyl iodoacetate
(25) that was coupled with 13C-labeled acetonitrile (10). The
use of ester 25 instead of ethyl bromoacetate, as is mentioned
in the literature, enables us to lower the amount of 25 that is
preferred due to the fact that 13C-labeled starting compounds
are relatively expensive.34 Ester 25 was synthesized in three
steps from [2-13C]-acetic acid (18b). First, the acid was
converted into [2-13C]-ethyl bromoacetate (20b) in a yield of
88% (two steps), using the aforementioned method. Subse-
quently, the bromide was replaced by an iodide via an Sn2
reaction with NaI to give [2-13C]-ethyl iodoacetate (25) in 98%
yield. In the presence of activated zinc dust in refluxing THF,
ester 25 was converted into the zinc enolate. Addition of [13C2]-
acetonitrile (10) gave the enamino ester that was hydrolyzed to
give [2,3,4-13C3]-ethyl acetoacetate (7) in a yield of 80% (two
steps).
As depicted in Scheme 3B, all positions and combinations
of positions of the â-keto ester can be labeled by using different
13C-labeled starting materials. However, the labeling of both
methyl groups C16 and C17 forms the exception. These labels
are introduced by using [13C3]-acetone (22) (Scheme 3A),
leading to the unfeasibility of the discrimination between these
two positions. Nevertheless, the above-described schemes enable
the synthesis of nearly the whole cassette of isotopomers of
all-trans-retinal. Next to this, we are also able to prepare a broad
range of chemically modified retinals via small modifications
of the developed synthetic scheme .28,29
Characterization of [U-13C20]-all-trans-Retinal (1). The
characterization of the compounds will be focused on the final
product, [U-13C20]-all-trans-retinal (1), whereas the spectro-
scopic data of all other synthesized intermediates can be found
in the Experimental Section. Preparative HPLC was used for
the purification of all-trans-retinal. The purified all-trans isomer
was subsequently characterized by using mass spectroscopy and
high-resolution NMR spectroscopy.
Mass Spectrometry. The single focus electron-impact mass
spectrum of the [U-13C20]-all-trans-retinal (1) appears as a
typical retinoid mass spectrum with the base peak at m/z 304,
the molecular ion peak. Compared to the natural abundance all-
trans-retinal (m/z 284) the base peak has shifted 20 mass units.35
This shift is due to the substitution of all 20 12C nuclei for 13
C
nuclei. Next to the molecular ion peak a second large peak is
observed at m/z 286. This peak is also observed in the mass
spectrum of natural abundance all-trans-retinal and is due to
the expulsion of water.
Double focus mass spectroscopy was used to determine the
exact mass of the synthesized compounds. A mass of 304.28037
was determined for the [U-13C20]-retinal (1), whereas the natural
abundance all-trans-retinal has a mass of 284.21412. These
values are in close agreement with the calculated mass
(13C20H28O ) 304.28111; C20H28O ) 284.21402).
The degree of incorporation was determined by using the
computer simulation program Isopro 3.0 (Cornell University).
The isotope incorporation was determined via comparison of
the exact mass of the M+• peak with the [M - 1]+•, [M - 2]+•,
and [M - 3]+• peak of the simulated mass spectrum and the
spectrum of the [U-13C20]-retinal (1). On the basis of these results
an isotope incorporation of g99% was found. Consequently,
no isotope dilution took place during the synthesis of the retinal.
NMR Spectroscopy: (a) 1H NMR Spectroscopy. 1H NMR
spectroscopy was used to determine the structure and purity of
the synthesized products. Additional J-couplings occur due to
the 99% enriched 13C-label at each carbon position. However,
these 13C enrichments do not influence the chemical shifts of
both the protons and carbons. In Figure 1 the most significant
regions of the 1H NMR spectrum (600 MHz, CDCl3) of
[U-13C20]-all-trans-retinal (Figure 1, spectra A and B) and
natural abundance all-trans-retinal (Figure 1C) are shown. The
additional J-couplings between the carbon nuclei and the protons
result in complex splitting patterns (Figure 1A). However, by
using 13C decoupling these relatively strong carbon-proton
couplings are eliminated, resulting in a simplification of the 1H
spectrum (Figure 1B). Due to the strong decoupling power,
necessary for the 1H-13C spin-decoupling across the complete
spectrum, line broadening occurs, which is caused by heating
of the sample. Therefore, the J-couplings of the retinal where
defined by using the lower spectrum whereas the 13C decoupled
proton spectrum was used for the assignment of all proton
resonances. Comparison of the spectra of the [U-13C20]-all-trans-
retinal and the unlabeled retinal directly proves that the
introduction of the 13C-labels does not affect the isotropic
chemical shifts of the protons. These shifts and the relating
J-couplings of [U-13C20]-retinal are summarized in Table 1. The
determined spectral values are in complete agreement with the
published data of Liu et al.36
In addition, the intensity of 15-H shows that a high isotope
enrichment is achieved. The double-doublet resonance, marked
with an asterisk (*), occurs as a result of isotope dilution: the
incorporation of 13C at position C15 can be estimated at g99%,
within the experimental error.
(35) Lin, R. L.; Waller, G. R.; Mitchell, E. D.; Yang, K. S.; Nelson, E. C. Anal.
Chem. 1970, 35, 435-441.
(36) Liu, R. S. H.; Asato, A. E. Tetrahedron 1984, 40, 1931-1969.
(33) Blaise, E. E. C. R. Hebd. Seances Acad. Sci. 1901, 132, 478-480.
(34) Hannick, S. M.; Kishi, Y. J. Org. Chem. 1983, 48, 3833-3835.
9
6328 J. AM. CHEM. SOC. VOL. 124, NO. 22, 2002

[U-13C ]-all-trans-Retinal
A R T I C L E S
20
1
Figure 1. Significant regions of the H NMR spectrum (600 MHz, CDCl3) of the [U-13C20]-all-trans-retinal (1) (A + B) and the natural abundance all-
trans-retinal (C). The additional J-couplings between the carbon nuclei and the protons result in complex splitting patterns (part A). However, by using 13
C
decoupling these relatively strong carbon-proton couplings can be overcome, leading to a simplification of the 1H spectrum (part B). Due to strong decoupling
1
power line broadening occurs that is caused by heating of the sample. The lines demonstrate the large JC-H couplings. The doublet-doublet resonance,
marked with an asterisk (*), occurs as a result of isotope dilution.
(b) 13C NMR Spectroscopy. The significant regions of the
Conclusions
1H-noise-decoupled 13C NMR spectrum (150 MHz, CDCl3) of
We have synthesized [U-13C20]-all-trans-retinal (1) via a
[U-13C20]-all-trans-retinal (A) and natural abundance all-trans-
modular strategy in an economic and convergent way with high
1
retinal (B) are shown in Figure 2. Like the H-spectrum, the
isotope incorporation (>99%) and without isotope dilution.
13C spectrum of the 13C-labeled retinal also shows similarity
with the 13C-spectrum of the natural abundance retinal. Due to
all 13C-labels, complex splitting patterns are observed in the
13C NMR spectrum. Nevertheless, the chemical shift values of
all carbon nuclei can be assigned. These shifts are in close
agreement with the published data of Liu et al. and are
summarized in Table 2.36
Using 2.0 g of [13C2]-acetic acid, 4.4 g of [1,2,3,4-13C4]-ethyl
acetoacetate, 1.0 g of [13C3]-acetone, and 1.2 g [13C2]-acetonitrile
we have prepared 125 mg of [U-13C20]-all-trans-retinal (1). The
overall yield of the synthesis was 6% (17 steps) based on [13C2]-
acetic acid (18a). After optimization of the scheme with
unlabeled starting materials, the [U-13C20]-all-trans-retinal (1)
was synthesized in just two months time. As far as we know
9
J. AM. CHEM. SOC. VOL. 124, NO. 22, 2002 6329

A R T I C L E S
Creemers and Lugtenburg
Figure 2. Significant regions of the 13C NMR spectrum (150 MHz, CDCl3) of the [U-13C20]-all-trans-retinal (1) (A) and the natural abundance all-trans-
retinal (B). Due to all 13C-labels, complex splitting patterns are observed in the 13C NMR spectrum.
Table 1. 1H NMR Data of [U-13C20]-all-trans-Retinal (1)a
this is the first organic system of average size where all carbons
δ (ppm)
H
multiplicity
J (Hz)
intensity
derive from a single carbon source. In addition, the developed
scheme also enables the synthesis of the full cassette of site-
directed 13C-labeled isotopomers by using alternatively 13C-
labeled starting materials. In addition, modifications of the
described modules give access to a broad range of chemically
modified retinoids and carotenoids.28,29 The [U-13C20]-labeled
retinal was used for solid-state MAS NMR spectroscopic studies
of the spatial and electronic structure of the 11-cis-retinylidene
chromophore in rhodopsin and isorhodopsin. The structural
information will lead to a deeper insight of the mechanism of
the ultrafast photoconversion in rhodopsin.
10.11
CHO
ddd
1JC-H ) 169.7
2JC-H ) 24.5
3JH-H ) 8.2
1JC-H ) 150.1
-
1
7.14
6.36
6.17
5.97
2.33
2.03
2.01
1.72
1.68
1.47
1.03
H-11
dm
m
m
1
2
2
1
3
3
2
3
2
2
6
H-7/H-12
H-8/H-10
H-14
13-CH3
9-CH3
H-4
-
dm
dm
dm
m
dm
dm
dm
dm
1JC-H ) 157.6
1JC-H ) 127.8
1JC-H ) 126.7
-
5-CH3
H-3
1JC-H ) 125.7
1JC-H ) 127.5
1JC-H ) 128.2
1JC-H ) 125.1
H-2
1-CH3
On the basis of this work we think that organic synthesis is
now able to supply in an efficient and economic way all
cofactors and amino acids in a [U-13C]-labeled form and as a
a The 1H chemical shift values are in complete agreement with the
published data of Asato et al.36
9
6330 J. AM. CHEM. SOC. VOL. 124, NO. 22, 2002

[U-13C ]-all-trans-Retinal
A R T I C L E S
20
Table 2. 13C NMR Data of [U-13C20]-all-trans-Retinal (1)a
[2-13C]-Acetic acid, [13C2]-acetic acid, [13C2]-acetonitrile, [13C3]-
acetone, and [1,2,3,4-13C4]-ethyl acetoacetate were a kind gift from
Cambridge Isotope Laboratories Inc., USA. All other chemicals were
purchased from Aldrich, Fluka or Acros Chimica.
δ (ppm)
C
multiplicity
J (Hz)
1JC-C ) 57.4
191.1
154.9
141.3
137.5
137.0
CHO
C-13
C-9
C-6
C-8
d
m
m
m
dd
[U-13C15]-3-Methyl-5-(2,6,6-trimethylcyclohex-1-enyl)penta-2,4-
dienenitrile (4). To a solution of 303 mg (1.4 mmol) of [1,2,3,4,(3-
CH3)-13C5]-4-(diethylphosphono)-3-methyl-2-butenenitrile (3) in 15 mL
of dry THF was added at 0 °C via a syringe 0.75 mL (1.2 mmol) of
n-BuLi (1.6 M solution in hexane). Stirring was continued for 15 min
at room temperature. To the anion of the phosphonate was added 130
mg (0.80 mmol) of â-cyclocitral (2), dissolved in 10 mL of dry THF.
The mixture was stirred for another 4 h at room temperature. Quenching
of the reaction was accomplished by adding 50 mL of saturated NH4-
Cl solution. The aqueous layer was extracted three times with 50 mL
of diethyl ether. The combined organic layers were washed with brine,
dried with MgSO4, and filtered. The product was purified by chroma-
tography (silica gel, diethyl ether/40-60 light petroleum ether, 20:80)
to give 150 mg (0.65 mmol, 81%) of 4 as a mixture of the 9-cis and
all-trans compounds. For the analysis the all-trans isomer was isolated
from the other isomer via HPLC purification.
1JC-C ) 72.1
1JC-C ) 58.4
1JC-C ) 69.4
1JC-C ) 53.9
134.5
C-12
dddd
2
2JC-C ≈ JC-C ) 7.4
132.5
130.5
C-11
C-5
ddm
ddd
1JC-C ) 69.9
1JC-C ) 53.9
1JC-C ) 75.1
1
1JC-C ≈ JC-C ) 35.4
129.2
39.5
34.2
C-7/C-10/C-14
m
dd
dddd
1
C-2
C-1
1JC-C ≈ JC-C ) 35.4
1
1
1JC-C ≈ JC-C ≈ JC-C
≈
1JC-C ) 35.4
1
33.1
28.9
21.8
19.1
13.1
13.0
C-4
dd
d
dm
dd
dm
dm
1JC-C ≈ JC-C ) 35.4
1-CH3
5-CH3
C-3
1JC-C ) 35.4
1JC-C ) 41.9
1
1JC-C ≈ JC-C ) 35.4
1H NMR (600 MHz, CDCl3) of all-trans-[U-13C15]-3-methyl-5-(2,6,6-
trimethylcyclohex-1-enyl)penta-2,4-dienenitrile (4): δ 6.55 (ddm, 1JC-H
13-CH3
9-CH3
1JC-C ) 40.4
1JC-C ) 40.4
3
1
) 146.9 Hz, JH-H ) 15.2 Hz, H-5, 1H), 6.13 (ddm, JC-H ) 154.8
Hz, 3JH-H ) 15.2 Hz, H-4, 1H), 5.15 (ddd, 1JC-H ) 171.6 Hz, 2JC-H
≈
a The 13C chemical shift values are in complete agreement with the
published data of Liu et al.36
2JC-H ) 7.4 Hz, H-2, 1H), 2.20 (ddm, 1JC-H ) 128.2 Hz, 2JC-H ) 9.5
1
Hz, 3-CH3, 3H), 2.04 (dm, JC-H ) 127.3 Hz, H-3′, 2H), 1.70 (dm,
full cassette of site-directed isotopomers. The modular total
organic synthetic strategy will make a central contribution to
the already efficient expression methods. Together with the
isotope-sensitive NMR and vibrational spectroscopic methods
translation of the now available genetic information into
structural and functional information at the atomic level of the
proteins that are coded by the genes can be implemented. In
addition, these studies can also be extended without any problem
toward systems with site-directed modifications in the cofactors
and in the protein chain. These structural changes can be
compared with the native system at the atomic level leading to
an even deeper understanding of the function of the protein
system in question.
1JC-H ) 125.8 Hz, 2′-CH3, 3H), 1.61 (dm, JC-H ) 130.6 Hz, H4′,
1
2H), 1.43 (dm, 1JC-H ) 126.2 Hz, H-5′, 2H), 1.02 (dm, 1JC-H ) 125.5
Hz, 1′-CH3, 6H) ppm.
13C NMR (150 MHz, CDCl3) of all-trans-[U-13C15]-3-methyl-5-
(2,6,6-trimethylcyclohex-1-enyl)penta-2,4-dienenitrile (4): δ 157.3
1
1
1
(dddm, JC-C ) 73.8 Hz, JC-C ) 53.2 Hz, JC-C ) 42.2 Hz, C-3),
1
136.2 (m, C-5/C-1′), 132.6 (m, C-4/C-2′), 118.1 (ddm, JC-C ) 81.0
2
1
1
Hz, JC-C ) 8.8 Hz, CN), 96.2 (ddm, JC-C ) 81.0 Hz, JC-C ) 73.8
Hz, C-2), 39.4 (ddm, 1JC-C ≈ JC-C ) 32.9 Hz, C-5′), 34.1 (m, C-6′),
1
1
1
1
33.1 (dd, JC-C ≈ JC-C ) 36.8 Hz, C-3′), 28.8 (d, JC-C ) 35.6 Hz,
6′-CH3), 21.7 (dm, 1JC-C ) 43.4 Hz, 2′-CH3), 19.0 (dd, 1JC-C ≈ JC-C
1
1
) 36.8 Hz, C-4′), 16.5 (d, JC-C ) 42.2 Hz, 3-CH3) ppm.
HRMS (DIP - ESI): calcd for 13C15H22N 231.2255; found 231.2234.
[U-13C15]-3-Methyl-5-(2,6,6-trimethylcyclohex-1-enyl)penta-2,4-
dienal (5). A solution of 150 mg (0.65 mmol) of nitrile 4 in 25 mL of
dry light petroleum ether was cooled to -60 °C after which 0.98 mL
of 1 M (0.98 mmol) DIBAL-H was added via a syringe. The reaction
mixture was allowed to warm to -40 °C in 1 h. Subsequently, a
homogeneous mixture of 1.7 g of water absorbed on silica (0.3 g water/
gram silica) was added and stirring was continued for another 2 h at 0
°C. After drying of the mixture by adding MgSO4, all solids were
filtered off and thoroughly washed with diethyl ether. Evaporation of
the organic solvents in vacuo yielded a dark yellow liquid. The product
was purified by chromatography (silica gel, diethyl ether/40-60 light
petroleum ether, 5:95) to give 120 mg (0.51 mmol, 79%) of 5 as a
mixture of the 9-cis and all-trans compound. For the analysis the all-
trans isomer was isolated from the other isomer via HPLC purification.
Experimental Section
General. All experiments were carried out in a dry nitrogen
atmosphere, unless aqueous conditions were used. If necessary, reaction
vessels were flame-dried under nitrogen prior to use. The following
solvents were dried either by distillation (EtOH from Mg and light
petroleum ether (low boiling petroleum ether 40-60 °C) from P2O5)
or by storing over molecular sieves (4 Å) (THF). Saturated solutions
of Na2S2O3, NaOAc, NaHCO3, and NH4Cl refer to saturated solutions
of the salt in water. Brine refers to a saturated solution of NaCl in
water.
Reactions were monitored by using thin-layer chromatography (TLC,
on Merck F254 silica gel 60 aluminum sheets, 0.2 mm; spots were
visualized with UV-light (254 nm)) or treated with an oxidizing spray
(2 g of KMnO4 and 4 g of NaHCO3 in 100 mL of water). Column
chromatography was performed on Merck silica gel 60 (0.040-0.063
mm, 230-400 mesh).
1H NMR (600 MHz, CDCl3) of all-trans-[U-13C15]-3-methyl-5-(2,6,6-
1
trimethylcyclohex-1-enyl)penta-2,4-dienal (5): δ 10.13 (ddd, JC-H
)
2
3
169.7 Hz, JC-H ) 24.5 Hz, JC-H ) 8.1 Hz, CHO, 1H), 6.74 (ddm,
1JC-H ) 146.4 Hz, JH-H ) 15.0 Hz, H-5, 1H), 6.21 (m, H-4, 1H),
3
Melting points were measured on a Bu¨chi apparatus and are given
5.93 (dddd, 1JC-H ) 157.4 Hz, 2JC-H ≈ JC-H ≈ JH-H ) 7.4 Hz, H-2,
2
3
uncorrected.
1H), 2.31 (dm, 1JC-H ) 127.7 Hz, 3-CH3, 3H), 2.04 (dm, 1JC-H ) 125.2
1H NMR spectra were recorded on a Bruker AM-600 spectrometer
or a Bruker WM-300 with tetramethylsilane (TMS; δ ) 0.00 ppm) as
1
Hz, H-3′, 2H), 1.72 (dm, JC-H ) 125.9 Hz, 2′-CH3, 3H), 1.62 (dm,
1JC-H ) 128.7 Hz, H-4′, 2H), 1.48 (dm, 1JC-H ) 125.0 Hz, H-5′, 2H),
1
internal standard. H noise-decoupled 13C spectra were recorded on a
1
1.04 (dm, JC-H ) 125.5 Hz, 6′-CH3, 6H) ppm.
Bruker AM-600 at 151 MHz with chloroform (δ ) 77.0 ppm) as an
internal standard.
Mass spectra were recorded on a Finnigan MAT 900 equipped with
a direct insertion probe (DIP) or on a Finnigan MAT 700-TSQ equipped
with a custom-made electron-spray interface (ESI).
13C NMR (150 MHz, CDCl3) of all-trans-[U-13C15]-3-methyl-5-
(2,6,6-trimethylcyclohex-1-enyl)penta-2,4-dienal (5): δ 191.3 (dm,
1JC-C ) 56.8 Hz, CHO), 155.0 (m, C-3), 136.9 (m, C-1′), 135.6 (m,
C-4/C-5), 132.7 (ddd, JC-C ) 73.0 Hz, JC-C ≈ JC-C ) 41.7 Hz,
1
1
1
9
J. AM. CHEM. SOC. VOL. 124, NO. 22, 2002 6331

A R T I C L E S
Creemers and Lugtenburg
C-2′), 128.7 (ddm, 1JC-C ) 66.7 Hz, 1JC-C ) 56.8 Hz, C-2), 39.5 (dd,
by evaporation in vacuo of the formed SO2 and HCl. 8 (3.53 g, 20.9
mmol, 98%) was synthesized via this method as a slightly yellow
liquid.26
1
1
1JC-C ≈ JC-C ) 34.7 Hz, C-5′), 34.2 (m, C-6′), 33.2 (dd, JC-C
≈
1
1JC-C ) 34.7 Hz, C-3′), 28.9 (d, JC-C ) 35.5 Hz, 6′-CH3), 21.7 (dm,
1JC-C ) 74.2 Hz, 2′-CH3), 19.0 (dd, JC-C ≈ JC-C ) 34.7 Hz, C-4′),
1
1
1
Both the H NMR spectrum and the 13C NMR spectrum showed a
1
keto-enol mixture of [1,2,3,4-13C3]-ethyl 2-chloroacetoacetate.
1H NMR (600 MHz, CDCl3): δ 4.76 (ddd, 1JC-H ) 160.0 Hz, 2JC-H
12.9 (dm, JC-C ) 40.3 Hz, 3-CH3) ppm.
HRMS (DIP - ESI): calcd for 13C15H22O 233.2174; found 233.2123.
2
[U-13C20]-3,7-Dimethyl-9-(2,6,6-trimethylcyclohex-1-enyl)nona-
2,4,6,8-tetraenal (1). To a solution of 230 mg (1.0 mmol) of [1,2,3,4,-
(3-CH3)-13C5]-4-(diethylphosphono)-3-methyl-2-butenenitrile (3) in 15
mL of dry THF was added at 0 °C via a syringe 0.55 mL (0.9 mmol)
of n-BuLi (1.6 M solution in hexane). Stirring was continued for 15
min at room temperature. To the anion of the phosphonate was added
120 mg (0.51 mmol) of aldehyde 5 dissolved in 10 mL of dry THF.
The mixture was stirred for 1 h at room temperature. Quenching of
the reaction was accomplished by adding 50 mL of saturated NH4Cl
solution. The aqueous layer was extracted three times with 30 mL of
diethyl ether. The combined organic layers were washed with brine,
dried with MgSO4, and filtered. The product was purified by flash
chromatography (silica gel, diethyl ether/40-60 light petroleum ether,
20:80) to give 150 mg (0.49 mmol, 97%) of [U-13C20]-3,7-dimethyl-
9-(2,6,6-trimethylcyclohex-1-enyl)nona-2,4,6,8-tetraenenitrile (6) as a
mixture of isomers. A solution of 150 mg (0.49 mmol) of nitrile 6
dissolved in 20 mL of dry light petroleum ether was cooled to -60 °C
after which 0.75 mL of 1 M (0.75 mmol) DIBAL-H was added via a
syringe. The reaction mixture was allowed to warm to -40 °C in 1 h.
Subsequently, a homogeneous mixture of 1.3 g of water absorbed on
silica (0.3 g water/gram silica) was added and stirring was continued
for another 2 h at 0 °C. After the mixture was dried by adding MgSO4,
all solids were filtered off and thoroughly washed with diethyl ether.
Evaporation of the organic solvents in vacuo yielded a dark orange
liquid. The [U-13C20]-all-trans-retinal from the cis isomers was obtained
via column chromatography (silica gel, diethyl ether/40-60 light
petroleum ether, 20:80). Subsequently, a solution of the cis isomers in
40-60 light petroleum ether was irradiated for 2 h in the presence of
iodine crystals under a tungsten lamp. The organic layer, containing
the second batch of all-trans-retinal, was washed with saturated Na2S2O3
and dried with MgSO4. The all-trans-retinal was purified via column
chromatography purification (silica gel, diethyl ether/40-60 light
petroleum ether, 20:80). [U-13C20]-all-trans-retinal (1, 125 mg) was
synthesized via this method in a yield of 83% based on aldehyde 5.
≈ JC-H ) 5.6 Hz, H-2, 1H (keto)), 4.30 (m, H-1′, 4H (keto/enol)),
2.39 (dm, 1JC-H ) 129.4 Hz, H-4, 3H (keto)), 2.19 (dm, 1JC-H ) 129.5
Hz, H-4, 3H (enol)), 1.82 (s (br), OH, 1H (enol)), 1.32 (t, 3JH-H ) 7.1
Hz, H-2′, 6H (keto/enol)) ppm.
13C NMR (150 MHz, CDCl3): δ 196.6 (dd, 1JC-C ) 45.1 Hz, 1JC-C
1
1
) 38.9 Hz, C-3 (keto)), 172.5 (ddd, JC-C ) 86.1 Hz, JC-C ) 50.8
2
1
Hz, JC-C ) 4.7 Hz, C-3 (enol)), 169.3 (dm, JC-C ) 86.1 Hz, C-1
1
1
(enol)), 164.9 (d, JC-C ) 62.7 Hz, C-1 (keto)), 96.6 (ddd, JC-C
≈
1JC-C ) 86.1 Hz, JC-C ) 4.3 Hz, C-2 (enol)), 63.1 (s, C-1′ (keto/
enol)), 61.3 (ddd, 1JC-C ) 62.7 Hz, 1JC-C ) 38.9 Hz, 2JC-C ) 15.4 Hz,
C-2 (keto)), 26.2 (dd, 1JC-C ) 45.1 Hz, 2JC-C ) 15.4 Hz, C-4 (keto)),
19.7 (dm, 1JC-C ) 50.8 Hz, C-4 (enol)), 13.9 (s, C-2′ (keto/enol)) ppm.
2
HRMS (DIP - ESI): calcd for 13C4C2H9ClO3 168.0374; found
168.0432.
[U-13C3]-Chloroacetone (9). To 3.53 g (20.9 mmol) of [1,2,3,4-
13C4]-ethyl 2-chloroacetoacetate (8) dissolved in 20 mL of THF was
added 1.9 mL (0.1 mol) of water and 2.22 mL (41.8 mmol) of
concentrated H2SO4. The reaction mixture was refluxed for 40 h.
Subsequently, the mixture was cooled to room temperature followed
by the addition of 15 mL of water and 25 mL of diethyl ether. The
aqueous layer was extracted three times with 50 mL of diethyl ether.
The combined organic layers were washed with saturated NaHCO3
solution and brine. Drying with MgSO4 and filtration yielded 9 dissolved
in a mixture of diethyl ether and THF. Because of the volatility of the
product, only the diethyl ether was removed via distillation at
atmospheric pressure. The resulting solution of [1,2,3-13C3]-chloro-
acetone (9) in THF was used in the following step.
1H NMR (600 MHz, CDCl3): δ 4.09 (dd, 1JC-H ) 149.0 Hz, 2JC-H
1
) 4.1 Hz, H-1, 2H), 3.74 (m, 4H, THF), 2.31 (dd, JC-H ) 128.6 Hz,
2JC-H ) 4.1 Hz, H-3, 3H), 1.85 (m, 4H, THF) ppm.
13C NMR (150 MHz, CDCl3): δ 200.1 (dd, 1JC-C ) 43.2 Hz, 1JC-C
1
2
) 40.2 Hz, C-2), 67.7 (s, THF), 48.4 (dd, JC-C ) 40.2 Hz, JC-C
)
18.2 Hz, C-1), 26.7 (dd, 1JC-C ) 43.2 Hz, 2JC-C ) 18.2 Hz, C-3), 25.4
(s, THF) ppm.
1H NMR (600 MHz, CDCl3) of all-trans-[U-13C20]-retinal (1): δ
10.11 (ddd, 1JC-H ) 169.7 Hz, 2JC-H ) 24.5 Hz, 3JH-H ) 8.2 Hz, CHO,
1H), 7.14 (dm, 1JC-H ) 150.1 Hz, H-11, 1H), 6.36 (m, H-7/H-12, 2H),
6.17 (m, H-8/H-10, 2H), 5.97 (dm, 1JC-H ) 157.6 Hz, H-14, 1H), 2.33
[U-13C5]-4-Chloro-3-methyl-2-butenenitrile (12). A solution of 43.9
mmol of LDA was prepared at -60 °C from 6.42 mL (46.0 mmol) of
diisopropylamine dissolved in 75 mL of dried THF and 27.4 mL (43.9
mmol) of n-butyllithium (1.6 M solution in hexane). Subsequently, 1.00
g (23.0 mmol) of [13C2]-acetonitrile (10) dissolved in 20 mL of THF
was added dropwise. After the solution was stirred for 15 min at -60
°C, 3.62 mL (25.3 mmol) of diethyl chlorophosphate dissolved in 25
mL of THF was added slowly. The reaction mixture was allowed to
warm to 0 °C in 1 h. The solution of [1,2,3-13C3]-chloroacetone (9)
(maximum amount 20.9 mmol based on [1,2,3,4-13C4]-ethyl 2-chloro-
acetoacetate (8)) in THF was added slowly to the solution of the freshly
prepared phosphonate anion. Stirring was continued for 90 min at room
temperature followed by the addition of 50 mL of brine. The aqueous
layer was extracted three times with 50 mL of diethyl ether. The
combined organic layers were washed with brine, dried with MgSO4,
and filtered. Evaporation of the diethyl ether in vacuo yielded a yellow
liquid. The product was purified by chromatography (silica gel, diethyl
ether/40-60 light petroleum ether, 20:80) to give 1.76 g (14.7 mmol)
of 12 as a mixture of the cis and trans compound. The overall yield
starting from ester 8 was 70%.
1
1
(dm, JC-H ) 127.8 Hz, 13-CH3, 3H), 2.03 (dm, JC-H ) 126.7 Hz,
1
9-CH3, 3H), 2.01 (m, H-4, 2H), 1.72 (dm, JC-H ) 125.7 Hz, 5-CH3,
3H), 1.68 (dm, 1JC-H ) 127.5 Hz, H-3, 2H), 1.47 (dm, 1JC-H ) 128.2
1
Hz, H-2, 2H), 1.03 (dm, JC-H ) 125.1 Hz, 1-CH3, 6H) ppm.
13C NMR (150 MHz, CDCl3) of all-trans-[U-13C20]-retinal (1): δ
191.1 (d, JC-C ) 57.4 Hz, CHO), 154.9 (m, C-13), 141.3 (m, C-9),
1
1
1
137.5 (m, C-6), 137.0 (dd, JC-C ) 72.1 Hz, JC-C ) 58.4 Hz, C-8),
134.5 (dddd, 1JC-C ) 69.4 Hz, 1JC-C ) 53.9 Hz, 2JC-C ≈ JC-C ) 7.4
2
1
1
Hz, C-12), 132.5 (ddm, JC-C ) 69.9 Hz, JC-C ) 53.9 Hz, C-11),
1
1
1
130.5 (ddd, JC-C ) 75.1 Hz, JC-C ≈ JC-C ) 35.4 Hz, C-5), 129.2
1
1
(m, C-7/C-10/C-14), 39.5 (dd, JC-C ≈ JC-C ) 35.4 Hz, C-2), 34.2
(dddd, 1JC-C ≈ JC-C ≈ JC-C ≈ JC-C ) 35.4 Hz, C-1), 33.1 (dd, 1JC-C
1
1
1
≈ JC-C ) 35.4 Hz, C-4), 28.9 (d, 1JC-C ) 35.4 Hz, 1-CH3), 21.8 (dm,
1
1JC-C ) 41.9 Hz, 5-CH3), 19.1 (dd, JC-C ≈ JC-C ) 35.4 Hz, C-3),
13.1 (dm, 1JC-C ) 40.4 Hz, 13-CH3), 13.0 (dm, 1JC-C ) 42.1 Hz, 9-CH3)
ppm.
1
1
HRMS (DIP - ESI): calcd for 13C20H28O 304.2811; found 304.2804.
1
1H NMR (600 MHz, CDCl3): δ 5.50 (dm, JC-H ) 175.7 Hz, H-2
[1,2,3,4-13C4]-Ethyl 2-Chloroacetoacetate (8). To 2.86 g (21.3
mmol) of [1,2,3,4-13C4]-ethyl acetoacetate (7) was added at 0 °C via a
dropping funnel 1.80 mL (22.4 mmol) of sulfuryl chloride. The mixture
was stirred overnight at room temperature. The product was obtained
(trans), 1H), 5.27 (dm, JC-H ) 174.4 Hz, H-2 (cis), 1H), 4.27 (dm,
1
1
1JC-H ) 151.9 Hz, H-4 (cis), 2H), 4.08 (dm, JC-H ) 151.3 Hz, H-4
(trans), 2H), 2.16 (dm, 1JC-H ) 129.1 Hz, H-5 (trans), 3H), 2.07 (dm,
1JC-H ) 128.9 Hz, H-5 (cis), 3H) ppm.
9
6332 J. AM. CHEM. SOC. VOL. 124, NO. 22, 2002

[U-13C ]-all-trans-Retinal
A R T I C L E S
20
1
1
13C NMR (150 MHz, CDCl3): δ 157.9 (m, C-3 (cis/trans)), 115.9
42.3 Hz, C-7), 22.4 (ddm, JC-C ) 44.7 Hz, JC-C ) 38.7 Hz, C-4),
1
2
3
1
(ddd, JC-C ) 80.2 Hz, JC-C ) 8.5 Hz, JC-C ) 3.7 Hz, C-1 (trans)),
17.6 (dm, JC-C ) 42.3 Hz, C-8) ppm.
1
2
3
HRMS (DIP - ESI): calcd for 13C8H14O 134.1313; found 134.1351.
[U-13C10]-3,7-Dimethylocta-2,6-dienenitrile (15). A solution of 5.1
mmol of LDA was prepared at -60 °C from 1.48 mL (10.6 mmol) of
diisopropylamine dissolved in 30 mL of dried THF and 6.42 mL (10.2
mmol) of n-butyllithium (1.6 M solution in hexane). Subsequently, 0.22
g (5.1 mmol) of [13C2]-acetonitrile (10) dissolved in 5 mL of THF was
added dropwise. After the mixture was stirred for 15 min at -60 °C,
0.74 mL (5.1 mmol) of diethyl chlorophosphate dissolved in 5 mL of
THF was added slowly. The reaction mixture was allowed to warm to
0 °C in 1 h. A solution of 0.47 g (3.9 mmol) of [U-13C8]-6-methyl-5-
hepten-2-one (14) in 5 mL of THF was added slowly to the solution
of the phosphonate anion. Stirring was continued for 2 h at room
temperature. Adding 25 mL of brine quenched the reaction mixture.
The aqueous layer was extracted three times with 30 mL of diethyl
ether. The combined organic layers were washed with brine, dried with
MgSO4, and filtered. Evaporation of the diethyl ether in vacuo yielded
a yellow liquid. The product was purified by chromatography on silica
gel (diethyl ether/40-60 light petroleum ether, 10:90) to give 450 mg
(2.8 mmol, 72%) of 15 as a mixture of the cis and trans isomers.
115.1 (ddd, JC-C ) 79.7 Hz, JC-C ) 6.9 Hz, JC-C ) 4.6 Hz, C-1
1
1
(cis)), 99.0 (ddm, JC-C ) 79.7 Hz, JC-C ) 75.6 Hz, C-2 (cis)), 98.7
(ddm, 1JC-C ) 80.2 Hz, 1JC-C ) 77.6 Hz, C-2 (trans)), 46.8 (dm, 1JC-C
1
) 44.1 Hz, C-4 (trans)), 44.2 (dm, JC-C ) 44.5 Hz, C-4 (cis)), 20.9
(ddm, 1JC-C ) 43.5 Hz, 2JC-C ) 7.1 Hz, C-5 (cis)), 19.0 (dm, 1JC-C
)
43.0 Hz, C-5 (trans)) ppm.
HRMS (DIP - ESI): calcd for 13C5H6NCl 120.0357; found
120.0378.
[1,2,3,4,(3-CH3)-13C5]-4-(Diethylphosphono)-3-methyl-2-butene-
nitrile (3). To 1.76 g (14.7 mmol) of [1,2,3,4,(3-CH3)-13C5]-4-chloro-
3-methyl-2-butenenitrile (12) was added 3.31 mL (19.0 mmol) of
triethyl phosphite. The mixture was heated at 180 °C for 6 h. The
reaction was driven to completion by regular removal of the formed
chloroethane with vacuum suction. The mixture was allowed to cool
to room temperature and the product was isolated by using distillation
at reduced pressure. The yield was 2.95 g (13.3 mmol, 91%) of the
phosphonate 3 as a mixture of the cis and trans isomer (bp 118 °C, 0.2
mmHg).37
1
1H NMR (600 MHz, CDCl3): δ 5.28 (dm, JC-H ) 173.3 Hz, H-2
1
1H NMR (600 MHz, CDCl3): δ 5.10 (dm, JC-H ) 170.3 Hz, H-6
1
(cis/trans), 2H), 4.14 (m, POCH2CH3, 4H), 2.87 (ddm, JC-H ) 128.7
2
1
1
Hz, JP-H ) 23.9 Hz, H-4 (cis), 2H), 2.83 (ddm, JC-H ) 128.7 Hz,
(cis/trans), 2H), 5.02 (dm, JC-H ) 149.9 Hz, H-2 (trans), 1H), 4.93
2JP-H ) 23.5 Hz, H-4 (trans), 2H), 2.20 (dm, JC-H ) 128.8 Hz, H-5
1
1
1
(dm, JC-H ) 147.1 Hz, H-2 (cis), 1H), 2.55 (dm, JC-H ) 129.1 Hz,
(trans), 3H), 2.11 (dm, 1JC-H ) 128.6 Hz, H-5 (cis), 3H), 1.36 (t, 3JH-H
) 7.1 Hz, POCH2CH3, 6H) ppm.
1
H-5 (cis), 1H), 2.42 (dm, JC-H ) 129.3 Hz, H-5 (trans), 1H), 2.20
(dm, 1JC-H ) 128.8 Hz, H-9 (trans), 3H), 2.16 (dm, 1JC-H ) 128.0 Hz,
13C NMR (150 MHz, CDCl3): δ 155.5 (m, C-3 (cis/trans)), 116.3
(dm, 1JC-C ) 80.0 Hz, C-1 (cis/trans)), 99.0 (m, C-2 (cis/trans)), 62.5
(d, 2JC-P ) 6.8 Hz, POCH2CH3 (cis/trans)), 36.4 (ddm, 1JC-P ) 135.9
Hz, 1JC-C ) 40.6 Hz, C-4 (trans)), 34.6 (ddm, 1JC-P ) 135.9 Hz, 1JC-C
1
H-9 (cis), 3H), 2.05 (dm, JC-H ) 127.8 Hz, H-4 (trans), 2H), 1.90
1
1
(dm, JC-H ) 129.3 Hz, H-4 (cis), 2H), 1.69 (dm, JC-H ) 125.9 Hz,
1
H-8 (cis/trans), 6H), 1.60 (dm, JC-H ) 120.0 Hz, H-10, 6H) ppm.
13C NMR (150 MHz, CDCl3): δ 164.9 (m, C-3 (cis/trans)), 135.0
1
1
1
1
) 40.3 Hz, C-4 (cis)), 24.3 (dm, JC-C ) 42.0 Hz, C-5 (cis)), 22.3
(dddm, JC-C ) 74.2 Hz, JC-C ≈ JC-C ) 42.4 Hz, C-7 (cis)), 133.0
(dm, 1JC-C ) 42.0 Hz, C-5 (trans)), 16.3 (d, 3JC-P ) 6.1 Hz, POCH2CH3
(cis/trans)) ppm.
1
(m, C-7 (trans)), 122.0 (m, C-6 (cis/trans)), 117.1 (dm, JC-C ) 74.6
1
Hz, C-1 (cis)), 116.8 (dm, JC-C ) 74.9 Hz, C-1 (trans)), 95.7 (ddm,
HRMS (DIP - ESI): calcd for 13C5C4H16O3NP 222.1036; found
222.1002.
1JC-C ≈ JC-C ) 74.9 Hz, C-2 (trans)), 95.0 (ddm, JC-C ≈ JC-C
)
1
1
1
74.6 Hz, C-2 (cis)), 38.4 (ddm, 1JC-C ≈ JC-C ) 38.2 Hz, C-4 (trans)),
1
[U-13C8]-6-Methyl-5-hepten-2-one (14). To 1.40 g (10.5 mmol) of
[1,2,3,4-13C4]-ethyl acetoacetate (7) in 30 mL of dry ethanol was added
202 mg (8.8 mmol) of sodium in small portions. As soon as all sodium
was dissolved the mixture was cooled to 0 °C and 1.23 g (8.0 mmol)
of [U-13C5]-4-bromo-2-methyl-2-butene (13) dissolved in 25 mL of
ethanol was added dropwise. The reaction mixture was stirred at room
temperature for 2 h and subsequently refluxed for 4 h. During this time
a brown solid (NaBr) was formed. After removal of the EtOH by
evaporation, 20 mL of 10% NaOH solution was added. The mixture
was stirred overnight at room temperature after which it was heated to
60 °C for another 3 h to complete the saponification. Decarboxylation
was accomplished by acidification to a pH > 4. The aqueous layer
was extracted three times with 50 mL of diethyl ether. The combined
ether layers were washed with brine and dried over MgSO4. Concentra-
tion in vacuo yielded a dark yellow liquid. The product was purified
by chromatography on silica gel (diethyl ether/40-60 light petroleum
ether, 10:90) to give 0.72 g (6.0 mmol, 75%) of 14 as a slightly yellow
liquid.
36.1 (ddm, 1JC-C ≈ JC-C ) 36.2 Hz, C-4 (cis)), 25.7 (m, C-5 and C-8
1
(cis/trans)), 22.7 (dm, 1JC-C ) 41.3 Hz, C-9 (cis)), 20.8 (dm, 1JC-C
)
50.3 Hz, C-9 (trans)), 17.5 (m, C-10 (cis/trans)) ppm.
HRMS (DIP - ESI): calcd for 13C10H15N 159.1540; found 159.1519.
[U-13C10]-2,6,6-Trimethylcyclohex-2-ene-1-ylcarbonitrile (16). To
a solution of 1.43 mL of concentrated sulfuric acid in 15 mL of
nitromethane was added at 0 °C via a dropping funnel 450 mg (2.8
mmol) of [U-13C10]-3,7-dimethylocta-2,6-dienenitrile (15) dissolved in
5 mL of nitromethane. After the mixture was stirred for 45 min at 0
°C the reaction was quenched by adding 25 mL of ice-water. The
aqueous layer was extracted three times with 30 mL of diethyl ether.
The combined organic layers were washed with brine and saturated
NaOAc solution, dried with MgSO4, and filtered. Evaporation of the
diethyl ether in vacuo yielded a slightly yellow liquid. The product
was purified by chromatography (silica gel, diethyl ether/40-60 light
petroleum ether, 10:90) to give 372 mg (2.3 mmol, 83%) of 16 as a
mixture of [U-13C10]- 2,6,6-trimethylcyclohex-2-ene-1-ylcarbonitrile
(16a) and [U-13C10]-2,6,6-trimethylcyclohexene-1-ylcarbonitrile (16b)
(97:3).28
1H NMR (600 MHz, CDCl3): δ 5.06 (dm, 1JC-H ) 150.9 Hz, H-5,
1H), 2.46 (dm, 1JC-H ) 128.3 Hz, H-4, 2H), 2.25 (dm, 1JC-H ) 124.7
Hz, H-3, 2H), 2.14 (dd, 1JC-H ) 127.0 Hz, 2JC-H ) 5.7 Hz, H-1, 3H),
1H NMR (600 MHz, CDCl3) of [U-13C10]-2,6,6-trimethylcyclohex-
2-ene-1-ylcarbonitrile (16a): δ 5.58 (dm, 1JC-H ) 152.8 Hz, H-4, 1H),
2.76 (dm, JC-H ) 132.2 Hz, H-2, 1H), 2.08 (dm, JC-H ) 127.6 Hz,
H-5, 2H), 1.83 (dm, JC-H ) 126.2 Hz, 3-CH3, 3H), 1.55 (dm, JC-H
3
3
1.68 (dddd, 1JC-H ) 125.3 Hz, 2JC-H ≈ JC-H ≈ JC-H ) 5.7 Hz, H-7,
1
1
3H), 1.61 (dddd, 1JC-H ) 125.3 Hz, 2JC-H ≈ JC-H ≈ JC-H ) 5.7 Hz,
3
3
1
1
H-8, 3H) ppm.
1
) 127.5 Hz, H-6, 1H), 1.33 (dm, JC-H ) 128.9 Hz, H-6, 1H), 1.13
1
13C NMR (150 MHz, CDCl3): δ 208.9 (ddm, 1JC-C ≈ JC-C ) 38.7
1
1
(dm, JC-H ) 125.8 Hz, 1-CH3, 3H), 1.09 (dm, JC-H ) 125.6 Hz,
1-CH3, 3H) ppm.
13C NMR (150 MHz, CDCl3) of [U-13C10]-2,6,6-trimethylcyclohex-
1
1
1
Hz, C-2), 132.7 (dddd, JC-C ) 73.9 Hz, JC-C ≈ JC-C ) 42.3 Hz,
2JC-C ) 5.1 Hz, C-6), 122.5 (ddm, 1JC-C ) 73.9 Hz, 1JC-C ) 44.7 Hz,
1
1
2
C-5), 43.7 (dddm, JC-C ≈ JC-C ) 38.7 Hz, JC-C ) 13.8 Hz, C-3),
1
1
2-ene-1-ylcarbonitrile (16a): δ 126.4 (dddm, JC-C ) 73.3 Hz, JC-C
29.9 (dd, 1JC-C ) 38.7 Hz, 2JC-C ) 13.8 Hz, C-1), 25.6 (dm, 1JC-C
)
) 43.5 Hz, 1JC-C ) 39.9 Hz, C-3), 124.5 (ddm, 1JC-C ) 73.3 Hz, 1JC-C
1
1
) 39.8 Hz, C-4), 119.6 (d, JC-C ) 55.2 Hz, CN), 43.8 (dddm, JC-C
(37) Gebhard, R.; Van der Hoef, K.; Lefeber, A. W. M.; Erkelens, C.;
1
1
1
Lugtenburg, J. Recl. TraV. Chim. Pays-Bas 1990, 109, 378-387.
) 55.2 Hz, JC-C ) 43.5 Hz, JC-C ) 34.3 Hz, C-2), 32.7 (dd, JC-C
9
J. AM. CHEM. SOC. VOL. 124, NO. 22, 2002 6333

A R T I C L E S
Creemers and Lugtenburg
1
1
1
1
≈ JC-C ) 34.3 Hz, C-6), 31.8 (dddd, 1JC-C ≈ JC-C ≈ JC-C ≈ JC-C
1H NMR (600 MHz, CDCl3): δ 5.65 (dm, 1JC-H ) 159.7 Hz, H-2,
1H), 4.14 (m, H-1′, 2H), 2.16 (dm, JC-H ) 127.6 Hz, H-4 (Z), 3H),
1
1
1
) 34.3 Hz, C-1), 26.8 (d, JC-C ) 34.3 Hz, 1-CH3), 25.5 (d, JC-C
34.3 Hz, 1-CH3), 22.4 (m, C-5 and 3-CH3) ppm.
)
1
3
1.89 (dm, JC-H ) 126.7 Hz, H-4 (E), 3H), 1.27 (t, JH-H ) 7.1 Hz,
H-2′, 3H) ppm.
HRMS (DIP - ESI): calcd for 13C10H15N 159.1540; found 159.1562.
[U-13C10]-2,6,6-Trimethyl-cyclohex-2-ene-1-yl-carbaldehyde (17).
A solution of the mixture of both the R-nitrile 16a and â-nitrile 16b
(330 mg, 2.1 mmol) in 30 mL of dry light petroleum ether was cooled
to -60 °C and 3.15 mL of 1 M (3.2 mmol) DIBAL-H was added via
a syringe. The reaction mixture was allowed to warm to -40 °C in 1
h. TLC analysis showed complete conversion of R-nitrile 16a whereas
â-nitrile 16b was unaffected. After addition of a homogeneous mixture
of 5.5 g of water adsorbed on silica (0.3 g of water/g of silica), the
reaction mixture was stirred for 2 h at 0 °C. Subsequently, MgSO4
was added and the solids were filtered off and thoroughly washed with
diethyl ether. Evaporation of the organic solvents in vacuo yielded a
dark yellow liquid. The product was purified by chromatography (silica
gel, diethyl ether/40-60 light petroleum ether, 10:90) to give 249 mg
(1.5 mmol, 73%) of aldehyde 17 and 12 mg (0.08 mmol, 4%) of
â-nitrile 16b.
13C NMR (150 MHz, CDCl3): δ 166.7 (ddm, 1JC-C ) 76.0 Hz, 2JC-C
1
1
1
) 5.5 Hz, C-1), 156.3 (dddd, JC-C ) 72.1 Hz, JC-C ≈ JC-C ) 40.2
2
1
1
Hz, JC-C ) 5.5 Hz, C-3), 116.0 (dd, JC-C ) 76.0 Hz, JC-C ) 72.1
1
Hz, C-2), 59.4 (s, C-1′), 27.3 (dm, JC-C ) 40.2 Hz, C-4 (E)), 20.1
1
(dm, JC-C ) 40.2 Hz, C-4 (Z)), 14.3 (s, C-2′) ppm.
HRMS (DIP - ESI): calcd for 13C5C2H12O2 133.1005; found
133.1047.
[U-13C5]-3-Methyl-2-buten-1-ol (24). A solution of 1.85 g (13.9
mmol) of [1,2,3,4,(3-CH3)-13C5]-ethyl-3-methyl-2-butenoate (23) in 50
mL of dry light petroleum ether was cooled to -60 °C and 30.6 mL of
1 M (30.6 mmol) DIBAL-H was added via a syringe. Stirring was
continued for 30 min at -60 °C, followed by the addition of a
homogeneous mixture of 53.5 g of water adsorbed on silica (0.3 g of
water/g of silica). The mixture was stirred for 2 h at 0 °C. Subsequently,
MgSO4 was added and the solids were filtered off and thoroughly
washed with diethyl ether. Evaporation of the organic solvents in vacuo
yielded 24: 1.62 g of a slightly yellow liquid. The product was used
without further purification.
1H NMR (600 MHz, CDCl3) of [U-13C10]-2,6,6-trimethylcyclohex-
2-ene-1-ylcarbaldehyde (17): δ 9.47 (ddd, 1JC-H ) 171.2 Hz, 2JC-H
20.9 Hz, JH-H ) 5.2 Hz, CHO, 1H), 5.72 (dm, JC-H ) 151.9 Hz,
H-3, 1H), 2.36 (dm, JC-H ) 130.3 Hz, H-1, 1H), 2.14 (dm, JC-H
)
3
1
1H NMR (600 MHz, CDCl3): δ 5.40 (dm, 1JC-H ) 153.2 Hz, H-2,
1H), 4.12 (dm, 1JC-H ) 141.7 Hz, H-1, 2H), 1.74 (dddd, 1JC-H ) 125.7
1
1
)
1
127.4 Hz, H-4, 2H), 1.59 (dm, JC-H ) 127.2 Hz, 2-CH3, 3H), 1.49
Hz, 2JC-H ≈ JC-H ≈ JC-H ) 5.1 Hz, H-4 (E), 3H), 1.68 (dddd, 1JC-H
3
3
(dm, 1JC-H ) 125.1 Hz, H5, 2H), 0.99 (dm, 1JC-H ) 124.8 Hz, 1-CH3,
2
3
3
1
) 125.7 Hz, JC-H ≈ JC-H ≈ JC-H ) 5.1 Hz, H-4 (Z), 3H), 1.54 (s
3H), 0.91 (dm, JC-H ) 125.3 Hz, 1-CH3, 3H) ppm.
13C NMR (150 MHz, CDCl3) of [U-13C10]-2,6,6-trimethylcyclohex-
(br), OH, 1H) ppm.
13C NMR (150 MHz, CDCl3): δ 136.3 (ddd, 1JC-C ) 72.5 Hz, 1JC-C
) 42.8 Hz, 1JC-C ) 41.4 Hz, C-3), 123.5 (ddm, 1JC-C ) 72.5 Hz, 1JC-C
2-ene-1-ylcarbaldehyde (17): δ 202.4 (d, 1JC-C ) 38.4 Hz, CHO), 127.1
1
1
1
(dm, JC-C ) 72.5 Hz, C-2), 125.4 (ddm, JC-C ) 72.5 Hz, JC-C
)
) 47.5 Hz, C-2), 59.3 (dm, 1JC-C ) 47.5 Hz, C-1), 25.7 (dm, 1JC-C
)
38.4 Hz, C-3), 63.3 (m, C-1), 31.6 (m, C-5/C-6), 27.4 (d, 1JC-C ) 34.1
Hz, 6-CH3), 26.9 (d, 1JC-C ) 33.4 Hz, 6-CH3), 23.0 (m, C-4), 22.5 (d,
1JC-C ) 42.4 Hz, 2-CH3) ppm.
1
42.8 Hz, C-4 (E)), 17.7 (dm, JC-C ) 41.4 Hz, C-4 (Z)) ppm.
HRMS (DIP - ESI): calcd for 13C5H10O 91.0914; found 91.0898.
[U-13C5]-4-Bromo-2-methyl-2-butene (13). A solution of 1.62 g
of [1,2,3,4,(3-CH3)-13C5]-3-methyl-2-buten-1-ol (24) in 15 mL of
dichloromethane was cooled to 0 °C and 6.63 mL of 47% hydrobromic
acid dissolved in dichloromethane was added slowly. Stirring was
continued for 2 h with exclusion of light. To the mixture was added
25 mL of a saturated NaHCO3 solution followed by some drops of
1,2-epoxybutane. The aqueous layer was extracted three times with 50
mL of diethyl ether. The combined organic layers were washed with
brine, dried with MgSO4, and filtered. Because of the volatility of the
product, most of the organic solvent was removed by distillation at
atmospheric pressure. Only the last part of the solvent was removed
by evaporation in vacuo yielding 13: 1.23 g (8.0 mmol) of a yellow
liquid. The overall yield starting from ester 23 was 57%. The product
was used without further purification.
HRMS (DIP - ESI): calcd for 13C10H16O 162.1537; found 162.1573.
[U-13C10]-2,6,6-Trimethylcyclohex-1-enylcarbaldehyde (2). To a
solution of aldehyde 17 (180 mg, 1.1 mmol) in 5 mL of MeOH was
added at 0 °C 1.5 mL of 5% KOH in MeOH. After the mixture was
stirred for 45 min at 0 °C the reaction was quenched by adding 5 mL
of brine. The aqueous layer was extracted three times with 15 mL of
diethyl ether. The combined organic layers were washed with brine,
dried with MgSO4, and filtered. Evaporation of the diethyl ether in
vacuo yielded 2: 142 mg (0.86 mmol, 80%) of a slightly yellow liquid.
1H NMR (300 MHz, CDCl3): δ 10.13 (ddm, JC-H ) 169.8 Hz,
1
2JC-H ) 21.5 Hz, CHO, 1H), 2.19 (dm, JC-H ) 127.3 Hz, H-3, 2H),
1
2.09 (ddm, 1JC-H ) 127.1 Hz, 2JC-H ) 10.0 Hz, 2-CH3, 3H), 1.62 (dm,
1JC-H ) 129.6 Hz, 4-H, 2H), 1.44 (dm, JC-H ) 125.1 Hz, H-5, 2H),
1
1
1.19 (dm, JC-H ) 125.6 Hz, 6-CH3, 6H) ppm.
1H NMR (600 MHz, CDCl3): δ 5.53 (dm, 1JC-H ) 158.7 Hz, H-2,
13C NMR (75 MHz, CDCl3): δ 192.2 (d, JC-C ) 52.4 Hz, CHO),
1
1
1H), 4.01 (dm, 152.8 Hz, H-1, 2H), 1.78 (dddd, JC-H ) 126.1 Hz,
1
1
1
159.9 (ddd, JC-C ) 68.4 Hz, JC-C ≈ JC-C ) 40.2 Hz, C-2), 140.5
3
3
1
2JC-H ≈ JC-H ≈ JC-H ) 5.6 Hz, H-4 (E), 3H), 1.73 (dddd, JC-H
)
(ddd, 1JC-C ) 68.4 Hz, 1JC-C ) 52.4 Hz, 1JC-C ) 41.5 Hz, C-1), 40.5
2
3
3
126.1 Hz, JC-H ≈ JC-H ≈ JC-H ) 5.6 Hz, H-4 (Z), 3H) ppm.
1
1
1
1
(dd, JC-C ≈ JC-C ) 33.2 Hz, C-5), 35.7 (dd, JC-C ≈ JC-C ) 40.2
13C NMR (150 MHz, CDCl3): δ 140.1 (ddd, 1JC-C ) 73.5 Hz, 1JC-C
1
Hz, C-3), 33.0 (m, C-6), 27.7 (d, JC-C ) 35.1 Hz, 6-CH3), 18.8 (m,
≈ JC-C ) 42.5 Hz, C-3), 120.7 (ddm, 1JC-C ) 73.5 Hz, 1JC-C ) 47.7
1
C-4/2-CH3) ppm.
1
1
Hz, C-2), 29.7 (dm, JC-C ) 47.7 Hz, C-1), 25.8 (dm, JC-C ) 42.5
HRMS (DIP - ESI): calcd for 13C10H16O 162.1537; found 162.1569.
[1,2,3,4,(3-CH3)-13C5]-Ethyl 3-Methyl-2-butenoate (23). To 5.56
g (24.6 mmol) of ethyl [1,2-13C2]-2-(diethylphosphono)acetate (21)
dissolved in 75 mL of dry of THF was added at 0 °C via a syringe
14.3 mL (22.9 mmol) of n-BuLi (1.6 M solution in hexane). Stirring
was continued for 15 min at room temperature. To the anion of the
phosphonate was added 1.00 g (16.4 mmol) of [1,2,3-13C3]-acetone (22),
dissolved in dry THF. The mixture was stirred for another 3 h at room
temperature. Quenching of the reaction was accomplished by adding
50 mL of saturated NH4Cl solution. The aqueous layer was extracted
three times with 50 mL of diethyl ether. The combined organic layers
were washed with brine, dried with MgSO4, and filtered. Evaporation
of the diethyl ether in vacuo yielded 23: 1.99 g (14.9 mmol, 91%) of
a slightly yellow liquid.
1
Hz, C-4 (E)), 17.5 (dm, JC-C ) 42.5 Hz, C-4 (Z)) ppm.
HRMS (DIP - ESI): calcd for 13C5H9Br 153.0055; found 153.0100.
Acknowledgment. The authors are very grateful to Cam-
bridge Isotope Laboratories Inc., USA for their kind gift of all
13C-labeled starting materials. The authors wish to thank C.
Erkelens and F. Lefeber for recording the NMR spectra and B.
Hofte and B. Karabatak for recording the mass spectra.
Supporting Information Available: Additional experimental
details (PDF). This material is available free of charge via the
Internet at http://pubs.acs.org.
JA012368H
9
6334 J. AM. CHEM. SOC. VOL. 124, NO. 22, 2002