Synthesis of isotopically labeled all-trans retinals for DNP-enhanced solid-state NMR studies of retinylidene proteins

Article abstract of DOI:10.1002/jlcr.3576

Three all-trans retinals containing multiple ¹³C labels have been synthesized to enable dynamic nuclear polarization enhanced solid-state magic angle spinning NMR studies of novel microbial retinylidene membrane proteins including proteorhodpsi

Full text of DOI:10.1002/jlcr.3576

Received: 1 June 2017
DOI: 10.1002/jlcr.3576
Accepted: 17 October 2017
S P E C I A L I S S U E A R T I C L E
Synthesis of isotopically labeled all‐trans retinals for DNP‐
enhanced solid‐state NMR studies of retinylidene proteins
Alexander J. Leeder1
Clemens Glaubitz2,3
| Lynda J. Brown1
| Richard C.D. Brown1
| Johanna Becker‐Baldus2,3 | Michaela Mehler2,3
|
1 Department of Chemistry, University of
Southampton, Southampton, UK
2 Institute of Biophysical Chemistry,
Goethe University Frankfurt, Frankfurt,
Germany
3 Centre for Biomolecular Magnetic
Resonance, Goethe University Frankfurt,
Frankfurt, Germany
Three all‐trans retinals containing multiple 13C labels have been synthesized to
enable dynamic nuclear polarization enhanced solid‐state magic angle spinning
NMR studies of novel microbial retinylidene membrane proteins including
proteorhodpsin and channelrhodopsin. The synthetic approaches allowed spe-
cific introduction of 13C labels in ring substituents and at different positions
in the polyene chain to probe structural features such as ring orientation and
interaction of the chromophore with the protein in the ground state and in
photointermediates. [10‐18‐13C9]‐All‐trans‐retinal (1b), [12,15‐13C2]‐all‐trans‐
retinal (1c), and [14,15‐13C2]‐all‐trans‐retinal (1d) were synthesized in in 12,
8, and 7 linear steps from ethyl 2‐oxocyclohexanecarboxylate (5) or β‐ionone
(4), respectively.
Correspondence
Richard C. D. Brown, Department of
Chemistry, University of Southampton,
Highfield, Southampton SO17 1BJ, UK.
Email: rcb1@soton.ac.uk
Clemens Glaubitz, Institute of Biophysical
Chemistry, Goethe University Frankfurt,
Max‐von‐Laue‐Str. 9, 60438 Frankfurt,
Germany.
Email: glaubitz@em.uni‐frankfurt.de
Funding information
Engineering and Physical Sciences
Research Council, Grant/Award Numbers:
EP/K039466/1 and EP/L505067/1;
Deutsche Forschungsgemeinschaft, Grant/
Award Number: Project SFB 807 (Trans-
port and communication acros; European
Regional Development Fund
1 | INTRODUCTION
and channels, was for a long time dominated by extensive
studies on bacteriorhodopsin. This field has attracted
Retinoids, or vitamin A derivatives, are ubiquitous in
nature and have been studied extensively due to their
wide‐ranging biological roles,1 such as in vision,2 immune
function,3 and tumor suppression.4 The incorporation of
retinals into rhodopsins, through a protonated Schiff's
base linkage, is a fundamental requirement for the func-
tion of these light‐sensitive membrane proteins, which
are found in all phyla of life. A well‐known example is
the G‐protein coupled receptor rhodopsin, which is
responsible for dim light vision in mammals. The field
of microbial rhodopsins, which act as sensors, ion pumps,
recent attention with the discovery of many new rhodop-
sins such as proteorhodopsin (PR), channelrhodopsin
(ChR), and Krokinobacter eikastus rhodopsin 2 (KR2).
PRs form a family of light‐driven proton pumps,5 ChRs
function as light‐gated ion channels,6 and KR2 is the
first‐discovered light‐driven Na+ pump.7 These proteins
have stimulated much recent interest, with possible appli-
cations in areas such as optogenetics and energy harvest-
ing.8-10 A significant structural feature of PR, ChR, and
KR2 is that they contain an all‐trans‐retinylidene chromo-
phore in their unactivated states; in contrast, the visual
J Label Compd Radiopharm. 2017;1–12.
wileyonlinelibrary.com/journal/jlcr
Copyright © 2017 John Wiley & Sons, Ltd.
1

2
LEEDER ET AL.
receptor rhodopsin contains the polyene in its 11‐Z
configuration.
insight into light‐driven conformational changes in
retinylidene proteins by using DNP‐enhanced solid‐state
NMR with 15N‐labeled and 13C‐labeled proteins.12 The
synthetic routes to polyenes 1b and 1c were planned to
make use of inexpensive and readily available 13C‐
enriched (99 atm % 13C) building blocks and ultimately
permit flexible access to other labeling patterns
(Figure 2). Stereocontrol in the final olefination was not
critical here, due to the fact that labeled 13E/Z retinal
isotopomers are separable, and the 13Z isomers also rep-
resent useful reference compounds when reconstituted
into rhodopsins. A more convergent approach applied to
the synthesis of 1d (see Scheme 4) does allow for high
levels of 13E selectivity but was considered less conve-
nient for the labeling patterns present in 1b and 1c.
Accordingly, singly and doubly 13C‐labeled diethyl
Much effort has been directed toward structural inves-
tigation of rhodopsins and their photocycle intermediates,
to further advance understanding of how irradiation of
the retinylidene chromophore induces structural changes
that trigger the enzymatic cascade ultimately leading to
vision, or enable ion pumping or channel gating.1 In this
regard, solid‐state magic angle spinning (MAS) 13C NMR
at low temperatures has provided high‐ resolution struc-
tural information such as bond lengths and torsion angles
in the chromophore, which together with quantum chem-
ical calculation, connects ground state crystallographic
data and kinetic and functional data derived from optical
spectroscopy and electrophysiology.11
One key advantage of solid‐state NMR is certainly the
fact that membrane proteins can be analyzed directly
embedded within a lipid bilayer, which is essential for mech-
anistic studies under conditions resembling their native
environment.12,13 However, the concentration of the protein
in the lipid bilayer is restricted due to its stability and optical
density of the sample, limiting the amount of observable
protein for biophysical studies. Successful application of
13C MAS NMR to these systems has therefore relied on stra-
tegically inserted 13C labels within the retinylidene chromo-
phore to achieve useful signal to noise ratios. Even with 13C
labeling, NMR signals remain fairly weak, especially for
photocycle intermediates. Cross effect dynamic nuclear
polarization (DNP)14 has emerged as a technique to increase
sensitivity in solid‐state NMR, highlighting its value in the
field of structural determination. Here, we report the synthe-
sis of 3 all‐trans retinal isotopomers containing multiple 13C
labels at positions defined to enable DNP‐enhanced solid‐
state NMR structural studies of retinylidene proteins such
as PR, ChR, and KR2 (Figure 1).
(cyanomethyl)phosphonates
3
and
triethyl
phosphonoacetates 2 were prepared from labeled acetoni-
trile and ethyl bromoacetate respectively, and in high
yields (Scheme 1).15,16
The
synthesis
of
the
13C‐labeled
(1b),
[10,11,12,13,14,15,16,17,18‐13C9]‐all‐trans‐retinal
containing 9 positions of 13C‐enrichment, began by
trimethylation of ethyl 2‐oxocyclohexanecarboxylate
(5)17 with [13C]‐CH3I, to afford the β‐ketoester 6
(Scheme 2). From 6, acid‐catalyzed hydrolysis and decar-
boxylation afforded a relatively volatile cyclohexanone,
which was converted to vinyl triflate 7 prior to a Heck
reaction with methylvinyl ketone18 that delivered triply‐
labeled β‐ionone [13C3]‐4. The first olefination reaction
using doubly 13C‐labeled triethyl phosphonoacetate
([13C2]‐2) yielded the trienoate [13C5]‐8 as an unseparated
mixture of E/Z isomers (~9:1 from 1H NMR) in 78%
yield.19 Following LiAlH4 reduction of the isomeric mix-
ture to the allylic alcohols and subsequent TPAP oxida-
tion, all‐E‐trienal stereoisomer [13C5]‐9 was isolated by
column chromatography.
Olefination of trienal [13C5]‐9 using phosphonate
[13C2]‐2 for the second time introduced the C12 and C13
labeled carbon atoms, affording the tetraenoate as its all‐
E‐stereoisomer. Formation of Weinreb amide [13C7]‐10
2 | RESULTS AND DISCUSSION
Three all‐trans‐retinals, 1b to d (Figure 1), containing
multiple 13C labels were designed to enable structural
FIGURE 1 Retinal structures showing synthetic 13C‐labeling
FIGURE 2 Synthesis plan for labeled retinal isotopomers 1b and
1c. (* indicates position of 13C)
patterns (* indicates position of 13C)

LEEDER ET AL.
3
SCHEME 1 Synthesis of 13C‐labeled phosphonates [13C2]‐2,
[13C1]‐2, [13C2]‐3, and [13C1]‐3. (* indicates position of 13C)
SCHEME 3 Synthesis of [12,15‐13C2]‐all‐trans‐retinal (1c) and
[12,15‐13C2]‐(13Z)‐retinal (12). (* indicates position of 13C)
and the all‐E isomers, 12 and 1c, respectively, were both
isolated as pure compounds.
A more convergent strategy was applied to the synthe-
sis of 13C‐labeled all‐E‐retinal 1d, by combining 2 major
fragments 9 and 13 (Figure 3).1b, 20, 21 The fragment con-
taining 2 13C labels required the phosphonium ylide 14,
which was combined with hydroxyacetone to give the E
enoate 15 (Scheme 4). The allylic alcohol 15 was
converted to the phosphonate 13 by bromination and
Arbuzov reaction.20 The final olefination proceeded with
high E selectivity, and the ensuing reduction‐oxidation
sequence secured [14,15‐13C2]‐all‐trans‐retinal (1d).21
The labeled [14,15‐13C2]‐retinal (1d) has been
incorporated in 15N‐labeled channelrhodopsin‐2 (ChR2)
reconstituted into lipid bilayers, and the results of DNP‐
enhanced solid‐state NMR spectroscopy for the
ground and intermediate states have been reported.12
Proteorhodopsin containing [10,11,12,13,14,15,16,17,18‐
13C9]‐all‐trans‐retinal (1b) was also reconstituted as pro-
teoliposomes, and DNP‐enhanced solid‐state 2D 13C‐13C
correlation spectroscopy shows the 6 labeled carbon
SCHEME 2 Synthesis of [10,11,12,13,14,15,16,17,18‐13C9]‐all‐
trans‐retinal (1b). (* indicates position of 13C)
facilitated the subsequent high‐yielding conversion to
methyl ketone [13C7]‐11.15 The final olefination with
diethyl (cyanomethyl)phosphonate [13C2]‐3 resulted in a
mixture of 13E/Z isomers, which following DIBAL‐H
reduction and aqueous work‐up,15 delivered the
[10‐18‐13C9]‐all‐trans‐retinal as a 2:1 mixture with the
minor 13Z isomer in 70% yield. This isomeric mixture
was separated by column chromatography and prepara-
tive HPLC on silica gel to provide a pure sample of the
all‐E stereoisomer 1b.
FIGURE 3 Synthesis plan for labeled retinal 1d. (* indicates
position of 13C)
The synthesis of [12,15‐13C2]‐all‐trans‐retinal (1c) was
achieved similarly, but starting from commercial β‐
ionone and using the singly 13C‐labeled phosphonates
[13C1]‐2 and [13C1]‐3 (Scheme 3).19 In this case, the 13Z
SCHEME
4
Synthesis of [14,15‐13C2]‐all‐trans‐retinal (1d).
(* indicates position of 13C)

4
LEEDER ET AL.
4.1 | Ethyl 1,3,3‐tri(methyl‐13C)‐2‐
oxocyclohexane‐1‐carboxylate (6)
atoms present in the polyene chain (Figure 4). [12,15‐
13C2]‐All‐trans‐retinal was incorporated into ChR2 and
will be used for precise conformational analysis of the
chromophore. Full assignments and biophysical studies
of these and other retinylidene proteins will be reported
elsewhere.
By adaption of a procedure by Stevans et al,17 to a suspen-
sion of NaH (60% in mineral oil, 5.60 g, 140 mmol) in THF
(80 mL) under N2 at 0°C was added a solution of ethyl 2‐
oxocyclohexane‐1‐carboxylate (5.91 mL, 34.5 mmol) in
THF (80 mL) dropwise over 90 minutes. The mixture
was stirred on ice for 30 minutes before addition of
13CH3I (6.47 mL, 104 mmol) dropwise over 20 minutes.
The mixture was stirred at rt for 36 hours with the forma-
tion of a white precipitate after 1 hour. The mixture was
diluted with Et2O (100 mL) and cooled to 0°C before
quenching with ice‐cold H2O (25 mL). The aqueous layer
was extracted with Et2O (3 × 30 mL), and the combined
organic solution was washed with brine (30 mL), dried
(MgSO4), and the solvent removed under reduced pres-
sure. Purification by silica gel chromatography (EtOAc:
hexane = 2:98) afforded the product as a colorless oil
(6.01 g, 27.9 mmol, 81%). FT‐IR (neat) νmax 2939 (m),
3 | CONCLUSIONS
The synthesis of 3 all‐E retinal isotopomers, 1b to 1d,
containing multiple 13C‐labels, has been achieved
in 12, 8, and
7
linear steps from ethyl 2‐
oxocyclohexanecarboxylate (5) or β‐ionone (4) respec-
tively. For 1b and 1c, the final olefination delivered a
2:1 mixture of all‐trans‐retinals and their 13Z isomers,
respectively, which were separated by preparative HPLC.
The labeled chromophores have been incorporated in
retinylidene proteins reconstituted as proteoliposomes to
provide samples suitable for solid‐state DNP‐enhanced
NMR studies. The biophysical studies of this intriguing
group of light‐responsive proteins containing site‐specifi-
cally 13C‐labeled synthetic chromophores are ongoing12
and will be disclosed in due course.
1732 (m), 1703 (s), 1423 (m), 1074 (m) cm−1. H NMR
1
(400 MHz, CDCl3): δ 4.23 to 4.04 (m, 2H), 2.58 to 2.50
(m, 1H), 2.06 to 1.91 (m, 1H), 1.76 to 1.68 (m, 1H), 1.65
to 1.49 (m, 2H), 1.45 to1.35 (m, 2H), 1.29 (d, JCH = 131.0 Hz,
3H), 1.23 (t, JHH = 7.1 Hz, 3H), 1.09 (dd, JCH = 128.0,
JCH = 5.0 Hz, 3H), 1.07 (dd, JCH = 127.7, JCH = 5.0 Hz, 3H)
ppm (Only peaks for 13C labeled carbons reported). LRMS
(ES+) m/z 216 [M + H]+, 238 [M + Na]+. HRMS (ES+)
For C913C3H20O3Na+ calculated 238.1405, found
238.1402 Da.
4 | EXPERIMENTAL
Supporting information includes general experimental
details and copies of 1H and 13C NMR spectra.
4.2 | 2,2,6‐Tri(methyl‐13C)cyclohexan‐1‐one
A
solution
of
ethyl
1,3,3‐tri(methyl‐13C)‐2‐
oxocyclohexane‐1‐carboxylate (6) (5.48 g, 25.5 mmol)
and conc. HCl (40 mL) in EtOH (80 mL) was heated
under reflux for 3 days. The mixture was then cooled
and diluted with H2O (20 mL) and neutralized with 1 M
NaOH (170 mL). The aqueous layer was extracted with
pentane (4 × 50 mL) and the combined organic solution
washed with brine and dried (MgSO4). The solvent was
removed under reduced pressure (400 mBar, 20°C) to
afford the product as a colorless oil (3.64 g, 32.1 mmol,
100%). FT‐IR (neat) νmax 2967 (w), 2929 (m), 2868 (w),
1
1704 (s), 1454 (m) cm−1. H NMR (CDCl3, 400 MHz): δ
2.72 to 2.59 (m, 1H), 2.10 to 2.00 (m, 1H), 1.88 (qt,
JHH = 13.4, JHH = 3.8 Hz, 1H), 1.81 to 1.72 (m, 1H), 1.69
to 1.61 (m, 1H), 1.60 to 1.49 (m, 1H), 1.38 to 1.22 (m,
1H), 1.18 (dd, JCH = 127.0, JCH = 4.9 Hz, 3H), 1.04
(dd, JCH = 126.4, JCH = 4.9 Hz, 3H), 0.99 (dd, JCH = 126.8,
JHH = 6.4 Hz, 3H) ppm. 13C NMR (CDCl3, 101 MHz): δ
25.6 (13CH3), 25.3 (13CH3), 15.0 (13CH3) ppm (Only peaks
for 13C labeled carbons reported). LRMS (EI) m/z 143 [M]
FIGURE 4 DNP‐enhanced 13C‐13C MAS‐NMR spectrum of PR
incorporating 1b showing the 6 labeled carbon atoms in the chain.
(Bruker 400 MHz DNP system with 3.2‐mm HCN Cryo MAS probe,
~107 K, PDSD mixing time 20 ms, 192 scans, 292 increments with a
33‐μs delay, 1.5‐mg protein in DMPC:DMPA lipid bilayers (lipid‐to‐
protein ratio 2 w/w), pH 9, radical: 20 mM AMUpol, 30% d‐6‐
glycerol, 60% D2O, 10% H2O (enhancement: ~32)

LEEDER ET AL.
5
+•
(33%), 84 (100%). HRMS (EI) For C613C3H16O+• calcu-
(ES+) m/z = 196 [M + H]+. HRMS (ES+) For C1013C3H21O
+ calculated 196.1688, found 196.1692 Da.
lated 143.1296, found 143.1300 Da.
4.3 | 2,6,6‐Tri(methyl‐13C)cyclohex‐1‐en‐1‐
yl trifluoromethanesulfonate (7)
4.5 | Triethylphosphonoacetate‐13C2
([13C2]‐2)
By adaption of a procedure by Breining et al,18 to a solu-
tion of LDA (1.90 M, 21.3 mL, 40.5 mmol) in THF
(35 mL) under N2 at −78°C was added 2,2,6‐tri‐(methyl‐
13C)cyclohexan‐1‐one (3.62 g, 25.3 mmol) in THF (35 mL)
dropwise over 45 minutes. The mixture was stirred at
−78°C for 2 hours before dropwise addition of a solution
of N‐phenyl‐bis(trifluoromethanesulfonimide) (9.02 g,
25.3 mmol) in THF (40 mL). The mixture was allowed to
warm to rt over 24 hours. The reaction was quenched with
saturated aqueous NH4Cl (80 mL) and the aqueous phase
extracted with pentane (3 × 40 mL). The combined organic
solution was washed with brine, dried (MgSO4), and the sol-
vent removed under reduced pressure (200 mbar, 23°C) to
give an orange oil. Purification by silica gel chromatography
(pentane) afforded the vinyl triflate as a colorless oil (3.47 g,
12.6 mmol, 50%). FT‐IR (neat) νmax 2940 (w), 1458 (w), 1400
By adaption of a procedure by Kiddle et al,16 a solution of
2‐13C‐ethyl bromoacetate (2.152 g, 12.74 mmol) and
P(OEt)3 (2.05 g, 12.4 mmol) under N2 was heated at
130°C in a microwave for 10 minutes. Purification by sil-
ica gel chromatography (EtOAc:hexane = 85:15) afforded
the product as a colorless oil (2.62 g, 11.6 mmol, 94%). 1H
and 13C NMR spectroscopic data were consistent with
those reported.22 FT‐IR (neat) νmax 2980 (m), 1692
(s), 1252 (s), 1020 (s), 963 (s) cm−1
.
1H NMR
(CDCl3, 400 MHz): δ 4.29 to 4.09 (m, 6H), 2.96
(ddd, JCH = 129.9, JHP = 21.5, JHH = 7.3 Hz, 2H), 1.36
(t, JHH = 7.2 Hz, 6H), 1.29 (t, JHH = 7.2 Hz, 3H) ppm.
13C NMR (CDCl3, 101 MHz): δ 165.7 (13C, dd, JCC = 58.7,
JCP = 5.9 Hz), 34.3 (13CH2 dd, JCC = 134.3, JCP = 59.4 Hz)
ppm. (Only peaks for 13C labeled carbons reported) LRMS
(ES+) m/z = 227 [M + H]+.
1
(s), 1243 (s), 1201 (s), 1139 (s), cm−1. H NMR (CDCl3,
400 MHz): δ 2.16 (t, JHH = 5.9 Hz, 2H), 1.71 to 1.58
(m, 4H), 1.76 (d, JCH = 128.0 Hz, 3H), 1.16 (dd, JCH = 126.9,
JCH = 4.7 Hz, 6H) ppm. 13C NMR (CDCl3, 101 MHz): δ 26.4
(13CH3), 17.6 (13CH3) ppm (Only peaks for 13C labeled
carbons reported). LRMS (EI) m/z 275 [M]+• (27%), 259
[M − CH3]+ (33%), 69 [CF3]+ (100%). HRMS (EI) For
C713C3H15F3O3S+• calculated 275.0789, found 275.0790 Da.
4.6 | Ethyl (2E,4E)‐3‐methyl‐5‐(2,6,6‐
tri(methyl‐13C)cyclohex‐1‐en‐1‐yl)penta‐2,4‐
dienoate ‐1,2‐13C2 ([13C5]‐8)
Following a procedure by McLean et al,19 to a suspension
of NaH (630 mg, 15.7 mmol) in Et2O (12 mL) under N2 at
0°C was added a solution of triethylphosphonoacetate‐
13C2 ([13C2]‐2) (3.51 mL, 15.5 mmol) in Et2O (8 mL)
dropwise, and the solution was stirred for 2 hours at rt.
A solution of (E)‐4‐(2,6,6‐tri(methyl‐13C)cyclohex‐1‐en‐1‐
yl)but‐3‐en‐2‐one ([13C3]‐4) (1.54 g, 7.87 mmol) in Et2O
(8 mL) was added dropwise, and the resulting solution
was stirred for 96 hours. The reaction was quenched with
H2O (10 mL) and the aqueous phase was extracted with
Et2O (3 x 10 mL). The combined organic solution was
washed with brine and dried (Na2SO4) before concentrat-
ing under reduced pressure. Purification by silica gel chro-
4.4 | (E)‐4‐(2,6,6‐Tri(methyl‐13C)cyclohex‐
1‐en‐1‐yl)but‐3‐en‐2‐one ([13C3]‐4)
By adaption of a procedure by Breining et al,18 a solution of
vinyl triflate 7 (3.33 g, 12.1 mmol), NEt3 (6.75 mL,
48.4 mmol), methyl vinyl ketone (1.97 mL, 48.4 mmol),
and [Pd(PPh3)2Cl2] (425 mg, 0.605 mmol) in DMF
(60 mL) under N2 was heated at 75°C for 18 hours. The
reaction was quenched with H2O (50 mL) and the aqueous
phase extracted with pentane (4 × 50 mL). The combined
organic solution was washed with brine, dried, (Na2SO4)
and the solvent removed under reduced pressure. Purifica-
tion by silica gel chromatography (Et2O:hexane = 3:97)
afforded the ketone as a pale yellow oil (1.58 g, 8.09 mmol,
67%). FT‐IR (neat) νmax 2930 (m), 2863 (m), 1692 (w), 1670
matography (Et2O:hexane
= 0.5:99.5) afforded the
product as a pale yellow oil and a mixture of inseparable
isomers (1.645 g, 6.153 mmol, 78%, 9E:9Z = 9:1). FT‐IR
(neat) νmax 2957 (m), 2928 (m), 2865 (m), 1709 (s), 1606
(m), 1443 (m), 1233 (m) 1155 (s), 1047 (m), 967 (m), 875
(w) cm−1 1H NMR (CDCl3, 400 MHz): δ 7.64 (dd,
.
(s) cm−1
.
1H NMR (CDCl3, 400 MHz):
δ
7.28
JHH = 16.4, JHH = 2.3 Hz, 1H, Z), 6.56 (d, JHH = 16.1 Hz,
1H, E), 6.10 (dd, JHH = 16.1, JCH = 5.3 Hz, 1H, E), 5.74
(d, JCH = 159.3 Hz, 1H, E), 5.65 (d, JCH = 160.3 Hz,
1H, Z), 4.18 (qd, JHH = 7.1, JCH = 3.1 Hz, 2H), 2.34
(d, JCH = 4.6 Hz, 3H), 2.07 to 2.00 (m, 2H), 1.70 (d,
JCH = 125.8 Hz, 3H), 1.66 to 1.58 (m, 2H), 1.52 to 1.43
(m, 2H), 1.29 (t, JHH = 7.1 Hz, 3H), 1.07 (dd, JCH = 125.3,
(d, JHH = 16.5 Hz, 1H), 6.12 (d, JHH = 16.5 Hz, 1H), 2.30
(s, 3H), 2.05 to 2.12 (unresolved t, 2H), 1.77
(d, JCH = 126.1 Hz, 3H), 1.62 to 1.67 (m, 2H), 1.46 to 1.53
(m, 2H), 1.08 (dd, JCH = 125.4, JCH = 4.9 Hz, 6H) ppm.
13C NMR (CDCl3, 101 MHz) δ 28.7 (13CH3), 21.7 (13CH3)
ppm (Only peaks for 13C labeled carbons reported). LRMS

6
LEEDER ET AL.
JCH = 4.8 Hz, 6H, Z), 1.02 (dd, JCH = 125.3,
JCH = 4.8 Hz, 6H, E) ppm. 13C NMR (CDCl3, 101 MHz):
δ 167.3 (13C, d, JCC = 77.0 Hz), 166.3 (13C, d,
JCC = 77.0 Hz), 118.0 (13CH, d, JCC = 77.0), 116.2
(13CH, d, JCC = 77.0), 28.9 (13CH3), 28.9 (13CH3), 21.8
(13CH3), 21.6 (13CH3) ppm. (Only peaks for 13C labeled
carbons reported) LRMS (ES+) m/z = 262 [M + H]+.
solution of 13C2‐triethylphosphonoacetate ([13C2]‐2)
(2.01 g, 8.86 mmol) in Et2O (5 mL) dropwise and the sus-
pension was stirred for 1 hour at rt. A solution of (2E,4E)‐
3‐methyl‐5‐(2,6,6‐tri(methyl‐13C)cyclohex‐1‐en‐1‐yl)
penta‐2,4‐dienal‐1,2‐13C2 ([13C5]‐9) (1.03 g, 4.65 mmol) in
Et2O (5 mL) was added dropwise and stirred at rt for
18 hours. The reaction was then quenched with H2O
(6 mL) and the aqueous phase was extracted with Et2O
(3 × 6 mL). The combined organic solution was washed
with brine, dried (Na2SO4) and the solvent removed under
reduced pressure. Purification by silica gel chromatogra-
phy (EtOAc:hexane = 2:97) afforded the product as a yel-
low/green oil (1.24 g, 4.19 mmol, 90%). FT‐IR (neat) νmax
2927 (m), 2864 (m), 2361 (m), 1707 (s), 1619 (m), 1597 (m)
cm−1. 1H NMR (CDCl3, 400 MHz): δ 7.92 to 7.52 (m, 1H),
6.40 (d, JHH = 16.2 Hz, 1H), 6.16 (br dd, JHH = 16.2,
JCH = 5.3 Hz, 1H), 6.15 (dd, JCH = 153.9, JHH = 12.5 Hz,
1H), 5.87 (dddd, JCH = 162.3, JHH = 15.0, JCH = 7.0,
JCH = 2.9 Hz, 1H), 4.22 (qd, JHH = 7.1, JCH = 3.1 Hz,
2H), 2.05 (d, JCH = 4.9 Hz, 3H), 2.03 (t, JHH = 6.2 Hz,
2H), 1.72 (d, JCH = 125.7 Hz, 3H), 1.66 to 1.57 (m, 2H),
1.50 to 1.45 (m, 2H), 1.31 (t, JHH = 7.1 Hz, 3H), 1.03 (dd,
JCH = 125.3, JCH = 4.8 Hz, 6H) ppm. 13C NMR (CDCl3,
101 MHz): δ 167.5 (ddd, JCC = 76.5, JCC = 7.9, JCC = 1.5 Hz,
13C), 140.6 (ddd, JCC = 70.0, JCC = 57.6, JCC = 1.5 Hz,
13CH), 127.2 (ddd, JCC = 57.6, JCC = 8.1, JCC = 1.0 Hz,
13CH), 120.0 (ddd, JCC = 76.5, JCC = 70.0, JCC = 1.5 Hz,
13CH), 28.9 (13CH3), 21.7 (13CH3) ppm. (Only peaks for
13C labeled carbons reported) LRMS (ES+): m/z = 296
[M + H]+. HRMS (ES+) For C1213C7H28O2Na+ calculated
318.2216, found 318.2218 Da.
HRMS (ES+) For C1213C5H26NaO2 calculated 290.1993,
+
found 290.2000.
4.7 | (2E,4E)‐3‐Methyl‐5‐(2,6,6‐tri(methyl‐
13C)cyclohex‐1‐en‐1‐yl)penta‐2,4‐dienal‐1,2‐
13C2 ([13C5]‐9)
Following a procedure by McLean et al,19 To a LiAlH4
(1 M in THF, 7.15 mL, 7.15 mmol) in Et2O (15 mL) under
N2 at −78°C was added a solution of ethyl (2E,4E)‐3‐
methyl‐5‐(2,6,6‐tri(methyl‐13C)cyclohex‐1‐en‐1‐yl)penta‐
2,4‐dienoate‐1,2‐13C2 ([13C5]‐8) (1.59 g, 5.95 mmol) in
Et2O (30 mL) dropwise and stirred at −78°C for
30 minutes. The mixture was then warmed to rt and
stirred for 20 minutes. The reaction was quenched with
H2O (6 mL), 1 M NaOH (2 mL) and H2O (2 mL) sequen-
tially and the white solid was filtered through celite. The
filtrate was dried (MgSO4) and the solvent removed under
reduced pressure to afford a colorless oil which was re‐dis-
solved in CH2Cl2 (40 mL). Crushed molecular sieves
(2.00 g), NMO (1.40 g, 11.9 mmol) and TPAP (105 mg,
2.98 mmol) was added and stirred at rt for 30 minutes.
The black mixture was concentrated and the resulting
black oil purified by silica gel chromatography (EtOAc:
hexane = 4:96) to afford the product as a yellow oil
(1.16 g, 5.17 mmol, 87%). FT‐IR (neat) νmax 2928 (m),
4.9 | (2E,4E,6E)‐N‐methoxy‐N,5‐dimethyl‐
7‐(2,6,6‐tri(methyl‐13C)cyclohex‐1‐en‐1‐yl)
hepta‐2,4,6‐trienamide‐1,2,3,4‐13C4 ([13C7]‐
10)
1
2861 (m), 1627 (s), 1601 (s), 1442 (m) cm−1. H NMR
(CDCl3, 400 MHz): δ 10.13 (ddd, JCH = 169.7, JCH = 24.6,
JHH = 8.1 Hz, 1H), 6.74 (d, JHH = 16.1 Hz, 1H), 6.22
(dd, JHH = 16.1, JCH = 4.9 Hz, 1H), 5.94 (dd, JCH = 157.5,
JHH = 8.1 Hz, 1H), 2.32 (d, JCH = 4.0 Hz, 3H), 2.09 to 2.02
(m, 2H), 1.73 (d, JCH = 125.8 Hz, 3H), 1.68 to 1.59 (m, 2H),
1.53 to 1.45 (m, 2H), 1.05 (dd, JCH = 125.3, JCH = 4.9 Hz, 6H)
ppm. 13C NMR (CDCl3, 101 MHz): δ 191.3 (13CH, d,
JCC = 77.0 Hz), 128.7 (13CH, d, JCC = 77.0 Hz), 28.9
(13CH3), 21.7 (13CH3) ppm. (Only peaks for 13C labeled
carbons reported) LRMS (ES+) m/z 224 [M + H]+. HRMS
(ES+) For C1013C5H22ONa+ calculated 246.1731, found
246.1739 Da.
By adaption of a procedure by Groesbeek et al,15 to a solu-
tion of N,O‐dimethylhydroxylamine hydrochloride
(1.83 g, 18.7 mmol) in THF (30 mL) under N2 at −15°C
was added nBuLi (2.27 M in hexanes, 16.1 mL, 36.5 mmol)
dropwise over 15 minutes. After stirring at −15°C for
1 hour a solution of ethyl (2E,4E,6E)‐5‐methyl‐7‐(2,6,6‐
tri(methyl‐13C)cyclohex‐1‐en‐1‐yl)hepta‐2,4,6‐trienoate‐
1,2,3,4‐13C4 (1.23 g, 4.25 mmol) in THF (20 mL) was
added dropwise over 15 minutes at −15°C. After
30 minutes at −15°C, the reaction was quenched with sat-
urated aqueous NH4Cl and the aqueous phase was
extracted with Et2O (3 × 10 mL). The combined organic
solution was washed with brine, dried (Na2SO4) and the
solvent removed under reduced pressure. Purification by
silica gel chromatography (EtOAc:hexane = 15:85)
afforded the product as a yellow/green oil (1.13 g,
4.8 | Ethyl (2E,4E,6E)‐5‐methyl‐7‐(2,6,6‐
tri(methyl‐13C)cyclohex‐1‐en‐1‐yl)hepta‐
2,4,6‐trien oate‐1,2,3,4‐13C4
To a suspension of NaH (60% in mineral oil, 354 mg,
8.86 mmol) in Et2O (5 mL) under N2 at 0°C was added a

LEEDER ET AL.
7
4.11 | Diethyl ((cyano‐13C)methyl‐13C)
phosphonate ([13C2]‐3)
3.72 mmol, 88%). FT‐IR (neat) νmax 2932 (m), 2861 (w),
1603 (s), 1537 (s), 1364 (m) cm−1
.
1H NMR
(CDCl3, 400 MHz):
δ
8.02 to 7.52 (m, 1H),
By adaption of a procedure by Groesbeek et al,15 to a solu-
tion of n‐BuLi (2.32 M in hexanes, 4.76 mL, 11.0 mmol) in
THF (8 mL) under N2 at −78°C was added a solution of
HMDS (2.45 mL, 11.6 mmol) in THF (6 mL) dropwise
over 15 minutes. After stirring for 15 minutes a solution
of 13CH313CN (250 mg, 5.81 mmol) in THF (6 mL) was
added dropwise over 15 minutes and the solution was
6.47 (dddd, JCH = 161.6, JHH = 14.9, JCH = 7.0,
JCH = 3.7 Hz, 1H), 6.37 (br d, JHH = 16.5 Hz, 1H), 6.22
(br dd, JCH = 152.7, JHH = 12.0 Hz, 1H), 6.16
(dd, JHH = 16.5, JCH = 5.4 Hz, 1H), 3.72 (s, 3H), 3.27 (d,
JCH = 2.0 Hz, 3H), 2.08 to 2.00 (m, 5H), 1.72 (d,
JCH = 125.6 Hz, 3H), 1.66 to 1.58 (m, 1H), 1.53 to 1.42
(m, 2H), 1.03 (dd, JCH = 125.3, JCH = 5.1 Hz, 6H) ppm.
13C NMR (CDCl3, 101 MHz): δ 167.6 (dd, JCC = 71.2,
JCC = 7.0 Hz, 13C), 139.5 (ddd, JCC = 71.2, JCC = 58.0,
JCC = 1.5 Hz, 13CH), 127.9 (ddd, JCC = 71.2, JCC = 69.0,
JCC = 2.2 Hz, 13CH), 117.8 (ddd, JCC = 71.2, JCC = 69.0,
JCC = 2.2 Hz, 13CH), 28.9 (13CH3), 21.7 (13CH3) ppm.
(Only peaks for 13C labeled carbons reported) LRMS
stirred for
a
further 40 minutes.
A
solution
diethylchlorophosphate (0.92 mL, 6.40 mmol) in THF
(6 mL) was added dropwise over 10 minutes and then
stirred for a further 40 minutes. The mixture was allowed
to warm to rt and stirred for 40 minutes before pouring
into a stirred mixture of 2 M HCl (10 mL) and CH2Cl2
(15 mL). The aqueous phase was extracted with CH2Cl2
(3 × 10 mL) and then the combined organic solution
was washed with water before drying (MgSO4) and con-
centrating under reduced pressure to afford an orange
oil. Purification by silica gel chromatography (EtOAc:hex-
ane = 7:3 to 100:0) afforded the product as an orange oil
(926 mg, 5.17 mmol, 89%). Spectroscopic data were consis-
tent with those reported.23 FT‐IR (neat) νmax 2984 (w),
(ES+) m/z 311 [M
+
H]+. HRMS (ES+) For
C1213C7H30NO2+ calculated 311.2506, found 311.2513 Da.
4.10 | (3E,5E,7E)‐6‐Methyl‐8‐(2,6,6‐
tri(methyl‐13C)cyclohex‐1‐en‐1‐yl)octa‐3,5,7‐
trien‐2‐one‐2,3,4,5‐13C4 ([13C7]‐11)
2909 (w), 1260 (m), 1012 (s) cm−1 1H NMR (CDCl3,
.
400 MHz): δ 4.30 to 4.20 (m, 4H), 2.87 (ddd, JCH = 135.0,
JHP = 21.0, JCH = 10.5 Hz, 2H), 1.39 (t, JHH = 7.1 Hz, 6H)
ppm. 13C NMR (CDCl3, 101 MHz): δ 112.6 (13C, d,
JCC = 57.9, JCC = 11.4 Hz), 16.5 (13CH2, d, JCC = 143.8,
JCP = 60.2) ppm. (Only peaks for 13C labeled carbons
reported) LRMS (ES+): m/z 179 [M + H]+.
By adaption of a procedure by Groesbeek et al.15 To a
solution of (2E,4E,6E)‐N‐methoxy‐N,5‐dimethyl‐7‐(2,6,6‐
tri(methyl‐13C) cyclohex‐1‐en‐1‐yl)hepta‐2,4,6‐trienamide‐
1,2,3,4‐13C4 ([13C7]‐10) (289 mg, 0.93 mmol) in THF
(15 mL) at −78°C was added methyl lithium (1.48 M in
Et2O, 0.75 mL, 1.12 mmol) dropwise over 10 minutes.
After 10 minutes at −78°C the reaction was quenched
by addition of a slurry of silica (1 g) and H2O (2 mL)
and the resulting suspension was stirred for 15 minutes.
The mixture was dried (Na2SO4), the solids filtered and
the filtrate concentrated under reduced pressure. Purifi-
cation by silica gel chromatography (EtOAc:hexane = 1:9)
afforded the product as a viscous green oil (222 mg,
4.12 | [10,11,12,13,14,15,16,17,18‐13C9]‐all‐
trans‐retinal (1b)
By adaption of a procedure by Groesbeek et al,15 to a solu-
tion of diethyl ((cyano‐13C)methyl‐13C)phosphonate
([13C2]‐3) (205 mg, 1.14 mmol) in THF (1.5 mL) under
N2 atm. at 0°C was added n‐BuLi (2.32 M in hexanes,
0.45 mL, 1.00 mmol) dropwise over 5 minutes and the
solution was stirred at rt for 90 minutes. A solution of
(3E,5E,7E)‐6‐methyl‐8‐(2,6,6‐tri(methyl‐13C)cyclo hex‐1‐
1
8.37 mmol, 90%). H NMR (C6D6, 400 MHz): δ 7.70 to
7.29 (m, 1H), 6.34 (d, JHH = 16.0 Hz, 1H), 6.18 (dd,
JHH = 16.0, JCH = 5.4 Hz, 1H), 6.05 (dddd, JCH = 156.7,
JHH = 15.0, JCH = 7.2, JCH = 3.3 Hz, 1H), 6.01 to 5.53
(m, 1H), 1.93 (m, 2H), 1.79 (d, JCH = 125.3 Hz, 3H),
1.71 (d, JCH = 5.0 Hz, 3H), 1.60 to 1.52 (m, 2H), 1.47
to 1.41 (m, 2H), 1.06 (dd, JCH = 125.3, JCH = 4.8 Hz,
6H) ppm. 13C NMR (C6D6, 101 MHz): δ 196.55 (ddd,
JCC = 54.3, JCC = 6.6, JCC = 1.5 Hz, 13C), 138.4 (dd,
JCC = 68.0, JCC = 57.2 Hz, 13CH), 130.4 (dd, JCC = 68.0,
JCC = 54.3 Hz, 13CH), 129.0 (dd, JCC = 57.2, JCC = 6.6 Hz,
13CH), 29.4 (13CH3), 22.2 (13CH3) ppm. (Only peaks for
13C labeled carbons reported) LRMS (ES+) m/z 266
en‐1‐yl)octa‐3,5,7‐trien‐2‐one‐2,3,4,5‐13C4
([13C7]‐11)
(138 mg, 0.52 mmol) in THF (5 mL) was added
dropwise and stirred at rt for 18 hours. The reaction was
quenched with saturated aqueous NH4Cl and the aqueous
phase extracted with EtOAc (3 × 8 mL). The combined
organic solution was washed with brine, dried (Na2SO4)
and concentrated under reduced pressure to afford an
orange oil. Purification by silica gel chromatography
(EtOAc:hexane = 4:96, silica deactivated with NEt3)
afforded the impure nitrile intermediate as an orange
oil (145 mg).
[M
+
H]+. HRMS (ES+) For C1113C7H26NaO+
calculated 288.2111, found 288.2115 Da.

8
LEEDER ET AL.
The orange oil was taken up in CH2Cl2 (3.5 mL)
4.14 | Ethyl (2E,4E,6E)‐5‐methyl‐7‐(2,6,6‐
under N2 and DIBAL (1 M in CH2Cl2, 0.76 mL,
0.76 mmol) was added dropwise at −60°C. After stirring
at −60°C for 15 minutes, a slurry of SiO2 and H2O was
added and stirred for 15 minutes at rt. The solids were
filtered and the filtrate dried twice (Na2SO4) before con-
centrating under reduced pressure to afford an orange
oil. Purification by silica gel chromatography (EtOAc:
hexane = 4:96 to 1:9, silica deactivated with NEt3)
afforded the product as an orange oil (106 mg,
0.36 mmol, 70%, 13E:13Z = 2:1). The isomers were sep-
trimethylcyclohex‐1‐en‐1‐yl)hepta‐2,4,6‐
trienoate‐2‐13C
To a suspension of NaH (60% in mineral oil, 427 mg,
10.7 mmol) in Et2O (8 mL) under N2 at 0°C was added a
solution of triethylphosphonoacetate‐2‐13C ([13C1]‐2)
(2.25 g, 10.0 mmol) in Et2O (4 mL) dropwise, and the solu-
tion was stirred for 90 minutes at rt. A solution of dienal 9
(1.10 g, 5.04 mmol) in Et2O (4 mL) was added dropwise,
and the solution was stirred at rt for 2 hours. The reaction
was then quenched with H2O (10 mL), and the aqueous
phase was extracted with Et2O (3 × 8 mL). The combined
organic solution was washed with brine, dried (Na2SO4),
and the solvent removed under reduced pressure. Purifi-
cation by silica gel chromatography (EtOAc:hexane = 2:97)
afforded the product as a yellow/green oil (1.37 g,
4.73 mmol, 94%). FT‐IR (neat) νmax 2929 (m), 2861 (m),
arated
Ether = 3.5:96.5). FT‐IR (neat) νmax 2928 (w), 1716
(w), 1662 (w), 971 (m) cm−1 1H NMR (C6D6,
by
preparative
HPLC
(EtOAc:pet.
.
500MHz):δ10.01(ddd,JCH =169.1,JCH =25.4,JHH =7.8Hz,
1H), 7.03 to 6.62 (m, 1H), 6.37 (br d, JHH = 16.1 Hz, 1H), 6.26
(dd, JHH = 16.1, JCH = 4.9 Hz, 1H), 6.21 to 5.83 (m, 1H), 5.96
(dt, JCH = 157.1, JHH = 7.8 Hz, 1H), 1.99 to 1.93 (m, 2H), 1.78
(d, JCH = 4.4 Hz, 3H), 1.75 to 1.70 (m, 3H), 1.76 (d,
JCH = 125.6 Hz, 3H), 1.62 to 1.55 (m, 2H), 1.50 to 1.44 (m,
2H), 1.12 (dd, JCH = 125.2, JCH = 4.7 Hz, 6H) ppm. 13C
NMR (C6D6, 126 MHz): δ 189.4 (ddd, JCC = 56.7, JCC = 6.8,
JCC = 2.9 Hz, 13C), 152.6 (dddd, JCC = 67.1, JCC = 54.2,
JCC = 6.8, JCC = 2.9 Hz, 13C), 135.2 (ddddd, JCC = 67.1,
JCC = 54.2, JCC = 6.8, JCC = 3.3, JCC = 1.7 Hz, 13CH), 131.2
(ddd, JCC = 67.1, JCC = 58.4, JCC = 7.6 Hz, 13CH), 130.3 to
129.6 (m, 13CH), 130.0 to 128.8 (m, 13CH), 28.8 (13CH3),
21.6 (13CH3) ppm. (Only peaks for 13C labeled carbons
reported) LRMS (ES+) m/z 294 [M + H]+. HRMS (ES+)
For C1113C9H29NaO+ calculated 294.2515, found
294.2519 Da.
1
1663 (s), 1565 (m) cm−1. H NMR (CDCl3, 400 MHz) δ
7.72 (ddd, JHH = 15.0, JHH = 12.1, JCH = 2.5 Hz, 1H),
6.40 (br d, JHH = 16.0 Hz, 1H), 6.20 to 6.12 (m, 2H), 5.88
(dd, JCH = 162.4, JHH = 15.0 Hz, 1H), 4.22 (d,
JHH = 7.1 Hz, 2H), 2.05 (s, 3H), 2.03 (t, JHH = 5.9 Hz,
2H), 1.72 (s, 3H), 1.67 to 1.59 (m, 2H), 1.52 to 1.45
(m, 2H), 1.32 (t, JHH = 7.1 Hz, 3H), 1.04 (s, 6H) ppm.
13C NMR (CDCl3, 101 MHz): δ 120.1 (13CH2) ppm. (Only
peaks for 13C labeled carbons reported) LRMS (ES+) m/z
290 [M + H]+. HRMS (ES+) For C1813CH28NaO2 calcu-
+
lated 312.2015, found 312.2018 Da.
4.15 | (2E,4E,6E)‐N‐methoxy‐N,5‐dimethyl‐
7‐(2,6,6‐trimethylcyclohex‐1‐en‐1‐yl)hepta‐
2,4,6‐trienamide‐2‐13C
4.13 | Triethylphosphonoacetate‐2‐13C
([13C1]‐2)
By adaption of a procedure by Grosbeek et al,15 to a solution
of N,O‐dimethylhydroxylamine hydrochloride (1.83 g,
18.7 mmol) in THF (30 mL) under N2 at −15°C was added
n‐BuLi (2.27 M in hexanes, 16.1 mL, 36.5 mmol) dropwise
over 15 minutes. After stirring the solution at −15°C for
1 hour, a solution of ethyl (2E,4E,6E)‐5‐methyl‐7‐(2,6,6‐
trimethylcyclohex‐1‐en‐1‐yl)hepta‐2,4,6‐trienoate‐2‐13C
(1.23 g, 4.25 mmol) in THF (20 mL) was added dropwise
over 15 minutes at −15°C. After 30 minutes at −15 °C, the
reaction was quenched with saturated aqueous NH4Cl,
and the aqueous phase was extracted with Et2O
(3 × 10 mL). The combined organic solution was washed
with brine, dried (Na2SO4), and the solvent removed under
reduced pressure. Purification by silica gel chromatography
(EtOAc:hexane = 15:85) afforded the product as a yellow/
green oil (1.13 g, 3.72 mmol, 88%). FT‐IR (neat) νmax 2928
By adaption of a procedure by Kiddle et al,16 a solution
of 2‐13C‐ethyl bromoacetate (1.77 g, 10.5 mmol) and
P(OEt)3 (1.70 g, 10.3 mmol) under N2 was heated at
130°C in a microwave for 10 minutes. Purification by sil-
ica gel chromatography (EtOAc:hexane = 85:15) afforded
the product as a colorless oil (2.28 g, 10.1 mmol, 99%).
1H and 13C NMR spectroscopic data were consistent with
those reported.24 FT‐IR (neat) νmax 2983 (m), 1773 (s),
1253 (s), 1017 (s), 963 (s) cm−1 1H NMR (CDCl3,
.
400 MHz): δ 4.18 (dq, JHP = 14.6, JHH = 7.2 Hz, 6H),
2.95 (dd, JCH = 130.0, JHP = 21.5 Hz, 2H), 1.34 (t,
JHH = 7.2 Hz, 6H), 1.28 (t, JHH = 7.2 Hz, 3H) ppm.
13C NMR (CDCl3, 101 MHz):
δ
34.3 (13CH3, d,
JCP = 134.3 Hz) ppm. (Only peaks for 13C labeled car-
bons reported) 31P NMR (CDCl3, 162 MHz): δ 19.81 (d,
JCP = 134.6 Hz) ppm. LRMS (ES+) m/z = 226
[M + H]+.
(w), 2963 (m), 1645 (s), 1589 (m), 1408 (m), 1371 (s) cm−1
.
1H NMR (400 MHz, CDCl3) δ 7.77 (ddd, JHH = 14.9,
JHH = 12.0, JCH = 2.9 Hz, 1H), 6.47 (br dd, JHH = 161.9,

LEEDER ET AL.
9
JHH = 14.9 Hz, 1H), 6.37 (br d, JHH = 16.1 Hz, 1H), 6.22 (dd,
JHH = 12.0, JHH = 4.4 Hz, 1H), 6.15 (d, JHH = 16.1 Hz, 1H),
3.71 (s, 3H), 3.27 (s, 3H), 2.06 (s, 3H), 2.03 (t, JHH = 6.0 Hz,
2H), 1.71 (s, 3 H), 1.67 to 1.58 (m, 2H), 1.51 to 1.45 (m,
2H), 1.03 (s, 6H) ppm. 13C NMR (CDCl3, 101 MHz):
δ = 117.9 (13CH) ppm. (Only peaks for 13C labeled carbons
before pouring into a stirred mixture of 2 M HCl
(20 mL) and CH2Cl2 (30 mL). The aqueous phase was
extracted with CH2Cl2 (3 × 20 mL), and then the com-
bined organic solution washed with H2O before drying
(MgSO4) and concentrating under reduced pressure to
afford an orange oil. Purification by silica gel chromatog-
raphy (EtOAc:hexane = 7:3 to 100:0) afforded the product
reported) LRMS (ES+) m/z 305 [M + H]+. HRMS (ES+)
For C1813CH30NO2
calculated 305.2305, found
as an orange oil (1.885 mg, 10.58 mmol, 89%). 1H and 13
C
+
305.2303 Da.
NMR spectroscopic data were consistent with those
reported.15 FT‐IR (neat) νmax 2984 (w), 2909 (w), 2256
(w), 1260 (m), 1012 (s) cm−1
.
1H NMR (CDCl3,
4.16 | (3E,5E,7E)‐6‐Methyl‐8‐(2,6,6‐
trimethylcyclohex‐1‐en‐1‐yl)octa‐3,5,7‐trien‐
2‐one‐3‐13C ([13C1]‐11)
400 MHz): δ 4.31 to 4.19 (m, 4H), 2.87 (dd, JHP = 21.1,
JCH = 10.6 Hz, 2H), 1.39 (t, JHH = 7.1 Hz, 6H) ppm. 13C
NMR (CDCl3, 101 MHz): δ 112.6 (13C, d, JCP 11.0 Hz)
ppm. LRMS (ES+) m/z 178 [M + H]+.
To a solution of (2E,4E,6E)‐N‐methoxy‐N,5‐dimethyl‐7‐
(2,6,6‐trimethylcyclohex‐1‐en‐1‐yl)hepta‐2,4,6‐
trienamide‐2‐13C (964 mg, 3.18 mmol) in THF (50 mL) at
−78°C was added methyl lithium (1.45 M in Et2O,
2.62 mL, 3.80 mmol) dropwise over 10 minutes. After
10 minutes at −78°C, the reaction was quenched by
addition of a slurry of silica (2 g) and H2O (10 mL) and
the resulting suspension was stirred for 15 minutes at rt.
The mixture was dried (MgSO4), the solids filtered, and
the filtrate concentrated under reduced pressure to afford
the product as viscous green oil (788 mg, 3.04 mmol, 96%).
FT‐IR (neat) νmax 2927 (m), 2864 (w), 1657 (s), 1585 (s),
4.18 | [12‐15‐13C2]‐all‐trans‐retinal (1c)
By adaption of a procedure by Groesbeek et al,15 to a solu-
tion of diethyl ((cyano‐13C)methyl)phosphonate ([13C1]‐3)
(250 mg, 1.41 mmol) in THF (1.5 mL) under N2 atm at 0°C
was added n‐BuLi (2.31 M, 0.55 mL, 1.3 mmol) dropwise
over 5 minutes, and the solution was stirred at rt for
75 minutes. A solution of (3E,5E,7E)‐6‐methyl‐8‐(2,6,6‐
trimethylcyclohex‐1‐en‐1‐yl)octa‐3,5,7‐trien‐2‐one‐3‐13C
([13C1]‐11) (165 mg, 0.64 mmol) in THF (5 mL) was added
dropwise and stirred at rt for 18 hours. The reaction was
quenched with saturated aqueous NH4Cl and the aqueous
phase extracted with EtOAc (3 × 8 mL). The combined
organic solution was washed with brine, dried (Na2SO4),
and concentrated under reduced pressure to afford an
orange oil. Purification by silica gel chromatography
(EtOAc:hexane = 3:97) afforded the impure nitrile inter-
mediate as an orange oil (121 mg). The orange oil was
taken up in CH2Cl2 (3 mL) under N2, and DIBAL (1 M
in CH2Cl2, 0.64 mL, 0.64 mmol) was added dropwise at
−60°C. After stirring at −60°C for 1 hour, a slurry of
SiO2 and H2O was added and stirred for 15 minutes at
rt. The solids were filtered and the filtrate dried twice
(Na2SO4) before concentrating under reduced pressure
to afford an orange oil. Purification by silica gel chroma-
tography (EtOAc:hexane = 4:96 to 1:9, silica deactivated
with NEt3) afforded the product as an orange oil
(120 mg, 0.42 mmol, 30%, 13E:13Z = 2:1). The isomers
were separated by preparative HPLC (EtOAc:pet.
ether = 3.5:96.5). All‐trans‐ (1c): FT‐IR (neat) νmax 2928
1445 (w), 1359 (m), 1020 (s) cm−1 1H NMR (CDCl3,
.
400 MHz): δ 7.58 (ddd, JHH = 15.1, JHH = 11.9,
JCH = 1.2 Hz, 1H), 6.43 (br d, JHH = 16.0 Hz, 1H), 6.18
(br d, JHH = 16.0 Hz, 1H), 6.17 (br dd, JHH = 11.9,
JCH = 4.5 Hz, 1H), 6.17 (dd, JCH = 157.5, JHH = 15.1 Hz,
1H), 2.30 (d, JCH = 1.0 Hz, 3H), 2.07 (s, 3H), 2.04 (br
t, JHH = 6.2 Hz, 2H), 1.72 (s, 3H), 1.67 to 1.59 (m, 2H),
1.51 to 1.45 (m, 2H), 1.04 (s, 6H) ppm. 13C NMR (CDCl3,
101 MHz): δ 129.3 (13CH) ppm. LRMS (ES+) m/z = 260
[M + H]+. HRMS (ES+) For C1713CH26NaO+ calculated
282.1909, found 282.1918 Da.
4.17 | Diethyl ((cyano‐13C)methyl)
phosphonate ([13C1]‐3)
By adaption of a procedure by Groesbeek et al,15 to a solu-
tion of n‐BuLi (2.17 M in hexanes, 10.79 mL, 23.42 mmol)
in THF (16 mL) under N2 at −78°C was added a solution
of HMDS (5.02 mL, 23.78 mmol) in THF (12 mL)
dropwise over 15 minutes. After stirring for 15 minutes,
a solution of 13CH3CN (500 mg, 11.89 mmol) in THF
(12 mL) was added dropwise over 15 minutes and then
1
(w), 1716 (w), 1662 (w), 971 (m) cm−1. H NMR (C6D6,
400 MHz): δ 10.01 (dd, JCH = 169.2, JHH = 8.1 Hz, 1H),
6.83 (dd, JHH = 15.2, JHH = 11.5 Hz, 1H), 6.37 (d,
JHH = 16.1 Hz, 1H), 6.26 (d, JHH = 16.1 Hz, 1H), 6.02
(dd, JHH = 11.5, JCH = 4.8 Hz, 1H), 6.02 (dd, JCH = 154.4,
JHH = 15.2 Hz, 1H), 5.96 (apparent t, JHH/CH = 7.9 Hz,
1H), 1.96 (t, JHH = 6.0 Hz, 2H), 1.78 (s, 3H), 1.77 (s, 3H),
stirred for
a
further 40 minutes.
A
solution
diethylchlorophosphate (1.89 mL, 13.1 mmol) in THF
(12 mL) was added dropwise over 10 minutes, and the
solution was stirred for a further 40 minutes. The mixture
was allowed to warm to rt and stirred for 40 minutes

10
LEEDER ET AL.
1.73 (d, JCH = 4.0 Hz, 3H), 1.62 to 1.55 (m, 2H), 1.51 to
1.44 (m, 2H), 1.12 (s, 6H) ppm. 13C NMR (C6D6,
101 MHz): δ 190.1 (13CH, d, JCC = 7.0 Hz), 135.9 (13CH,
d, JCC = 7.0 Hz) ppm. (Only peaks for 13C labeled carbons
reported) LRMS (ES+) m/z = 287 [M + H]+. HRMS (ES+)
For C1813C2H28NaO+ calculated 309.2099, found
309.2102 Da.
13C2 (15) (0.450 g, 3.10 mmol) and Ph3P (0.815 g,
3.10 mmol) in MeCN (1 mL) was added CBr4 (1.03 g,
3.10 mmol) portionwise, and the resulting solution was
stirred at rt for 2 hours. The reaction mixture was concen-
trated under reduced pressure, and purification by silica
gel column chromatography (CH2Cl2:hexane = 0:1 to
1:1) afforded the title compound as a clear colorless oil
(0.480 g, 2.30 mmol, 74%, E:Z > 98:2). FT‐IR (neat) νmax
13Z‐ (12): 1H NMR (C6D6, 400 MHz): δ 10.15
(dd, JCH = 169.7, JHH = 7.2 Hz, 1H), 7.07 (dd,
JCH = 153.8, JHH = 15.0 Hz, 1H), 6.74 (dd, JHH = 15.0,
JHH = 11.4 Hz, 1H), 6.37 (d, JHH = 16.1 Hz, 1H), 6.28
(d, JHH = 16.1 Hz, 1H), 6.05 (dd, JHH = 11.4,
JCH = 4.9 Hz, 1H), 5.76 (apparent t, JCH/HH = 8.3 Hz, 1H),
1.96 (br t, JHH = 6.1 Hz, 2H), 1.82 to 1.75 (m, 6H), 1.59
(br d, JCH = 4.0 Hz, 3H), 1.62 to 1.55 (m, 2H), 1.50 to
1.45 (m, 2H), 1.13 (s, 6H) ppm. 13C NMR (C6D6,
101 MHz): δ = 189.0 (d, JCC = 5.1 Hz, 13C), 127.9 (d,
JCC = 5.1 Hz, 13CH) ppm (Only peaks for 13C labeled
carbons reported) LRMS (ES+) m/z = 287 [M + H]+.
HRMS (ES+) For C1813C2H28NaO+ calculated 309.2099,
found 309.2102 Da.
1
1732 (m), 1674 (m), 1221 (m), 1143 (m) cm−1. H NMR
(400 MHz, CDCl3): δ 5.96 (d, JCH = 161.1 Hz, 1H), 4.18
(qd, JHH = 7.1, JCH = 3.1 Hz, 2H), 3.95 (dd, JHH = 5.7,
JHH = 0.7 Hz, 2H), 2.28 (dt, JCH = 4.6, JCH = 1.2 Hz,
3H), 1.27 ‐ 1.23 (t, JHH = 7.1 Hz, 3H) ppm. 13C NMR
(CDCl3, 101 MHz): δ 165.92 (13CO, d, JCC = 76 Hz),
119.52 (13CH, d, JCC = 76 Hz) ppm. (Only peaks for 13C
labeled carbons reported). LRMS (ES+) m/z 226
[M + NH4]+.
4.21 | Ethyl (E)‐4‐(diethoxyphosphoryl)‐3‐
methylbut‐2‐enoate‐1,2‐13C2 (13)
By adaption of a procedure by Magoulas et al,20 neat
(EtO)3P (0.42 mL, 2.47 mmol) and ethyl (E)‐4‐bromo‐3‐
methylbut‐2‐enoate‐1,2‐13C2 (463 mg, 2.22 mmol) were
heated in a sealed pressure vial at 160°C for 4 hours. After
cooling, the reaction mixture was purified by silica gel col-
umn chromatography (EtOAc) to afford the title com-
pound as a pale yellow oil (0.572 g, 2.15 mmol, 96%, E:
Z > 98:2). FT‐IR (neat) νmax 1670 (m), 1619 (m), 1242
4.19 | Ethyl (E)‐4‐hydroxy‐3‐methylbut‐2‐
enoate‐1,2‐13C2 (15)
By adaption of a procedure by Magoulas et al,20 a sus-
pension of hydroxyacetone (0.38 g, 5.1 mmol) and [1,2‐
13C2](ethoxycarbonylmethylene) triphenylphosphorane25
(1.87 g, 5.34 mmol) in MeCN (10 mL) was heated under
reflux for 12 hours. The reaction mixture was allowed to
cool, concentrated to a yellow oil and triturated with
Et2O, and placed in the freezer overnight. The resultant
white solid was removed by filtration and discarded. The
filtrate was concentrated under reduced pressure, and
purification by silica gel column chromatography
(EtOAc:hexane = 4:1) afforded the title compound as a
pale yellow oil (0.540 g, 3.72 mmol, 70%, E:Z > 98:2).
FT‐IR (neat) νmax 3373 (m), 1651 (s), 1631 (s), 1209
1
(m), 1129 (m) cm−1. H NMR (400 MHz, CDCl3): δ 5.79
(dd, JPH = 5.1 Hz, JCH = 160.5 Hz, 1H), 4.15 to 4.09 (m,
6H), 2.68 (ddd, JHH = 0.7 Hz, JPH = 6.1 Hz, JCH = 23.5 Hz,
2H), 2.32 to 2.30 (m, 3H), 1.35 to 1.26 (m, 9H) ppm. 13C
NMR (CDCl3, 101 MHz):
δ
166.0 (13CO, dd,
JCC = 76 Hz, JPC = 4 Hz), 120.0 (13CH, dd, JCC = 76 Hz,
JPC = 12.5 Hz) ppm. (Only peaks for 13C labeled
carbons reported). LRMS (ES+) m/z 289 [M + Na]+.
HRMS (ESI+) for C913C2H21NaO5P+ calculated 289.1086,
found 289.1085 Da.
(m) cm−1 1H NMR (400 MHz, CDCl3): δ 5.98 (ddt,
.
JHH = 1.5, 3.1 Hz, JCH = 161.1 Hz, 1H), 4.20 ‐ 4.11 (m,
4H), 2.09 (d, JCH = 4.6 Hz, 3H), 1.99 (br t, JHH = 5.7 Hz,
1H), 1.73 (br s, 1H), 1.29 (t, JHH = 7.2 Hz, 3H) ppm. 13C
NMR (CDCl3, 101 MHz): δ 166.84 (13CO, d, JCC = 76 Hz),
113.82 (13CH, d, JCC = 76 Hz) ppm. (Only peaks for 13C
labeled carbons reported). LRMS (ES+) m/z 185
[M + K]+. HRMS (ESI+) for C513C2H13O3 calculated
147.0926, found 147.0927 Da.
4.22 | [14,15‐13C2]‐trans‐retinal (1d)
By adaption of a procedure by Wada et al,21b to a solu-
tion of ethyl (E)‐4‐(diethoxyphosphoryl)‐3‐methylbut‐2‐
enoate‐1,2‐13C2 (13) (268 mg, 1.00 mmol) in THF
(2 mL) at 0°C was added DMPU (0.48 mL, 0.51 g,
4.0 mmol) followed by nBuLi (2.3 M in hexanes,
0.52 mL, 1.20 mmol) dropwise. The reaction was kept
at 0°C for 40 minutes then cooled to −78°C. Aldehyde
(9) (200 mg, 0.916 mmol) in THF (1 mL) was added
dropwise slowly, and the reaction was allowed to warm
to 0°C and maintained below 5°C for 2 hours. The reac-
tion was quenched with saturated aqueous NH4Cl and
4.20 | Ethyl (E)‐4‐bromo‐3‐methylbut‐2‐
enoate‐1,2‐13C2
By adaption of a procedure by Magoulas et al,20 to a sus-
pension of ethyl (E)‐4‐hydroxy‐3‐methylbut‐2‐enoate‐1,2‐

LEEDER ET AL.
11
the aqueous layer extracted with EtOAc (× 2). The
combined organic solution was washed with water,
ORCID
Alexander J. Leeder http://orcid.org/0000-0001-9271-6038
Lynda J. Brown http://orcid.org/0000-0002-5678-0814
Clemens Glaubitz http://orcid.org/0000-0002-3554-6586
Richard C.D. Brown http://orcid.org/0000-0003-0156-7087
brine,
and
then
dried
(MgSO4)
before
concentrating under reduced pressure. Careful purifica-
tion of the mixture of olefin isomers (13E/13Z, esti-
mated 9:1 by 1H NMR spectroscopy) by silica gel
chromatography (EtOAc:hexane = 3:97) afforded the
ester
(ethyl
(2E,4E,6E,8E)‐3,7‐dimethyl‐9‐(2,6,6‐
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trimethylcyclohex‐1‐en‐1‐yl)nona‐2,4,6,8‐tetraenoate‐1,2‐
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SUPPORTING INFORMATION
Additional Supporting Information may be found online
in the supporting information tab for this article.
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Synthesis of isotopically labeled all‐trans retinals for
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