Synthesis of all-trans-[10′-3H]-8′-apo-β-carotenoic acid

Article abstract of DOI:10.1002/jlcr.536

The enzyme, 15, 15′-β-carotene dioxygenase (BCDOX), facilitates the oxidation of β-carotene to yield retinal. This is a remarkable process in which one of 11 double bonds in β-carotene is selectively oxidized. To further probe the mechanistic aspects of BCDOX, the synthesis of all-trans-[10′-³H]-8′-apo-β-carotenoic acid is reported. This compound will be used as a photoaffinity labeling reagent to probe the β-carotene binding pocket within BCDOX. The synthesis outlines a simple and efficient route for the incorporation of tritium at the 10′ olefinic carbon of 8′-apo-β-carotenoic acid. Copyright

Full text of DOI:10.1002/jlcr.536

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Labelled Cpd Radiopharm 2002; 45: 79–89.
DOI: 10.1002/jlcr.536
Research Article
Synthesis of all-trans-[100-3H]-80-apo-b-
carotenoic acid
Pulgam Veera Reddy, Monsterrat Rabago-Smith and Babak Borhan*
Department of Chemistry, Michigan State University, East Lansing,
MI 48824, USA
Summary
The enzyme, 15,150-b-carotene dioxygenase (BCDOX), facilitates the oxidation
of b-carotene to yield retinal. This is a remarkable process in which one of 11
double bonds in b-carotene is selectively oxidized. To further probe the
mechanistic aspects of BCDOX, the synthesis of all-trans-[100-3H]-80-apo-b-
carotenoic acid is reported. This compound will be used as a photoaffinity
labeling reagent to probe the b-carotene binding pocket within BCDOX. The
synthesis outlines a simple and efficient route for the incorporation of tritium at
the 100 olefiniccarbon of 8 0-apo-b-carotenoic acid. Copyright # 2002 John
Wiley & Sons, Ltd.
Key Words: 15,150-b-carotene dioxygenase; 80-apo-b-carotenoic acid; photo-
affinity labeling
Introduction
The need for carotenoids in the human diet has been well established.
Either the carotenoids or their oxidation products, particularly retinoid
compounds, are essential in various pathways such as the visual
transduction, gene regulatory control, cancer, and other disease
prevention.1–7 Although animals cannot produce retinoids such
as vitamin A, they can convert b-carotene (1) (a tetraterpenoid
*Correspondence to: B. Borhan, Department of Chemistry, Michigan State University, East
Lansing, MI 48824 USA. E-mail: borhan@cem.msu.edu
Contract/grant sponsor: Research Corporation; Contract/grant number RIO410
Copyright # 2002 John Wiley & Sons, Ltd.
Received 1 August 2001
Revised 10 October 2001
Accepted 12 October 2001

80
P. VEERA REDDY ET AL.
Scheme 1.
hydrocarbon with 11 double bonds) via a dioxygenase, which with
remarkable selectivity cleaves the central olefin to yield retinal (2)
(Scheme 1).8–10 This enzyme, 15,15’-b-carotene dioxygenase (BCDOX),
was first isolated by Goodman and Huang, but has not received a great
deal of attention until very recently.11 The past few years has witnessed
a resurgence of activity in this area, mainly as a result of the
identification and isolation of the gene responsible for BCDOX in a
number of organisms.12–17 In view of the growing interest in the
physiological implication of retinoids in human health,1,2,7,18–20 and the
intricate interplay of the concentration of different retinoids and their
physiological role,1,18,21–24 it seems prudent to understand fully the
nature, function, regulation, and mechanism of BCDOX.
Oxidative cleavage of b-carotene is an unprecedented process for a
dioxygenase enzyme. Typically, dioxygenases require a hydrophilic
handle such as a hydroxyl group to proceed, such as the case with intra-
and extra-diol catechol dioxygenases.25 Also, the catalytic mechanism
for the enzymatic cleavage of a somewhat featureless hydrocarbon such
as b-carotene (1) with such high regiospecificity is not well understood.
Enzyme-substrate interactions that lead to stereospecific catalytic action
through hydrogen bonding and dipolar communication are common.
Understanding the enzymatic action on hydrocarbon substrates can
shed light onto nature’s methodology for utilizing other molecular
interactions for recognition and specificity. Our interest in the
mechanistic aspects of BCDOX has led us to synthesize the tritiated
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SYNTHESIS OF CAROTENOIC ACID
81
photoaffinity label [100-3H]-80-apo-b-carotenoic acid (3) to probe the
binding pocket of b-carotene within BCDOX.
The choice of 3 as the photoaffinity probe stems from the fact that
apo-b-carotenoic acids are substrates for BCDOX, and thus will bind
the enzyme.26–28 On the other hand, a,bÀunsaturated polyene
carboxylic acids are capable of photoactivation and can be used as
photoaffinity probes. This was demonstrated by Rando and coworkers
during the isolation of retinoic acid binding proteins in which
29
radiolabeled retinoicacid was used as the photoaffinity labeling agent.
In a similar manner, our first attempt at photoaffinity labeling of
BCDOX will entail the use of 3, the synthesis of which is reported here.
Results and discussion
Synthesis of radiolabeled carotenoids are challenging for two reasons.
First, the polyolefinicnature of these compounds leads to their less than
desirable stability, and thus introduction of radiolabels is generally
limited to mild reactions. Although there is not a large body of literature
on the synthesis of radiolabeled carotenoids (mostly biosynthesized by
feeding of labeled precursors to various organisms),30,31 related
compounds such as retinoids have been synthesized by reduction of
intermediate carbonyl groups with Na/LiB3H4, or semihydrogenation
3
of intermediate acetylenes with H2.32–36 Secondly, their sensitivity to
light forces the need to work in darkrooms with minimal red lighting
and increases the technical difficulty of routine chemical manipulations.
Prudent with any radiolabeling, it is preferable to incorporate the
label as close to the final step as possible. This will reduce the need for
handling, and purification of radiolabeled intermediates, and increase
the radioactive yield of the labeled compound. Although there are a
range of positions present in 80-apo-b-carotenoic acid (3) that could be
considered as appropriate to incorporate tritium, we opted to place it on
the 100 carbon. We chose to use of NaB3H4 as the source of tritium, and
thus required a reductive step to incorporate the label. C100 was chosen
as a suitable site after several trial non-radioactive syntheses of 3 were
performed, in which the last reductive step was utilized for tritiation,
and the subsequent steps could be performed in high yields, without the
need for extensive manipulation and purification.
The synthesis of 3, depicted in Scheme 2, followed the C15+C10
approach used by a number of researchers for the synthesis of
Copyright # 2002 John Wiley & Sons, Ltd.
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P. VEERA REDDY ET AL.
Scheme 2.
analogous structures.37 Construction of the C15-ylide 4 and 2,7-
dimethyl-2, 4, 6-octatrienedial (5) was uneventful and followed reported
procedures.38,39 Wittig olefination of 5 with ylide 4 was initiated with
NaOMe in methanol and delivered the C25-aldehyde 6 in good yields
with 97% E stereoselectivity of the newly formed double bond. Double
olefination of 5 led to the formation of b-carotene (1) in 10% yield,
however, it was easily removed owing to its hydrophobicnature.
Treatment of aldehyde 6 with (carbethoxymethylene)triphenylpho-
sphorane in a sealed tube at 958C for 5 h led to 7 (>97% E) in 71%
yield. Ethyl ester 7 was reduced with DIBAL, and the resulting allylic
alcohol was oxidized with MnO2 to provide C27-aldehyde 8. In our
hands, commercially available oxidant did not yield any product, and
led to the decomposition of starting material. However, freshly prepared
activated MnO2 worked well.40 Also, maintainance of the temperature
at 08C was critical for the success of this oxidation.
The tritium label was incorporated by reduction of aldehyde 8 with
NaB3H4 at 08C, which was immediately reoxidized with MnO2 to yield
the radiolabeled C27-aldehyde 9. Although past reductions of a,b-
unsaturated carbonyls have sometimes given rise to other reductive
products such as 1,4-reduced compounds, none were observed in this
instance. Yields reported in Scheme 2 beyond the radioactive reduction
with NaB3H4 refer to isolated and purified products from the non-
radioactive reactions, which were used to optimize each step towards
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SYNTHESIS OF CAROTENOIC ACID
83
the synthesis of 3. During the radioactive synthesis, crude radioactive
intermediates were carried forward without isolation to minimize the
number of manipulations and avoid loss of radioactive compound. The
final compound was purified and fully characterized. Wittig olefination
of 9 with (carbethoxyethylidene)triphenyl-phosphorane in refluxing
THF proceeded well to deliver ester 10 as the sole product (Z isomer
was not detected by NMR spectroscopy). The resulting triphenylpho-
sphine oxide and remaining phosphonium salts were easily removed by
flushing the crude reaction mixture through a short pad of silica.
Hydrolysis of ethyl ester 10 proved to be more challenging than
expected and routine basic and acidic conditions did not lead to a high
yielding process. However, procedures detailed by a recent report by
Larsen et al.41 for hydrolysis of such esters was used successfully (see
experimental) to obtain the radiolabeled acid 3 (21 mCi/mmol) in 19%
overall radiochemical yield. With 3 in hand, we are presently in the
process of optimizing photoaffinity labeling conditions. In conclusion,
the present procedure provides a simple and efficient route for the
synthesis of all-trans-[100-3H]-80-apo-b-carotenoic acid (3).
Experimental
All reactions were carried under an atmosphere of nitrogen and removal
of solvents was performed under reduced pressure with a Buchi rotary
evaporator. THF and Et2O were freshly distilled from sodium/
benzophenone, and CH2Cl2 was distilled over CaH2 under a nitrogen
atmosphere. Radioactive NaB3H4 was purchased from American
Radiolabeled Chemicals, Inc (St. Louis, MO). Analytical TLC was
carried out using Merck 250 mm Silica gel 60 F254 and spots were
visualized under UV light. Column chromatography was conducted
using Silicycle silica gel (230–400 mesh). 300 MHz 1H-NMR and
75 MHz 13C-NMR spectra were recorded on a Varian Gemini-300
instrument, and the residual proticsolvent (CDCl or DMSO-d6) was
3
used as internal reference. UV-visible spectra were recorded on a
Perkin-Elmer Lambda 40 spectrometer. A Wallac WinSpectral 1414
liquid scintillation counter was used for quantification of radioactivity.
In a typical measurement 1–10 ml of sample was added to 3 ml of
Optiphase ‘HiSafe’ 3 liquid scintillation cocktail (Wallac) and the
solution was counted for 1 min. All the reactions were carried out in a
darkroom under minimal photographicsafety red lights.
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2,7,11-Trimethyl-13-(2,6,6-trimethyl-cyclohex-1-enyl)-trideca-
2,4,6,8,10,12-hexaenal (6)
Sodium methoxide (121.0 mg, 2.250 mmol, 1.5 eq.) was added to a
solution of 439 (817.0 mg, 1.5 mmol) in dry methanol (8 ml) at 08C, and
stirred until the phosphonium salt was completely converted to the
deep red phosphorane. A solution of 7-dimethyl-2,4,6-trienedial 538
(164.0 mg, 1 mmol) in dry methanol (1 ml) was added and the mixture
was stirred for 30 min. The reaction mixture was diluted with water
(10 ml) and extracted with diethyl ether (3 Â 5 ml). The organiclayer
was dried over Na2SO4 and the solvent was removed in vacuo to give the
C25-aldehyde 6 (217.0 mg, 62%, 97:3 E:Z), and b-carotene 1 (57.0 mg,
10%), which was purified by flash silica chromatography (2% ethyl
acetate in hexane).
UV: lmax (hexanes) 414 nm; 1H NMR (300 MHz, CDCl3) d 9.40 (1H,
s), 7.03 (1H, d, J¼ 12 HzÞ, 6.96 (1H, dd, J ¼ 14:4, 12.3 Hz), 6.77 (1H,
dd, J ¼ 14:4, 11.4 Hz), 6.41 (1H, dd, J ¼ 14:4, 11.7 Hz), 6.5 (1H, d,
J¼ 11:7 Hz), 6.7 (1H, d, J¼ 12:3 Hz), 6.12 (3H, m), 2.00 (3H, s, CH3),
1.97 (3H, s, CH3), 1.86 (3H, s, CH3), 1.67 (3H, s, CH3), 1.97–1.40 (6H,
m, 3 Â CH2Þ, 1.00 (6H, s, 2 Â CH3Þ; 13C NMR (75 MHz, CDCl3) d
194.38, 148.94, 141.72, 137.88, 137.71, 137.42, 136.66, 136.22, 130.73,
130.30, 129.74, 127.74, 127.63, 127.16, 39.51, 34.17, 33.03, 28.89, 26.81,
21.70, 19.13, 12.97, 12.77, 9.51.
4,9,13-Trimethyl-15-(2,6,6-trimethyl-cyclohex-1-enyl)-pentadeca-
2,4,6,8,10,12,14-heptaenoic acid ethyl ester (7)
A solution of 6 (110.0 mg, 0.310 mmol) and (carbethoxymethylene)-
triphenylphosphorane (323.0 mg, 0.930 mmol) in dry THF (4 ml) was
heated at 958C for 5 h in a sealed tube. The reaction mixture was cooled
to room temperature and diluted with water (10 ml) and Et2O (10 ml).
Phases were separated and the aqueous phase was extracted with Et2O
(2 Â 10 ml). The combined organics were dried over Na2SO4, filtered
and concentrated under reduced pressure. Crude product was purified
by flash silica chromatography (5% ethyl acetate in hexane) to yield 7
(93.0 mg, 71%, >97% E). The coupling constant for the newly formed
double bond confirms the E stereochemistry. The Z isomer was not
detected by NMR spectroscopy.
UV: lmax (hexanes) 423 nm; 1H NMR (300 MHz, CDCl3) d 7.35 (1H,
d, J¼ 15:6 Hz), 6.78 (1H, d, J¼ 12:9 Hz), 6.79 (1H, dd, J ¼ 14:7,
11.4 Hz), 6.65 (1H, dd, J ¼ 13:5, 11.7 Hz), 6.45 (1H, d, J¼ 11:1 Hz),
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SYNTHESIS OF CAROTENOIC ACID
85
6.33 (1H, d, J¼ 14:7 Hz), 6.23 (1H, d, J¼ 11:7 Hz), 5.96 (3H, m), 5.84
(1H, d, J¼ 15:6 Hz), 4.20 (2H, q, J¼ 7:2 Hz), 2.01, (2H, m, CH2), 1.97
(3H, s, CH3), 1.96 (3H, s, CH3), 1.89 (3H, s, CH3), 1.69 (3H, s, CH3),
1.60 (2H, m, CH2), 1.45 (2H, m, CH2), 1.28 (3H, t, J¼ 7:2 Hz,
OCH2 CH3 ), 1.00 (6H, s, 2 Â CH3); 13C NMR (75 MHz, CDCl3) d
167.51, 148.72, 139.25, 138.93, 137.79, 137.58, 136.91, 136.73, 133.74,
133.36, 131.57, 130.56, 129.54, 128.60, 127.16, 126.28, 116.23, 60.17,
39.55, 34.20, 33.07, 28.92, 21.75, 19.18, 14.31, 12.90, 12.76, 12.53.
4,9,13-Trimethyl-15-(2,6,6-trimethyl-cyclohex-1-enyl)-pentadeca-
2,4,6,8,10,12,14-heptaenal (8)
To a cold (08C) solution of 7 (90.0 mg, 0.215 mmol) in dry Et2O (6 ml)
was added 1 M solution of DIBAL in cyclohexane (0.43 ml, 0.430 mmol)
and stirred for 30 min. Saturated sodium–potassium tartrate solution
(3 ml) and glycerol (5 drops) were added. The reaction mixture was
stirred over night at room temperature; phases were separated and the
aqueous phase was extracted with Et2O (3 Â 10 ml). The combined
organiclayers were washed with brine (3 mL), dried over Na SO4,
2
filtered and concentrated in vacuo to give the corresponding allylic
alcohol, which was pure enough to be used without further purification
(80.0 mg, 99%).
1
UV: lmax (hexanes) 400, 422 nm; H NMR (300 MHz, CDCl3) d 6.67
(3H, m), 6.22 (7H, m), 5.84 (1H, dt, J ¼ 12, 6 Hz), 4.22 (2H, t,
J ¼ 6:0 Hz), 2.00 (2H, m, CH2), 1.94 (6H, s, 2 Â CH3Þ, 1.89 (3H, s,
CH3), 1.70 (3H, s, CH3), 1.60 (2H, m, CH2), 1.45 (2H, m, CH2), 1.00
(6H, s, 2 Â CH3Þ; 13C NMR (75 MHz, CDCl3) d 137.82, 137.68, 137.06,
136.68, 136.27, 136.09, 134.62, 132.32, 131.96, 130.68, 130.35, 129.35,
127.28, 126.66, 125.14, 63.91, 39.55, 34.21, 33.05, 28.92, 21.73, 19.20,
12.82, 12.78, 12.71.
MnO420 (100.0 mg, 1.160 mmol, 20 eq.) was added to a stirred solution
of the aforementioned allylicalcohol (22.0 mg, 0.058 mmol) in CH 2Cl2
(3 ml) at 08C and stirred for 5 h. The reaction mixture was filtered
through a pad of celite and washed with CH2Cl2 (4 Â 0.5 ml). Solvent
was removed under reduced pressure and the crude aldehyde was
purified by flash silica column chromatography (5% ethyl acetate in
hexane) to yield 8 (15.0 mg, 68%).
UV: lmax (hexanes) 436 nm; 1H NMR (300 MHz, CDCl3) d 9.57 (1H,
d, J=7.8 Hz), 7.13 (1H, d, J¼ 15:3 HzÞ, 6.82 (1H, dd, J ¼ 13:8,
11.7 Hz), 6.73 (1H, dd, J ¼ 14:7, 11.4 Hz), 6.53 (2H, m), 6.35 (1H, d,
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J ¼ 15 Hz), 6.25 (1H, d, J ¼ 12 Hz), 6.14 (4H, m), 2.01(2H, m, CH2),
2.00 (3H, s, CH3), 1.96 (3H, s, CH3), 1.94 (3H, s, CH3), 1.70 (3H, s,
CH3), 1.60 (2H, m, CH2), 1.45 (2H, m, CH2), 1.01(6H, s, 2 Â CH3);
13C NMR (75 MHz, CDCl3) d 193.74, 156.62, 141.21, 140.06, 137.81,
137.56, 137.44, 136.57, 135.36, 133.61, 131.43, 130.50, 129.73, 128.36,
127.53, 127.08, 126.93, 39.61, 34.25, 33.11, 28.95, 21.76, 19.21, 12.99,
12.82, 12.70.
NaB3H4 reduction of 8 and synthesis of 9
Aldehyde 8 (15.0 mg, 0.039 mmol) was added to a cold (08C) solution of
NaB3H4 (1.8 mCi, 255 mCi/mmol) in THF/MeOH (4 ml, 1 : 1). The
reaction mixture was stirred for 20 min at which time unlabeled NaBH4
(1.5 mg, 0.040 mmol) was added to the reaction mixture (stirred for
15 min). The reaction was quenched by addition of saturated NH4Cl
and extracted with Et2O (3 Â 2 ml). Phases were separated and the
organiclayer was washed with sat. NaCl, and dried over Na SO4.
2
Removal of solvent under reduced pressure gave the corresponding
allylic alcohol (13.0 mg, 93%) which was used without further
purification. After removal of solvent under reduced pressure, the
crude residue was dissolved in CH2Cl2 (2 ml), freshly prepared MnO240
(58.0 mg, 20 eq., 0.680 mmol) was added at 08C, and the reaction was
stirred for 5 h. The suspension was filtered through a pad of celite,
washed with CH2Cl2 (1 Â 2 ml), and concentrated under reduced
pressure followed by chromatographic purification over silica gel (5%
ethyl acetate in hexanes) to yield pure aldehyde 9 (9.6 mg, 73%). The
latter yield refers to product obtained from non-radioactive syntheses.
The radioactive material was used without purification following
extraction and removal of CH2Cl2 in the next step and the amount
isolated was assumed to be the same as for the non-radioactive reaction.
2,6,11,15-Tetramethyl-17-(2,6,6-trimethyl-cyclohex-1-enyl)-heptadeca-
2,4,6,8,10,12,14,16-octaenoic acid ethyl ester (10)
(Carbethoxyethylidene)triphenylphosphorane (18.0 mg, 0.050 mmol)
was added to a solution of 9 (9.6 mg, 0.025 mmol) in THF (3.5 ml)
and the reaction was refluxed for 5 h. The reaction mixture was diluted
with water (4 ml) and extracted with Et2O (3 Â 2 ml). The combined
ether extracts were dried over Na2SO4. The solvent was removed under
reduced pressure and the crude product was purified by flushing it
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SYNTHESIS OF CAROTENOIC ACID
87
through a small pad of silica in a Pasteur pipet (5% ethyl acetate in
hexane) to yield ethyl ester 10 (9.4 mg, 80%). During the preparation of
the radioactive material, the crude product was also flushed through a
small pad of silica, however, it was not isolated and characterized, but
was used directly in the next step.
UV: lmax (hexanes) 440 nm; 1H NMR (300 MHz, CDCl3) d 7.25 (1H,
d, J¼ 11:6 Hz), 6.59 (4H, m), 6.4 (2H, m), 6.25 (1H, d, J¼ 11:1 HzÞ, 6.4
(4H, m), 4.21 (2H, q, J¼ 7:2 HzÞ, 2.00 (2H, m, CH2), 1.97 (3H, s, CH3),
1.95 (3H, s, CH3), 1.69 (6H, s, 2 Â CH3Þ, 1.60 (2H, m, CH2), 1.54 (3H, s,
CH3), 1.45 (2H, m, CH2), 1.29 (3H, t, J¼ 7:2 Hz, OCH2 CH3 ), 1.00
(6H, s, 2 Â CH3); 13C NMR (75 MHz, CDCl3) d 168.52, 143.84, 138.74,
137.83, 137.80, 137.65, 136.94, 136.53, 135.71, 135.39, 131.99, 131.91,
130.67, 129.48, 129.32, 126.95, 126.15, 125.72, 123.14, 60.46, 39.58,
34.22, 33.07, 29.65, 28.92, 21.73, 19.19, 14.32, 12.86, 12.81, 12.75, 12.68.
[100-3H]-80-apo-b-carotenoic acid (3)
KOH (25%, 2 ml) was added to a solution of 10 (9.4 mg, 0.020 mmol), in
iso-butyl alcohol (3 ml). The resulting mixture was heated at 958C for
2 h and cooled to room temperature. H2SO4 (20%, 2 ml) was added and
the reaction mixture was again heated to 708C for 30 min. The organic
layer was separated and washed with hot water (3 Â 1 ml). Dichloro-
methane (5 ml) was added to the extracted iso-butyl alcohol resulting in
two layers; the desired acid was extracted into CH2Cl2. The CH2Cl2
layer was separated, dried over Na2SO4, and concentrated under
reduced pressure. The crude product was purified through a small pad
of silica in a Pasteur pipet (5–15% ethyl acetate in hexane) to yield pure
acid 3 (7.0 mg, 80%, 21 mCi/mmol, 0.34 mCi, 19% of total radionuclei
incorporated) based on comparative TLC, and UV with authentic cold
material, and all spectroscopic data were consistent with reported
values.41
UV: lmax (acetone) 440 nm; 1H NMR (300 MHz, CDCl3) d 12.20 (1H,
bs,–CO2H), 7.20 (1H, d, J¼ 10:8 HzÞ, 6.80–6.16 (11H, m, olefinic), 2.00
(2H, m, CH2), 1.96 (6H, s, 2 Â CH3), 1.95 (3H, s, CH3), 1.92 (3H, s,
CH3), 1.69 (3H, s, CH3), 1.58 (2H, m, CH2), 1.44 (2H, m, CH2), 1.02
(6H, s, 2 Â CH3).
Acknowledgements
We are grateful to the Research Corporation (RI0410) for financial
support of this research.
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