Synthesis and characterization of quantum dot nanoparticles bound to the plant volatile precursor of hydroxy-apo-10′-carotenal

Article abstract of DOI:10.1021/jo500605c

This study is focused on the synthesis and characterization of hydroxy-apo-10′-carotenal/quantum dot (QD) conjugates aiming at the in vivo visualization of β-ionone, a carotenoid-derived volatile compound known for its important contribution to the flavor and aroma of many fruits, vegetables, and plants. The synthesis of nanoparticles bound to plant volatile precursors was achieved via coupling reaction of the QD to C₂₇- aldehyde which was prepared from α-ionone via 12 steps in 2.4% overall yield. The formation of the QD-conjugate was confirmed by measuring its fluorescence spectrum to observe the occurrence of fluorescence resonance energy transfer.

Full text of DOI:10.1021/jo500605c

Article
pubs.acs.org/joc
Synthesis and Characterization of Quantum Dot Nanoparticles
Bound to the Plant Volatile Precursor of Hydroxy-apo-10′-carotenal
Vo Anh Tu,† Atsushi Kaga,† Karl-Heinz Gericke,‡ Naoharu Watanabe,†,§ Tetsuo Narumi,† Mitsuo Toda,†
Bernhard Brueckner,⊥ Susanne Baldermann,*,⊥,∥ and Nobuyuki Mase*,†
†Department of Applied Chemistry and Biochemical Engineering, Graduate School of Engineering, and Green Energy Research
Division, Research Institute of Green Science and Technology, Shizuoka University, 3-5-1 Johoku, Hamamatsu, Shizuoka 432-8561,
Japan
‡Institute for Physical and Theoretical Chemistry, University of Technology, Braunschweig, Hans-Sommer-Strasse 10, D-38106,
Braunschweig, Germany
§Graduate School of Science and Technology, Shizuoka University, Shizuoka, 432-8561 Japan
⊥Leibniz-Institute of Vegetable and Ornamental Crops Großbeeren/Erfurt e.V., Theodor-Echternmeyer-Weg 1, 14979 Großbeeren,
Germany
∥Institute of Nutritional Science, University of Potsdam, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany
S
* Supporting Information
ABSTRACT: This study is focused on the synthesis and
characterization of hydroxy-apo-10′-carotenal/quantum dot
(QD) conjugates aiming at the in vivo visualization of β-ionone,
a carotenoid-derived volatile compound known for its important
contribution to the flavor and aroma of many fruits, vegetables,
and plants. The synthesis of nanoparticles bound to plant volatile
precursors was achieved via coupling reaction of the QD to C27-
aldehyde which was prepared from α-ionone via 12 steps in 2.4%
overall yield. The formation of the QD-conjugate was confirmed
by measuring its fluorescence spectrum to observe the occurrence
of fluorescence resonance energy transfer.
contributes to the fragrance in the flower of Boronia
megastigma,5 Osmanthus fragrans,6 Rosa damascena, and Camel-
lia sinensis.7 Although its concentration is not particularly high,
β-ionone is among the most potent flavor-active molecules
characterized by extremely low odor thresholds. Therefore, we
are interested in determining the formation pathway yielding β-
ionone as a secondary metabolite in vivo, which has not been
identified yet. We would like to propose a hypothesis that helps
to visualize β-ionone in vivo (Figure 1). The C27-apocarotenal
intermediate (apo-10′-carotenal) from the first CCD4-catalyzed
oxidative cleavage of C40-carotenoid in the plastid is prepared
for coupling to nanocrystal quantum dot (QD) to afford
apocarotenal−QD conjugate 1. QD is employed for the
coupling reaction owing to its unique properties such as
water solubility, photostability, and exceptional fluorescence.8
The apocarotenal−QD conjugate 1 is prepared and sub-
sequently administrated into the living plant cell. Conjugate 1
cleaves to β-ionone−QD conjugate 2, catalyzed by enzyme
CCD1. Because of the fluorescence property of QD, β-ionone−
QD conjugate 2 can be detected by two-photon microscopy, a
fluorescence imaging technique allowing the imaging of living
INTRODUCTION
■
Plant carotenoids are tetraterpenes with polyene chains that
may contain up to 15 conjugated double bonds; therefore, they
act as natural pigments and are responsible for the distinct
yellow to the red-orange color of fruits, flowers, vegetables, and
leaves. Further, they can be oxidized at almost every position
and cleaved enzymatically at distinct double bonds. The
carotenoid cleavage dioxygenase (CCD) is known to be a
specific nonheme enzyme for the oxidation of the rigid
backbone of carotenoids.1 CCD exhibits a high degree of
regio- and stereospecificity for the cleavage of specific double
bond positions in diverse substrates. The cleavage products of
CCDs play important roles in plant growth, protection against
light exposure, and as chemoattractants and repellents in plants
and cyanobacteria,2 as anti-fungal agents, and as the source of
fragrances for reproduction.3 Among these products, a total of
1700 volatile compounds have been isolated and identified
from more than 90 plant families.4 These volatile compounds
are released from leaves, flowers, and fruits into the atmosphere
and from roots into the soil; they defend plants against
herbivores and pathogens and provide a reproductive advantage
by attracting the pollinators and seed dispersers.
β-Ionone, one of the most abundant carotenoid-derived
compounds, is a significant volatile compound, which
Received: March 14, 2014
Published: July 15, 2014
© 2014 American Chemical Society
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First, the synthesis of C15-phosphonium ylide 5 was
accomplished as shown in Scheme 2.9 The carbonyl group of
a
Scheme 2. Synthesis of C15-Phosphonium Ylide 5
Figure 1. An approach for the labeling of carotenoid-derived volatile
compounds using quantum dots (QDs).
tissue up to a very high depth. Consequently, β-ionone can be
in vivo visualized. Herein, we report the synthesis and the
characterization of apocarotenal−QD conjugate 1 aiming at the
in vivo visualization of β-ionone.
a
Reagents and conditions: (i) ethylene glycol, p-TsOH, CH(OCH3)3,
RESULTS AND DISCUSSION
■
rt, overnight; (ii) t-BuOOH, bleach (5.25% NaOCl), 8 h, 40% yield
over two steps; (iii) DIBAL, −30 to −20 °C, 1 h; (iv) 0.3 M HCl,
acetone, rt, 30 min, 91% yield over two steps (34% de, cis major); (v)
KOH/MeOH, CH2Cl2, 50 °C, 1.5 h, 81% yield from the trans-isomer;
(vi) CH2CHMgBr, −20 °C, 30 min; (vii) PPh3·HBr, 0 °C to rt,
overnight.
To determine the biosynthetic pathway of β-ionone, we
designed the synthesis of C27-conjugate 1 from α-ionone 6 as
shown in Scheme 1 (retrosynthetic analysis). C27-conjugate 1
Scheme 1. Retrosynthesis of Apocarotenal−QD Conjugate 1
α-ionone 6 was protected with ethylene glycol to afford the
ketal 9, which was then oxidized at the allylic position using
tert-BuOOH. The regio- and stereoselective reduction of enone
10, followed by the deprotection afforded 3-hydroxy-α-ionone
12. Diverse reducing agents, such as sodium borohydride/
cerium chloride (86% yield, 54% de, cis major), K-selectride
(87% yield, 14% de, trans major), and DIBAL (91% yield, 34%
de, cis major), were examined for the stereoselective reduction
of the carbonyl group of 12. However, no significant
stereoselectivity could be achieved. Among these reducing
agents, DIBAL was chosen because it was inexpensive. The
conversion of 3-hydroxy-α-ionone 12 to 3-hydroxy-β-ionone 13
was achieved in 81% isolated yield using potassium hydroxide/
methanol. Next, the Grignard reaction of 3-hydroxy-β-ionone
13 with vinylmagnesium bromide afforded tertiary vinyl-β-ionol
14. The treatment of alcohol 14 with triphenylphosphine in
hydrobromic acid afforded the desired C15-phosphonium salt 5,
which was directly used for the next coupling reaction without
further purification.
could be prepared via C15 + C10 + C2 → C27 route, where α-
ionone 6 and trans-1,4-dichloro-2-butene 8 are commercially
available. The Wittig coupling reaction of C15-phosphonium
ylide 5 and C10-dialdehyde 7 can be employed for the efficient
synthesis of 3-hydroxy-β-apo-12′-carotenal 4 (C25-aldehyde).
Ylide 5 was synthesized by a four-step reaction from α-ionone
6, whereas C10-dialdehyde 7 was prepared via a three-step
reaction from trans-1,4-dichloro-2-butene 8.
Next, the synthesis of C10-dialdehyde 7 was accomplished as
shown in Scheme 3. (E)-Tetraethyl but-2-ene-1,4-diylbis-
(phosphonate) 15 was first prepared by the Arbuzov reaction.
trans-1,4-Dichloro-2-butene (8) was reacted with triethyl
phosphate in a sealed tube at 160 °C and 0.4 MPa pressure
to afford 15 in 92% yield.10 The Horner−Wadsworth−
Emmons reaction of phosphonate 15 with pyruvic aldehyde
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a
a
Scheme 3. Synthesis of C10-Dialdehyde 7
Scheme 5. Coupling Reaction of QD to C27-Aldehyde 17
a
Reagents and conditions: (i) P(OEt)3, 0.4 MPa, 160 °C, 2 h, 92%
yield; (ii) MeCOCH(OMe)2, NaH, THF, 0 to 60 °C, 3 h, then 20%
aqueous H2SO4, 0 to 50 °C, 2.5 h, 35% yield over two steps.
a
Reagents and conditions: (i) EDC, NHS, MES buffer; (ii) 17, DMF,
dimethyl acetal under the basic conditions afforded the desired
C10-dialdehyde 7 in 35% yield over two steps.
24 h.
The Wittig olefination of C15-phosphonium ylide 5 with C10-
dialdehyde 7 was initiated by adding 1,2-epoxybutane to
successfully afford C25-aldehyde 4 in 58% yield. The treatment
of C25-aldehyde 4 with carbethoxymethylene triphenylphos-
phorane afforded ester 16, which was reduced with DIBAL
followed by oxidation with manganese dioxide to afford the
desired C27-aldehyde 17 in 44% yield over two steps (Scheme
4).
hydrochloride (EDC, 1,000 equiv) and NHS (1,000 equiv) in
2-(N-morpholino)ethanesulfonic acid buffer (MES) at pH 6.
Then, ester 19 was reacted with C27-aldehyde 17 (100 equiv) in
DMF for 24 h to afford conjugate 1.
The formation of conjugate 1 was confirmed by measuring
its fluorescence spectrum to observe the change in the
maximum wavelength of the reaction mixture compared to
that of QD-COOH 18 and C27-aldehyde 17. The fluorescent
nanocrystal QD 510 used in this study with a CdTe core is
hydrophilic owing to the COOH groups on its surface. The
molar weight of the nanocrystal is approximately 3000. The
absorption spectrum shows that QD 510 absorbs energy across
a broad range of the spectrum; therefore, it can be excited at
different wavelengths. The QD 510 emits sharply with a defined
narrow emission peak at 515 nm in DMSO (Figure 2, the
absorption and fluorescence spectra are normalized). The
sample was also excited at 460 nm that is the maximum
wavelength for absorption of C27-aldehyde 17 in DMSO.
The absorption of C27-aldehyde 17 shows a low degree of
resolution in its vibronic bands because of the presence of one
bulky isoprenoid ring coupled to the conjugated π-electrons in
the double-bond chain, thereby increasing the conformational
disorder in the polyene chain. The fluorescence spectrum of
C27-aldehyde 17 was thought to be composed of two
a
Scheme 4. Synthesis of C27-Aldehyde 17
a
Reagents and conditions: (i) 1,2-epoxybutane, reflux, overnight, 58%
yield; (ii) Ph3PCHCOOEt, THF, 90 °C, 7 h; (iii) DIBAL, Et2O, 0
°C, 30 min, then MnO2, CH2Cl2, 0 °C, 3.5 h, 44% yield over two
steps.
The critical step was the coupling of commercially available
and colloidal carboxyl QD (QD-COOH, 18)11 to C27-aldehyde
17 via an ester bond formation (Scheme 5).12 First, the
carboxylic acid group of 18 was activated by preparing the
corresponding N-hydroxysuccinimide (NHS) ester 19 in the
presence of 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide
Figure 2. Room-temperature absorption and fluorescence spectra of
QD 510 in DMSO.
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components, centered at 585 and 768 nm (Figure 3).
Moreover, the excitation-wavelength dependence of the
Figure 5. Fluorescence spectra of the reaction mixture in DMF with
time (λex = 355 nm).
Figure 3. Room-temperature absorption and fluorescence spectra of
C27-aldehyde 17 in DMSO.
Furthermore, the fluorescence intensity was found to increase
with the progress of the reaction. The results remarkably
showed the occurrence of fluorescence resonance energy
transfer (FRET, Figure 6), in which the QD acts as the
fluorescence spectrum of C27-aldehyde 17 in DMSO was
carefully examined. It was found that the intensity of the short-
wavelength band at 585 nm fluorescence decreases with
increasing excitation wavelength. This behavior was accom-
panied by an increase in the long-wavelength emission band
centered at 768 nm.
The change in the fluorescence of the reaction mixture with
time was also examined (Figures 4 and 5). The reaction was
Figure 6. Energy transfer from QD to carotenal in DMSO.
donor fluorophore and transfers the excited energy to the C27-
aldehyde moiety that acts as the acceptor chromophore. The
fluorescence measurement with time showed the same result as
that in DMF; the fluorescence intensity of the band centered at
510 nm decreased with increasing intensity at 573 nm, which is
close to the maximum wavelength of C27-aldehyde 17 in DMF
(565 nm). Once more, it confirms the radiationless trans-
mission of energy from a donor molecule to an acceptor
molecule. The transfer of energy leads to a decrease in the
donor fluorescence intensity and excited-state lifetime and an
increase in the acceptor’s emission intensity.
Figure 4. Fluorescence spectra of the reaction mixture in DMSO with
time (λex = 360 nm).
There is a slow decrease in the fluorescence intensity at 515
nm in DMSO, which was not observed in DMF, indicating that
the reaction rate in DMF was faster than that in DMSO.
Although the rate of reaction in DMF is faster, DMSO should
be used for this reaction because of its lower toxicity for the
plant cells. DMSO has a low acute and chronic toxicity in
animal, plant, and aquatic life, and it has been applied widely for
cell introduction.
A concentration-dependent fluorescence intensity was
examined under the same coupling reaction conditions of
QD 510 with different equivalents of C27-aldehyde 17 (70, 90,
and 100 equiv) in DMSO. The reaction was stirred for 1 h, and
the fluorescence spectra were recorded. The results clearly
indicated that there is a decrease in the fluorescence intensity at
performed in the presence of EDC and NHS in MES buffer for
24 h. The results showed that in DMSO, the fluorescence
intensity centered at 515 nm decreased with the increase in the
intensity at 595 nm, which is close to the maximum wavelength
of C27-aldehyde 17 in DMSO (585 nm). For further evidence,
the coupling reaction in different solvent systems was also
investigated. The maximum fluorescence wavelength in DMF
blue-shifted to 573 nm compared to 595 nm in DMSO because
of the solvent effect.
The fluorescence spectra of the reaction mixture showed a
change in the maximum fluorescence wavelength from 515 nm
for the QD to 595 nm for the reaction mixture, which is close
to that of C27-aldehyde 17 in DMSO centered at 585 nm.
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515 nm and an increase at 595 nm (Figure 7). The number of
carotenal moieties bound to QD increased with the increasing
no changes in the fluorescence emission. At 30 °C after 3 h, at
70 °C, and at low pH values of 5 we determined small shifts in
the maximum wavelengths (See Supporting Information [SI]).
Even if such extremes were to occur in our in vivo experiments,
these variations would still enable us to determine the FRET
phenomena, where we found a shift of the maximum
wavelength from 515 to 595 nm (Figure 4).
The fluorescence emission spectrum of the donor QD
molecule overlaps with the absorption spectrum of the acceptor
carotenoid molecule, and the two are within a minimal spatial
radius in the range 10−100 Å; therefore, the donor molecule
can directly transfer its excitation energy to the acceptor
molecule through long-range dipole−dipole intermolecular
coupling (Figures 6 and 9). For the detailed investigation of
Figure 7. Concentration-dependent fluorescence intensity at different
contraction (λex = 360 nm, the reactions were performed for 1 h).
equivalence of C27-aldehyde 17. This leads to the change in the
intensities of the peaks at 515 and 595 nm with the change in
concentration of C27-aldehyde 17.
To investigate whether the nonspecific adsorption of the
carotenal moiety occurred on the QD surface, the control
experiments were carried out using the same coupling
procedure for QD 510 and C27-aldehyde 17 in DMF and
DMSO in the absence of EDC and NHS in MES buffer. The
results of the fluorescence measurements strongly showed that
the photophysical properties of QD 510 remained unchanged;
however, no characteristic bands of the carotenal moiety were
detected in the emission spectra (Figure 8). The blank
○
Figure 9. Absorption of C27-aldehyde 17 (- -) and fluorescence of
●
▲
reaction mixture with QD 510 (- -) and QD 620 (- -) in DMSO (λex
= 360 nm).
the methodology, the EDC-mediated coupling reaction was
repeated using a different QD: (CdTe) 620.11 The fluorescence
emission intensity of the reaction mixture at 630 nm (excited at
360 nm) after 24 h remained unchanged in DMSO (Figure 9).
This clearly demonstrates that there is no FRET between QD
620 and the apocarotenal moiety because of no overlap
between the absorption of apocarotenal and the emission
spectra of QDs.
Commercially available QD (CdSe/ZnS) 525 and 625 were
also examined for the coupling reaction.13 The fluorescence of
the reaction mixture after coupling for 24 h showed the
maximum intensities at 530 and 630 nm for QD (CdSe/ZnS)
525 and 625, respectively; no change in the fluorescence was
observed (Figure 10). The slight red shift can be attributed to
the solvent effect. Although the absorption of apocarotenal
overlapped with the fluorescence of QD 525, the FRET did not
occur. This is probably because of the small overlapping area
and different structure of QD 525, i.e., the core−shell material
of this nanocrystal is further coated with another polymer layer.
Therefore, it increased the distance between the QD and
apocarotenal.
Figure 8. Fluorescence spectra of reaction between QD 510 and C27-
aldehyde 17 in DMF (- -) and DMSO (- -) in the absence of
coupling reagents.
●
○
Finally, in preliminary experiments we could confirm the
uptake of the reaction mixture with QD 510 in Arapdospsis
seedlings by means of confocal laser microscopy (See SI).
experiment showed that the signal observed in the case of
apocarotenal−QD conjugate 1 is not because of the nonspecific
adsorption of the dye; it is attributed to the expected covalent
linking of apocarotenal to QD.
To explore potential changes in living plants the temper-
ature-, light-, and pH-dependence of QD 510 nm was measured
in an aqueous environment. At room temperature we observed
CONCLUSION
■
We reported the design and synthesis of apocarotenal−QD
conjugate 1. To the best of our knowledge, this is the first
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1.85 (d, J = 1.3 Hz, 3H), 1.43 (s, 3H), 0.99 (s, 3H), 0.92 (s, 3H); 13C
NMR (75 MHz, CDCl3) δ 199.1 (CO), 161.4 (C), 135.0 (CH),
127.7 (CH), 126.0 (CH), 107.0 (C), 64.5 (CH2), 54.9 (CH), 47.5
(CH2), 36.0 (C), 27.7 (CH3), 26.9 (CH3), 25.0 (CH3), 23.3 (CH3).
(E)-4-(4-Hydroxy-2,6,6-trimethylcyclohex-2-en-1-yl)but-3-en-2-
one (12). A solution of the enone 10 (50 mg, 0.2 mmol) in CH2Cl2
(0.36 mL) was cooled down to −30 °C under Ar, and a solution of
DIBAL (0.36 mL of 1 M in hexane, 0.36 mmol) was added with a
syringe in 5 min. The mixture was stirred at −30 °C to −20 °C for 1 h.
The reaction was quenched by adding water at −10 °C followed by
silica gel (0.25 g). The mixture was filtered through Celite, and
CH2Cl2 was removed in vacuo. The residue was dissolved in acetone
(0.5 mL) and water (0.26 mL), and 0.3 M HCl (20 μL) was added.
The mixture was stirred at rt for 30 min and extracted with ethyl
acetate. The combined organic layer was dried over Na2SO4 and
evaporated to dryness to give the crude product. The crude product
was purified by column chromatography (hexane/ethyl acetate =
60:40) to give a mixture of trans- and cis-enone 12 (38 mg, 91%) in
trans/cis diastereometric ratio of 33:67.
○
Figure 10. Absorption of C27-aldehyde 17 (- -) and fluorescence of
(E)-4-((1R*,4R*)-4-Hydroxy-2,6,6-trimethylcyclohex-2-en-1-yl)-
1
●
▲
reaction with QD 525(- -) and QD 625 (- -) in DMSO (λex = 360
but-3-en-2-one (trans-12): Registry no. 118015-38-6; H NMR (300
nm).
MHz, CDCl3) δ 6.55 (dd, J = 15.8, 10.2 Hz, 1H), 6.11 (d, J = 15.8 Hz,
1H), 5.63 (brs, 1H), 4.38−4.19 (m, 1H), 2.51 (d, J = 10.2 Hz, 1H),
2.27 (s, 3H), 2.13 (brs, 1H), 1.84 (dd, J = 13.4, 5.9 Hz, 1H), 1.62 (s,
3H), 1.41 (dd, J = 13.4, 6.6 Hz, 1H), 1.03 (s, 3H), 0.89 (s, 3H); 13C
NMR (75 MHz, CDCl3) δ 198.2 (CO), 147.3 (CH), 135.1 (C),
133.6 (CH), 126.0 (CH), 65.1 (CH), 54.1 (CH), 43.7 (CH2), 33.7
(C), 29.1 (CH3), 26.9 (CH3), 24.3 (CH3), 22.4 (CH3).
synthesis of nanoparticles bound to plant volatile precursors.
Although it would be most desirable to use purified conjugates
for the in vivo visualization of the formation of carotenoid-
derived plant volatiles, the findings of this study suggest that the
reaction mixtures containing freshly generated QD conjugate-1
should be sufficient for elucidating the primary and secondary
metabolic pathways in plants in the future. Moreover,
multicolor fluorescence imaging may be possible using QDs
of different sizes, bound to the precursors, intermediates, and
enzymes. This approach can be applied to study the
localization, transport, storage, and crosstalk between the
diverse and complex biosynthesis pathways utilizing distinct
QDs for the staining of precursors, reaction products, and
enzymes. Additional information on the enzymatic reaction will
be reported elsewhere in due course.
(E)-4-((1S*,4R*)-4-Hydroxy-2,6,6-trimethylcyclohex-2-en-1-yl)-
but-3-en-2-one (cis-12): Registry no. 118015-37-5; 1H NMR (300
MHz, CDCl3) δ 6.63 (dd, J = 15.8, 9.6 Hz, 1H), 6.08 (d, J = 15.8 Hz,
1H), 5.59 (s, 1H), 4.32−4.20 (m, 1H), 2.28 (d, J = 9.6 Hz, 1H), 2.26
(s, 3H), 1.70 (dd, J = 13.0, 6.5 Hz, 1H), 1.63 (t, J = 1.6 Hz, 3H), 1.59
(brs, 1H), 1.39 (dd, J = 13.0, 9.8 Hz, 1H), 0.98 (s, 3H), 0.89 (s, 3H);
13C NMR (75 MHz, CDCl3) δ 198.5 (CO), 147.8 (CH), 135.5 (C),
132.8 (CH), 126.5 (CH), 66.4 (CH), 54.3 (CH), 40.6 (CH2), 34.9
(C), 29.0 (CH3), 27.0 (CH3), 26.9 (CH3), 22.3 (CH3).
(E)-4-(4-Hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl)but-3-en-2-
one (13). A solution of trans-enone 12 (52 mg, 0.25 mmol) in THF
(0.2 mL) was treated with a solution of KOH in methanol (10 wt %/v,
14 μL) under Ar. The mixture was heated to 50 °C for 1.5 h, and the
product was partitioned between water and ethyl acetate. The organic
layer was washed with water, dried over Na2SO4, and evaporated to
dryness to give the crude product (84 mg). The crude product was
purified by column chromatography to give the 3-hydroxy-β-ionone 13
(42 mg, 81% yield) as a yellow oil. Registry no. 116296-75-4; 1H NMR
(300 MHz, CDCl3) δ 7.22 (d, J = 16.4 Hz, 1H), 6.12 (d, J = 16.4 Hz,
1H), 4.10−3.89 (m, 1H), 2.44 (dd, J = 17.4, 5.4 Hz, 1H), 2.31 (s, 3H),
2.10 (dd, J = 18.2, 10.2 Hz, 1H), 1.99 (brs, 1H), 1.85−1.78 (m, 1H),
1.78 (s, 3H), 1.12 (s, 3H), 1.11 (s, 3H); 13C NMR (75 MHz, CDCl3)
δ 198.7 (CO), 142.4 (CH), 135.7 (C), 132.4 (C), 132.3 (CH), 64.3
(CH), 48.3 (CH2), 42.7 (CH2), 36.8 (C), 29.9 (CH3), 28.4 (CH3),
27.1 (CH3), 21.4 (CH3).
((2E,4E)-5-(4-Hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl)-3-meth-
ylpenta-2,4-dien-1-yl)triphenylphosphonium Bromide (5). A solu-
tion of the 3-hydroxy-β-ionone 13 (54 mg, 0.26 mmol) in toluene (1.0
mL) was cooled down to −20 °C under Ar. A 1 M solution of vinyl
magnesium bromide (0.63 mL, 0.63 mmol) was added dropwise in 10
min, and the mixture was stirred at −20 °C for 2 h. The reaction was
quenched with addition of saturated ammonium chloride solution at
−20 °C and stirred at rt for 10 min. The reaction mixture was
partitioned between water and ethyl acetate. The organic layer was
washed with water, dried over Na2SO4, and evaporated to dryness. The
crude the tertiary vinyl-β-ionol 14 was dissolved in MeOH (0.3 mL)
and directly used in the next step for the preparation of the Wittig salt
5. Triphenylphosphine hydrobromide (1.72 g, 5 mmol) was added to
methanol (0.3 mL) at 0 °C. The solution was stirred at rt for 20 min
and then treated with a solution of the crude tertiary vinyl-β-ionol 14
in MeOH (0.3 mL) by dropwise addition at 0 °C. The reaction was
kept at 0 °C for 1 h and was allowed to stir to rt overnight. The
EXPERIMENTAL SECTION
■
Synthesis of C10-Triphenylphosphonium Ylide 5. (E)-3,5,5-
Trimethyl-4-(2-(2-methyl-1,3-dioxolan-2-yl)vinyl)cyclohex-2-en-1-
one (10). Freshly distilled α-ionone (6, 1.92 g, 10 mmol) was
transferred into a 50 mL round-bottom flask with hexane (1 mL) and
was treated with ethylene glycol (1.86 g, 30 mmol). p-Toluene sulfonic
acid monohydrate (29 mg, 0.15 mmol) was added, and the mixture
was stirred at rt under Ar overnight. The product was partioned
between water and hexane, and the organic layer was washed with
water (3 × 80 mL), dried over Na2SO4, and evaporated to dryness to
yield the crude acetal 9 (2.66 g) as a pale-yellow oil. The crude
product was used in the next step without purification. The crude
acetal 9 was transferred into a two-necked flask using acetonitrile (9.5
mL). K2CO3 (166 g, 1.2 mmol) was added, and the mixture was
cooled down in an ice−salt bath to 0 °C under Ar. A 70% solution of
tert-butyl hydroperoxide (TBHP) in water (10.7 mL, TBHP: 75
mmol) was added dropwise to the mixture under Ar at 0 °C in 30 min.
House bleach containing 5.25% NaOCl (49.6 g, NaOCl: 35 mmol)
was then added over a period of 5 h at −10 to 0 °C. After the addition
was completed, the reaction mixture was stirred at 0 °C for an
additional hour. The product was treated with NaHCO3 (185 mg) at 0
°C and then extracted with hexane. The combined organic layer was
dried over Na2SO4, and evaporated to dryness to give the crude
product (2.58 g). The crude product was purified by autocolumned
chromatography (hexane/ethyl acetate = 70:30) to yield the enone 10
(765 mg, 40% over two steps) as a pale-yellow oil. Registry no. 79709-
34-5; 1H NMR (300 MHz, CDCl3) δ 5.87 (s, 1H), 5.68 (dd, J = 15.4,
9.2 Hz, 1H), 5.53 (d, J = 15.4 Hz, 1H), 4.01−3.75 (m, 4H), 2.51 (d, J
= 9.2 Hz, 1H), 2.30 (d, J = 16.7 Hz, 1H), 2.05 (d, J = 16.7 Hz, 1H),
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product was partitioned between hexane (5 mL) and methanol−water
(1:1 v/v, 5 mL). The aqueous layer was washed with hexane to remove
the excess triphenylphosphine hydrobromide, and the aqueous layer
was extracted with CH2Cl2. The combined CH2Cl2 layer was washed
with water, dried over Na2SO4, and evaporated to dryness to give the
C15-phosphonium ylide 5 that was used directly to prepare the C25-
aldehyde 4 without purification.
Synthesis of C10-Dialdehyde 7. Tetraethyl But-2-ene-1,4-diyl
(E)-Bis(phosphonate) (15). Triethyl phosphite (0.53 g, 3.2 mmol) and
(E)-1,4-dichlorobut-2-ene (8, 125 mg, 1.0 mmol) were added to an
autoclave under Ar. The reaction was carried out under 0.4 MPa, 160
°C over 2 h. After removing the excess reagent by distillation at 160 °C
under 2.6 mmHg, the phosphonate 15 (301 mg, 92%) was obtained as
the dark-orange residue. Registry no. 16626-80-5; 1H NMR (300
MHz, CDCl3) δ 5.62 (brs, 2H), 4.24−3.97 (m, 8H), 2.61 (dd, J = 17.5,
4.1 Hz, 4H), 1.32 (t, J = 7.0 Hz, 12H); 13C NMR (75 MHz, CDCl3) δ
124.3 (d, J = 3.8 Hz, CH), 61.7 (CH2), 31.3 (d, J = 3.9 Hz, CH2), 29.4
(d, J = 3.9 Hz, CH2), 16.2 (CH3).
7.38 (d, J = 15.5 Hz, 1H), 7.00−6.44 (m, 4H), 6.43−6.04 (m, 5H),
5.88 (d, J = 15.5 Hz, 1H), 4.22 (q, J = 7.1 Hz, 2H), 4.11−3.90 (m,
1H), 2.39 (dd, J = 16.4, 4.9 Hz, 1H), 2.14−1.65 (m, 3H), 2.00 (s, 3H),
1.98 (s, 3H), 1.92 (s, 3H), 1.74 (s, 3H), 1.47 (t, J = 12.0 Hz, 1H), 1.31
(t, J = 7.1 Hz, 3H), 1.07 (s, 6H); 13C NMR (75 MHz, CDCl3) δ 167.6
(CO), 148.8 (CH), 139.3 (CH), 138.9 (C), 138.4 (CH), 137.7 (C),
137.2 (CH), 136.6 (C), 133.8 (CH), 133.6 (C), 131.9 (CH), 131.1
(CH), 128.8 (CH), 126.4 (C), 126.2 (2 × CH), 116.4 (CH), 65.0
(CH), 60.1 (CH2), 48.3 (CH2), 42.5 (CH2), 37.0 (C), 30.2 (CH3),
28.6 (CH3), 21.5 (CH3), 14.2 (CH3), 12.8 (CH3), 12.7 (CH3), 12.4
(CH3); ESI-TOF high resolution mass: calcld for C29H41O3 [M + H]+
437.3056, found 437.3054.
(2E,4E,6E,8E,10E,12E,14E)-15-(4-Hydroxy-2,6,6-trimethylcyclo-
hex-1-en-1-yl)-4,9,13-trimethylpentadeca-2,4,6,8,10,12,14-heptae-
nal (17). To a cold solution of the ester 16 (5.1 mg, 12 μmol) in
anhydrous Et2O (0.2 mL) was added DIBAL (1 M solution in hexane,
50 μL, 50 μmol) at 0 °C and stirred for 20 min. Roschell salt was
added, and glycerol (3 drops) were added for quenching. The reaction
mixture was stirred 2 h at rt. Phases were separated, and the aqueous
phase was extracted with Et2O. The combined organic layers were
washed with brine, dried over Na2SO4, filtered, and concentrated in
vacuo to give the corresponding allylic alcohol, which was used without
further purification. MnO2 (10 mg, 0.12 mmol) was added to a stirred
solution of the above allylic alcohol in CH2Cl2 (3 mL) at 0 °C and
stirred for 3.5 h. The reaction mixture was filtered through a pad of
Celite and washed with CH2Cl2. Solvent was removed in vacuo, and
the crude aldehyde was purified by auto silica column chromatography
to yield the C27-aldehyde 17 (2.0 mg, 44%). Registry no. 15486-31-4;
1H NMR (300 MHz, CDCl3) δ 9.57 (d, J = 7.8 Hz, 1H), 7.15 (d, J =
15.4 Hz, 1H), 7.06−6.49 (m, 4H), 6.42−6.05 (m, 6H), 4.05−3.88 (m,
1H), 2.38 (dd, J = 17.0, 5.5 Hz, 1H), 2.14−1.70 (m, 3H), 2.00 (s, 3H),
1.97 (s, 3H), 1.94 (s, 3H), 1.72 (s, 3H), 1.47 (t, J = 11.92 Hz, 1H),
1.06 (s, 6H); 13C NMR (75 MHz, CDCl3) δ 193.8 (CO), 156.7
(CH), 141.2 (CH), 140.0 (C), 138.3 (CH), 137.7 (C), 137.1 (C),
137.0 (CH), 135.3 (CH), 133.7 (C), 131.7 (CH), 131.0 (CH), 128.5
(CH), 127.1 (CH), 126.8 (CH), 126.6 (C), 126.5 (CH), 64.9 (CH),
48.3 (CH2), 42.4 (CH2), 37.0 (C), 30.1 (CH3), 28.6 (CH3), 21.5
(CH3), 12.8 (CH3), 12.7 (CH3), 12.5 (CH3).
Coupling Reaction between QDs and the C27-Aldehyde 17.
To a colloidal solution of QDs (1 mM, 20 μL, 20 nmol) in 2-(N-
morpholino)ethanesulfonic acid (MES) buffer was added 1-ethyl-3-(3-
(dimethylamino)propyl)carbodiimide hydrochloride (10 μg/μL in
MES, 385 μL, 20 μmol) and N-hydroxysuccinimide (NHS, 10 μg/ μL
in MES, 230 μL, 20 μmol) in MES buffer. The reaction mixture was
stirred for 15 min. The reaction solution was treated with a solution of
the C27-aldehyde 17 (785 μL in DMF, 1 μg/μL, 2 μmol) and stirred
overnight. Absorption and fluorescence spectra of the reaction mixture
were subsequently measured over the time.
(2E,4E,6E)-2,7-Dimethylocta-2,4,6-trienedial (7). Dispersion of
sodium hydride (60% dispersion, 64 mg, 1.6 mmol) in anhydrous
THF (0.80 mL) was cooled in ice bath under Ar. A solution of the
phosphonate 15 (66 mg, 0.2 mmol) dissolved in anhydrous THF (0.8
mL) was then added. The mixture was allowed to stir for 15 min. A
solution of 1,1-dimethoxypropan-2-one (118 mg, 1.0 mmol) in
anhydrous THF (0.40 mL) was added dropwise. The reaction mixture
was carried out under reflux condition. It was observed that the
solution changed color from colorless to light yellow. After 3.5 h, 20%
sulfuric acid (0.3 mL) was added at 0 °C, increasing the reaction
temperature to 50 °C over 2.5 h. The reaction mixture was extracted
with diethyl ether. The combined organic layer was dried over
Na2SO4, concentrated in vacuo, and purified by column chromatog-
raphy (hexane/AcOEt = 100:0 to 80:20) to yield the C10-dialdehyde 7
1
(11 mg, y. 35%) as a yellow solid. Registry no. 5056-17-7; H NMR
(300 MHz, CDCl3) δ 9.55 (s, 2H), 7.13−6.95 (m, 4H), 1.95 (s, 6H);
13C NMR (75 MHz, CDCl3) δ 194.5 (CO), 146.08 (CH), 141.1
(C), 134.4 (CH), 9.7 (CH3).
Synthesis of C27-Aldehyde 17. (2E,4E,6E,8E,10E,12E)-13-(4-
Hydroxy-2,6,6-trimethylcyclohex-1-en-1-yl)-2,7,11-trimethyltrideca-
2,4,6,8,10,12-hexaenal (4). A mixture of the crude C15-phosphonium
ylide 5 (209 mg, 0.37 mmol) and the C10-dialdehyde 7 (51 mg, 0.31
mmol) and 1,2-epoxybutane (0.56 mL) in ethanol (5.6 mL) was
refluxed under Ar. The reaction was carried out overnight. The
reaction mixture was then diluted with water (10 mL) and extracted
with diethyl ether (3 × 5 mL). The organic layer was dried over
Na2SO4, and the solvent was removed in vacuo. The crude product was
purified by autocolumn chromatography to give the C25-aldehyde 4
1
(53.4 mg, 56% yield over three steps). Registry no. 50837-94-0; H
NMR (300 MHz, CDCl3) δ 9.42 (s, 1H), 7.10−6.84 (m, 2H), 6.84−
6.54 (m, 2H), 6.45−6.21 (m, 2H), 6.20−6.05 (m, 3H), 4.04−3.87 (m,
1H), 2.36 (dd, J = 16.8, 4.6 Hz, 1H), 2.14−1.70 (m, 2H), 2.01 (s, 3H),
1.99 (s, 1H), 1.96 (s, 3H), 1.85 (s, 3H), 1.71 (s, 3H), 1.46 (t, J = 11.9
Hz, 1H), 1.05 (s, 6H); 13C NMR (75 MHz, CDCl3) δ 194.5 (CO),
149.0 (CH), 141.7 (C), 138.2 (CH), 137.8 (CH), 137.6 (C), 137.6
(C), 136.8 (C), 136.6 (CH), 131.0 (CH), 130.8 (CH), 127.5 (CH),
127.4 (CH), 126.8 (CH), 126.7 (C), 64.8 (CH), 48.2 (CH2), 42.4
(CH2), 36.9 (C), 30.1 (CH3), 28.6 (CH3), 21.4 (CH3), 12.9 (CH3),
12.6 (CH3), 9.4 (CH3).
ASSOCIATED CONTENT
■
S
* Supporting Information
The experimental details, and the copies of NMR spectra. This
material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
Ethyl (2E,4E,6E,8E,10E,12E,14E)-15-(4-Hydroxy-2,6,6-trimethylcy-
clohex-1-en-1-yl)-4,9,13-trimethylpentadeca-2,4,6,8,10,12,14-hep-
taenoate (16). A solution of ethyl (triphenylphosphoranylidene)-
acetate (21 mg, 0.06 mmol) and the C25-aldehyde 4 (7.3 mg, 0.02
mmol) in dry THF (0.25 mL) was heated at 95 °C for 7 h in a sealed
tube. The reaction mixture was cooled to room temperature and
diluted with water and Et2O. Phases were separated, and the aqueous
phase was extracted with Et2O. The combined organic layer was dried
over Na2SO4, filtered and concentrated in vacuo. The crude product
was purified by autochromatography (hexane/ethyl acetate = 95:5) to
yield the ester 16 (5.1 mg, 35%). The coupling constant for the newly
formed double bond confirms the E stereochemistry by 1H NMR
■
Corresponding Authors
*E-mail: tnmase@ipc.shizuoka.ac.jp (N.M.)
*E-mail: Baldermann@igzev.de (S.B.)
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was supported in part by Nanobio Project from
Shizuoka University.
1
spectroscopy. Registry no. unknown; H NMR (300 MHz, CDCl3) δ
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spherical particles are coated with proprietary low-molecular weight
thiocarboxylic acid residues (HS-R-COOH). The largest ligand R =
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(12) Wang, S.; Mamedova, N.; Kotov, N. A.; Chen, W.; Studer, J.
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(13) Details of QDs: Qdot ITK (Innovator’s Tool Kit, Invitrogen)
525, 625 carboxyl quantum dots are commercially available and
prepared from nanometer-scale crystals of a semiconductor material
(CdSe), which are shelled with an additional semiconductor layer
(ZnS) to improve their chemical and optical properties. According to
the supplier’s information (Life Technologies), the core−shell
materials are further coated with a polymer layer that allows the
facile dispersion of the quantum dots in aqueous solutions with
retention of their optical properties. The polymer coating has
−COOH surface groups available for modifications such as macro-
molecule attachment. Qdot ITK carboxyl quantum dots are about the
size of large macromolecules or proteins.
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