Design and biological evaluation of synthetic retinoids: Probing length vs. stability vs. activity

Article abstract of DOI:10.1039/c3mb70273a

All trans-retinoic acid (ATRA) is widely used to direct the differentiation of cultured stem cells. When exposed to the pluripotent human embryonal carcinoma (EC) stem cell line, TERA2.cl.SP12, ATRA induces ectoderm differentiation and the formation of neuronal cell types. We report in this study that novel polyene chain length analogues of ATRA require a specific chain length to elicit a biological responses of the EC cells TERA2.cl.SP12, with synthetic retinoid AH61 being particularly active, and indeed more so than ATRA. The impacts of both the synthetic retinoid AH61 and natural ATRA on the TERA2.cl.SP12 cells were directly compared using both RT-PCR and Fourier Transform Infrared Micro-Spectroscopy (FT-IRMS) coupled with multivariate analysis. Analytical results produced from this study also confirmed that the synthetic retinoid AH61 had biological activity comparable or greater than that of ATRA. In addition to this, AH61 has the added advantage of greater compound stability than ATRA, therefore, avoiding issues of oxidation or decomposition during use with embryonic stem cells.

Full text of DOI:10.1039/c3mb70273a

Molecular
BioSystems
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Design and biological evaluation of synthetic
retinoids: probing length vs. stability vs. activity
Cite this: Mol. BioSyst., 2013,
9
, 3124
a
b
a
c
Graeme Clemens, Kevin R. Flower, Peter Gardner, Andrew P. Henderson,
c
cd
c
Jonathan P. Knowles, Todd B. Marder, Andrew Whiting* and
ef
Stefan Przyborski*
All trans-retinoic acid (ATRA) is widely used to direct the differentiation of cultured stem cells. When
exposed to the pluripotent human embryonal carcinoma (EC) stem cell line, TERA2.cl.SP12, ATRA induces
ectoderm differentiation and the formation of neuronal cell types. We report in this study that novel
polyene chain length analogues of ATRA require a specific chain length to elicit a biological responses
of the EC cells TERA2.cl.SP12, with synthetic retinoid AH61 being particularly active, and indeed more so
than ATRA. The impacts of both the synthetic retinoid AH61 and natural ATRA on the TERA2.cl.SP12
cells were directly compared using both RT-PCR and Fourier Transform Infrared Micro-Spectroscopy
(FT-IRMS) coupled with multivariate analysis. Analytical results produced from this study also confirmed
that the synthetic retinoid AH61 had biological activity comparable or greater than that of ATRA. In
addition to this, AH61 has the added advantage of greater compound stability than ATRA, therefore,
avoiding issues of oxidation or decomposition during use with embryonic stem cells.
Received 10th July 2013,
Accepted 2nd October 2013
DOI: 10.1039/c3mb70273a
www.rsc.org/molecularbiosystems
The use of retinoic acids as inducers of cell differentiation
has some limitations, exemplified by ATRA which readily
Introduction
There are over 4000 retinoids, both natural and synthetic, isomerises resulting in mixtures of ATRA, 9CRA, 13CRA and
1
10
structurally and often biochemically related to vitamin A. All- other species. This instability derives from the five conjugated
trans-retinoic acid 1 (ATRA), the major metabolite of vitamin A, double bonds that absorb light in the 300–400 nm region, which
1
1–14
and its two isomers, 9-cis-retinoic acid 2 (9CRA) and 13-cis- can occur under laboratory lighting conditions.
Murayama
11
retinoic acid 3 (13CRA), have essential roles in many biological et al. reported that isomers of ATRA differently affect the ability
processes during both chordate embryogenesis and adult homeo- of mammalian stem cells to differentiate along alternative lineages.
2
,3
stasis.
These include cellular differentiation, proliferation Consequently this can lead to the differential regulation of alter-
4
5
6
and apoptosis, embryonic development and vision. Due to native key molecular pathways involved in cellular development,
the ability of retinoids to control differentiation and apoptosis which can result in increased heterogeneity within cultures of
in both normal and tumour cells, they have the potential to act differentiating stem cells.
as chemopreventative and chemotherapeutic agents, although in the differentiation process, stable retinoid derivatives are
15,16
In order to increase reproducibility
7
,8
10
toxicity has prevented widespread use.
ATRA and other desirable, an approach adopted by us. These types of stable
commercially available retinoids are, however, used to treat compounds can maintain a constant concentration in the culture
9
dermatological conditions.
system, and are readily stored and handled. This is preferable to
the alternative approach of attempting to reduce the sensitivity of
ATRA to isomerisation by the use of additives such as bovine
serum albumin (BSA), fibrogen, lysozyme, phosphatidylcholine
a
Manchester Institute of Biotechnology, Manchester University, 131 Princess Street,
Manchester, M1 7DN, UK
b
School of Chemistry, The University of Manchester, Oxford Road, Manchester,
1
7,18
N-ethylmaleimide and vitamin C.
None of these additives
M13 9PL, UK
c
completely suppress isomerisation, and indeed, they may affect
cellular processes on their own.
Department of Chemistry, Durham University, Science Laboratories, South Road,
Durham, DH1 3LE, UK. E-mail: andy.whiting@durham.ac.uk
d
Institut f u¨ r Anorganische Chemie, Julius-Maximilians-Universit a¨ t W u¨ rzburg,
Retinoic acids contain three major characteristic regions: a
bulky hydrophobic section; a conjugated variable linker; and a
carboxylic acid end group (Fig. 1). Extensive modifications to all
Am Hubland, 97074 W u¨ rzburg, Germany
e
Department of Biological Sciences, Durham University, Science Laboratories,
South Road, Durham, DH1 3LE, UK. E-mail: stefan.przyborski@durham.ac.uk
1
,19
f
three regions are possible
and we reported the incorporation
Reinnervate Ltd., NETPark Incubator, Thomas Wright Way, Sedgefield,
TS21 3FD, UK
of an aromatic ring to replace parts of the polyene chain,
3
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Scheme 1 Synthesis of iodide 8 from methyl tetrolate 6.
Fig. 1 Structures for ATRA 1, 9CRA 2, 13CRA 3, generic retinoid structure, EC23
(
2 2
4-position CO H) and EC19 (3-position CO H).
1
0,19
forming a class of retinoids sometimes termed arotinoids.
Another common alteration is the inclusion of a 1,1,4,4-tetra-
methyl-1,2,3,4-tetrahydronaphthalene unit as a substitute for
the trimethylcyclohexenyl ring and part of the polyene chain, as this
19–22
removes metabolic oxidation sites.
Also, an acetylene moiety
has been successfully employed to act as a non-isomerisable
conjugated linker unit while retaining, or even enhancing, bio-
Scheme 2 Synthesis of synthetic retinoids 9, 12 (AH61) and 14 starting with
iodide 8 and acetylene 5.
10
logical activity in systems such as EC23 4a (Fig. 1). Interestingly,
biological characterisation of EC23 4a demonstrated its ability to
induce neural tissues, which contrasts with EC19 4b which also
induced cell differentiation, the latter resulting primarily in
The E-isomer was subsequently used in two separate reactions:
(1) direct coupling to acetylene 5 allowing for the formation of
10
epithelial cells with only few neural phenotypes. These results
indicated that subtle differences in molecular structure can have
dramatic effects on the biological response. In this paper, we
contrast these findings with recent work on the design, synthesis,
stability and biological activity of a series of new synthetic
retinoids designed to be closer in structure to the natural systems
‘short’ retinoid-like analogue 9; and (2) Heck–Mizoroki coupling
to vinylboronate ester 10, followed by iododeboronation, leading
to a dienyliodide 11 for further elaboration to longer derivatives as
outlined in Scheme 2, which employs an iterative Heck–Mizoroki
1
6
coupling with boronate 10 and highly stereoselective iodode-
17
boronation methodology. Subsequent, Sonogashira couplings of
1–3, but with some of the increased stability of 4a. With these
26,27
iodides 11 and 13 to acetylene 5 under standard conditions
provided esters 12 (known as AH61) and 14 in 76 and 64% yields,
types of compounds we expected to be able to probe further the
effects polyene chain length and stability versus their effect on
cellular development processes, particularly in stem cells or
models of stem cell differentiation.
2
8,29
respectively. Saponification
the corresponding carboxylic acids 12 (AH61) and 14 in 85 and
7% yields, respectively (Scheme 2).
of the methyl esters produced
8
Stability of the synthetic retinoids versus natural ATRA
Results and discussion
Preparation of retinoid analogues
With the series of synthetic retinoids 9, 12 (AH61) and 14 in hand,
their photostability was tested and compared to that of ATRA.
1
It was envisaged that through a series of cross-coupling reac- We have previously shown, by using H NMR spectroscopy, that
tions, including Heck–Mizoroki and Sonogashira processes, ATRA was susceptible to photoisomerisation and degradation
we would be able to obtain compounds of varying size to under- when exposed to laboratory (fumehood) fluorescent light in the
take a focussed structure activity relationship (SAR) study. visible to near-UV range. After three days exposure, substantial
Alkyne 5 was prepared as a generic synthon, starting with isomerisation/degradation occurred with approximately 37% of
1
0
iodination of 1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphthalene, ATRA remaining. When the synthetic retinoids 9, 12 (AH61)
followed by cross-coupling to TMSA under Sonogashira condi- and 14 were exposed to the same wavelength of fluorescent light
1
tions and finally deprotection according to optimised procedures and their H NMR spectra compared with control (un-irradiated)
1
0
previously reported. The synthesis of the different polyene samples, the severity of the isomerisation depended greatly
chains with varying lengths began with the treatment of methyl upon the length of the conjugated linker. The short analogue 9
2
3,24
tetrolate 6 with sodium iodide in acetic acid
to provide remained completely unaltered, with 100% of the original
Z-3-iodobut-2-enoic acid methyl ester 7 in 89% yield and 100% compound present (Fig. 2). The ‘ATRA-like’ analogue 12 (AH61)
stereoselectivity. Generation of the E-isomer 8 was achieved showed signs of isomerisation to one other isomer identified as
through isomerisation of the Z-isomer with hydroiodic acid in (E,Z) but still retained 86% of the original compound and, more
2
4,25
benzene
to give 3-E-iodobut-2-enoic acid methyl ester 8 in a importantly, showed no signs of degradation unlike as previously
66% yield (Scheme 1).
reported for ATRA (Fig. 3). As one might predict, the extended
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carried out using a human embryonal carcinoma (EC) cell line.
EC cells are the stem cells of teratocarcinomas, and therefore,
retain their ability to differentiate into one or more of the
embryonic germ layers. One of the earliest and most frequently
used human EC cell lines is the TERA2 cell line, originally
isolated from a lung metastasis originating from a testicular
3
0
germ cell tumour. A subsequent sub-clone culture, termed
TERA2.cl.SP12, was developed in 2001 by immunomagnetic
3
1
isolation followed by single cell selection and culture. This
cell line was chosen for our study, because it has been shown to
differentiate in response to ATRA. After 21 days of co-culture with
ATRA a heterogeneous cell population was visualised containing
1
Fig. 2 H NMR at 400 MHz of 9 in DMSO-d
6
(5.0–8.5 ppm region) in: top – in
the absence of light and; bottom – after 3 days exposure to white fluorescent
light at a distance of 40 cm.
3
2–34
multiple mature functioning neurons.
Consequently, this
makes the TERA2.cl.SP12 cell line an effective model system for
the in vitro study of neurogenesis.
To compare the effect of the synthetic retinoids 9, 12 (AH61)
and 14 versus both known natural retinoid responses and other
synthetic retinoids previously synthesised within the group,
cultures of TERA2.cl.SP12 EC cells were incubated with 10 mM
concentrations of each compound synthesised. Simultaneously,
control cultures were assessed consisting of untreated, therefore
undifferentiated, cells along with cultures exposed to the vehicle,
DMSO. Induction of cellular differentiation in response to the
synthetic retinoids was initially evaluated by analysing the
expression profile of known markers for both stem cell and
differentiated phenotypes (Fig. 5). Flow cytometry analysis was
performed at 3, 7 and 14 days on cell samples, treated with
1
Fig. 3 H NMR at 400 MHz of 12 (AH61) in DMSO-d
6
(5.0–8.5 ppm region) in:
top – in the absence of light and; bottom – after 3 days exposure to white
fluorescent light at a distance of 40 cm.
10 mM ATRA, 9, 12 (AH61) and 14. The cells were analysed for
the expression of the stem cell antigens, SSEA-3 (globoseries
stage specific embryonic antigen-3) and TRA-1-60 (keratin-
sulfate-associated glycoprotein stem surface marker), whereby
a decrease in the expression level of SSEA-3 and TRA-1-60
indicates cells committing to differentiation. Simultaneously,
as cell lines of the TERA2 lineage are well characterised in their
ability to form neurons in response to ATRA (33,34), flow cytometry
was also used to assess the expression of A2B5 (ganglioseries
antigen marking early-stage neural cells), a marker which is
expressed during the early stages of neuronal development. Similar
expression profiles were visualised for 12 (AH61) as were seen for
ATRA over the 14 day period (Fig. 5). In cell cultures treated with
the short and extended analogues 9 and 14, expression levels of
SSEA-3 and TRA-1-60 remained relatively high with A2B5 staying
comparatively low. Consequently, cultures treated with 9 and
1
Fig. 4 H NMR at 400 MHz of 14 in DMSO-d
6
(5.0–8.5 ppm region) in: (A) the
absence of light and; (B) after 3 days exposure to white fluorescent light at a
distance of 40 cm.
analogue 14 was shown to be the least stable, with isomerisa- 14 were cultured for only 7 days and not through to 14 days due
tion not dissimilar to that seen for ATRA (Fig. 4). The inherent to overgrowth (Fig. 5). As both 9 and 14 did not induce any
instability of ATRA lies within the conjugated linker region, as significant degree of cellular differentiation, all further studies
previously established, and replacement of sections of the were centred on the ATRA-sized analogue 12 (AH61).
polyene chain with both an acetylene moiety and phenyl rings
Further assessment of cellular differentiation in response to
as for EC23 4a provides complete stability. The stability of the synthetic retinoid 12 was evaluated through the expression
systems 9, 12 (AH61) and 14 reported here varies as a function profiles of known genes. Samples from the cells analysed by
of polyene chain length, though there is a significant improve- flow cytometry at 3, 7 and 14 days after treatment with 10 mM
ment in stability compared to similarly sized retinoic acids.
concentrations of retinoids were simultaneously analysed
through RT-PCR. Three genes of interest were chosen,
i.e. Nanog and Oct-4, as gatekeepers of pluripotency, and Pax-6
Effect of the novel retinoids on human pluripotent stem cells
Having prepared the series of synthetic retinoids 9, 12 (AH61) as a motor and ventral neural phenotype. The expression of
and 14, they were assessed for their ability to induce differentiation Nanog and Oct-4 was suppressed in cultures treated with both
3
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Fig. 6 RT-PCR analysis of the induction of TERA2.cl.SP12 cell differentiation in
response to retinoids. Genes associated with pluripotency (Nanog and Oct 4,
A and B respectively) and motor and ventral neural phenotype (Pax 6, C) were
differentially regulated during the 14 day culture period in response to 10 mM
ATRA and 12 (AH61). Both ATRA and 12 (AH61) resulted in a rapid decrease in
expression of the pluripotency genes and reciprocal increase in the motor and
ventral neural gene after 14 days. Data represent mean Æ SEM, n = 3.
to up to and just after day 7, after which it decreased slightly
to day 14. Again both natural retinoid and synthetic analogue
showed a similar profile (Fig. 6).
IR imaging of human pluripotent stem cells treated with novel
retinoid derivatives
Results from the biological assays, Fig. 5 and 6, clearly show
that the synthetic retinoid 12 (AH61) and ATRA induce the
differentiation of the TERA2.cl.SP12 cells in a similar manner,
whereas, retinoids 9 and 14 do not. Recently, Clemens et al.
successfully used Fourier Transform Infrared Microspectroscopy
Fig. 5 Flow cytometric analysis of the induction of TERA2.cl.SP12 cell differen-
tiation in response to retinoids. Markers of stem cells (SSEA-3 and TRA-1-60,
A and B respectively) and neural differentiation (A2B5, C) were differentially
regulated during the 14 day culture period in response to 10 mM ATRA, 9, 12 and
3
5
1
4. Both ATRA and 12 (AH61) resulted in a rapid decrease in expression of the (FT-IRMS) coupled with multivariate analysis to distinguish
stem cell markers and reciprocal increase in neural marker expression after
4 days. Treatment with 9 and 14 did not induce differentiation; cells continued
TERA2.cl.SP12 cells from their differentiating derivatives. An
infrared absorption spectrum of a molecule is characteristic
of the structure and bonding within the molecule and thus
represents a molecular fingerprint. The infrared spectrum of a
biological cell contains a superposition of spectra from all the
1
to proliferate and the experiment was terminated after 7 days as a results of
being over-confluent. Data represent mean Æ SEM, n = 3.
ATRA and analogue 12 (AH61), all in a similar manner (Fig. 6). molecules within the cell and, although these cannot be
Consistent with these observations, Pax-6 expression increased separated, the overall pattern is nevertheless characteristic of
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Fig. 7(a) shows that cells, 7 days after treated synthetic
retinoid 12 (AH61), can be distinguished from the pluripotent
stem cells using FT-IRMS coupled with Principle Component
Linear Discriminant Analysis (PC-LDA). The biochemical
changes of the differentiating cells are described in Fig. 7(b).
In the LDA 1 spectral loading, Fig. 7(b), absorption increases
at wavenumbers associated with proteins (1234, 1286, 1315,
À1
1
337 cm ) can be seen from cells treated with 12 (AH61) when
3
6
compared to those of the undifferentiated stem cells. Protein
changes can also be seen from the mean spectral differences
shown as second derivatives in Fig. 7(c). The negative ‘‘peaks’’
À1
at 1640 and 1660 cm (components of the amide I band which
is very sensitive to secondary protein structure) are indicative of
beta sheet and alpha helix structure, respectively. Clearly, the
cells treated with 12 (AH61) have an increased level of alpha helix
structure proteins compared with the control TERA2.cl.SP12
stem cells.
The LDA 1 spectral loading also shows decreases in absorp-
tion associated with RNA at 1120 (symmetric stretching of
3
7
À1
the phosphodiester band of RNA) and 1367–1369 cm
Fig. 7 (a) PC-LDA analysis, 2 PCs extracted, spectra transformed to second
derivative and then auto-scaled. Histograms display where the DMSO and
synthetic retinoid 12 (AH61) treated TERA2.cl.SP12 cells lie in the LDA 1 scores
plot after 7 days; (b) (upper curve) LDA 1 spectral loading, which shows the
wavenumbers that contribute to the separation seen from the PC-LDA analysis
(
lower curves) mean spectra of the DMSO and 12 (AH61) treated cells (fingerprint
spectral range); (c) mean spectral comparison of the DMSO and 12 (AH61)
treated cells (protein spectral range). (Note that a positive absorption band will
appear as a negative ‘‘peak’’ in the second derivative but at the same wave-
number position as the original.)
the cell. The technique can therefore be used to identify different cell
types or to identify induced cellular changes such as differentiation.
Accordingly, we used FT-IRMS to further assess and validate the
biological activity of synthetic retinoid 12 (AH61).
Fig. 8 (a) PC-LDA of TERA2.cl.SP12 cells treated with ATRA (green), EC23 (blue)
and 12 (AH61) (cyan) at day 7, 3 PCs extracted (b) PC-LDA of TERA2.cl.SP12 cells
treated DMSO (black), ATRA (green), EC23 (blue) and 12 (AH61) (cyan) at day 7,
10 PCs extracted.
3
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C–N stretching vibration associated with cytosine and guanine).
Molecular BioSystems
36
(
synthetic retinoid 12 (AH61) are biochemically similar, 7 days
These overall changes in net RNA and protein content from the after the introduction of the retinoids indicated by the virtual
cells treated with 12 (AH61) may be a sign of mRNA stores being overlap of the cyan and green data. This in agreement with both
used up and transcription occurring to direct the synthesis of the flow cytometry and RT-PCR analysis results seen in Fig. 5
specific proteins as a result of induction and progression of and 6, respectively.
cellular differentiation.
Biological assays, such as flow cytometry and RT-PCR, are
Spectra recorded from the TERA2.cl.SP12 cells treated with seen as the industry standard and are commonly used when
12 (AH61) were also compared against cells treated with ATRA, discriminating stem cells from their differentiating derivatives.
and EC23, at 7 days using PC-LDA, Fig. 8. The LDA scores plot However, these biological assays can be laborious and have
3
8
clearly shows that spectra recorded from the retinoid treated cells been shown to have some drawbacks. Results seen from the
blue, cyan, green) can be distinguished from the pluripotent FT-IRMS analysis are in agreement with those seen from the
(
stem cells (black) in LDA scores space. Fig. 8 and 9 also show biological assays and these were achieved without intensive
that the TERA2.cl.SP12 cells treated with both ATRA and the sample preparation, relatively cheaply, and very importantly,
quickly. As an infrared absorption spectrum of a cell contains
information of the macromolecular composition, protein, lipid,
carbohydrate and nucleic acid changes from differentiating cells can
be explained. This therefore shows the potential of FT-IRMS in
screening stem cells and their differentiating derivatives. The infor-
mation obtained can be seen as complementary to biological assays
when investigating cell differentiation and understanding cellular
development. Because of the speed of FT-IRMS, it is possible that
it may be favoured when looking at the effect of new synthetic
retinoids on known embryonic model systems.
Summary and conclusions
Our findings further underline the value of understanding how
the structure of retinoid analogues relates to their biological
activity. In this study we report on the importance of polyene
chain length in relation to compound stability and their effect on
cellular differentiation by human pluripotent stem cells. Increasing
the polyene chain length resulted in enhanced compound
instability and levels of isomerisation proportional to the
degree of such structural modification. Lengthening of the
polyene linker region resulted in isomerisation not dissimilar
to that seen for ATRA. In terms of induction of biological
activity, a short chain length did not provoke a biological
response (compound 9). The longest chain length we tested
(compound 14) induced only a small partial cellular response
that was most likely the result of a mixture of compounds
produced from the isomerised parent molecule. In relation to
the molecular structures tested, the optimal structure of the
polyene linker region appears to be of medium chain length
[compound 12 (AH61)] in between that of retinoids 9 and 14.
Using conventional molecular methods and FT-IRMS, we showed
that synthetic retinoid 12 (AH61) showed biological activities com-
parable to ATRA. However unlike ATRA, compound 12 (AH61)
remained mostly intact during the compound stability tests
and showed only mild isomerisation.
In conclusion, we have further demonstrated how under-
standing the structure activity relationship of synthetic retinoids
plays an important role in compound design. Moreover, com-
pound 12 (AH61) represents a potentially useful tool for biolo-
gical investigations avoiding the issues of severe compound
isomerisation experienced by ATRA whilst conserving a high
level of biological activity.
Fig. 9 Mean spectral comparisons of ATRA, 12 and DMSO treated TER-
À1
A2.cl.SP12 cells: (a) lipid spectral range (2830–3010 cm ); (b) fingerprint spectral
À1
À1
range (1000–1475 cm ) (c) protein spectral range (1475–1760 cm ).
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Experimental
General synthetic chemistry
reflux for 48 h and then allowed to cool to room temperature.
The mixture was partitioned between water (100 mL) and ether
(150 mL), followed by the aqueous phase being extracted with
All cross-couplings were carried out under an argon atmosphere
using standard Schlenk techniques unless otherwise stated. Other
reactions were performed in the air in reagent-grade solvents. The
compound 1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphthalene acetyl-
ene 5 was prepared according to the literature. Trimethyl-
silylacetylene was obtained from Fluorochem, methyl propiolate
and methyl tetrolate from Aldrich, and 4,4,6-trimethyl-2-vinyl-
ether (2Â 25 mL). The combined organic phases were then
washed with saturated NaHCO (50 mL), 5% NaS O (50 mL)
3
2 5
4
and brine (50 mL). Drying (MgSO ) and evaporation yielded the
crude as a pale yellow oil. Purification by silica gel chromato-
graphy (EtOAc : pet. ether, 5 : 95, as eluent) yielded (E)-3-iodo-
but-2-enoic acid methyl ester as a colourless oil (1.10 g, 25%)
and recovered (Z)-3-iodobut-2-enoic acid methyl ester (2.90 g,
1
0
1,3,2-dioxaborane 10 from Frontier Scientific.
6
6%). All spectral and analytical properties were identical to
Dry solvents were dried by the use of a commercial solvent
39
those reported in the literature.
2E,4E)-3-Methyl-5-(4,4,6-trimethyl-[1,3,2]-dioxaborinan-2-yl)-
purification system (SPS). Petroleum ether (40 : 60) refers to the
fraction of petroleum ether that boils between 40 and 60 1C.
Where mixtures of solvents have been used for chromatogra-
phy, the ratios given refer to volumes used. Thin layer chroma-
tography (TLC) was performed on Polygram SIL G/UV254
plastic backed silica gel plates. Visualisation was achieved
using a UV lamp at 254 nm, or staining with basic potassium
permanganate. Column chromatography was carried out at
(
penta-2,4-dienoic acid methyl ester. To a dried Schlenk tube
under a positive pressure of argon was added (E)-3-iodobut-2-
enoic acid 8 methyl ester (0.941 g 4.16 mmol), dry MeCN
(
(
1
15 mL), Pd(OAc)
128 mg, 0.42 mmol), AgOAc (758 mg, 4.54 mmol) and vinylboronate
0 (752 mg, 0.81 mL, 4.88 mmol), the mixture degassed using
2
(47 mg, 0.21 mmol), tri(o-tolyl)phosphine
freeze–pump–thaw method (3Â) and heated to 55 1C with
medium pressure using silica gel, 40–60 mm, 60 Å obtained
vigorous stirring. After 22 h the mixture was cooled, diluted
1
from Alfa Aesar. H NMR spectra were obtained in CDCl
3
,
with Et
2
O (80 mL), passed through Celite, washed with 5% HCl
) and
unless otherwise stated, at 400, 500 or 700 MHz on Varian
Mercury-400, Varian Inova-500 or Varian VNMRS 700 MHz
spectrometers respectively. Spectra are reported as chemical
shift d (ppm) (number of protons, multiplicity, coupling
constant J (Hz), assignment). All peaks are reported using the
residual protic solvent peak of CHCl (d = 7.26 ppm), as an
(20 mL), water (40 mL) and brine (40 mL). Drying (MgSO
4
evaporation gave the crude product as a orange oil. Purification
by silica gel chromatography (EtOAc : pet. ether, 1 : 9 as eluent)
gave (2E,4E)-3-methyl-5-(4,4,6-trimethyl-[1,3,2]-dioxaborinan-2-
yl)-penta-2,4-dienoic acid methyl ester (780 mg, 74%) as a clear
À1
3
H
oil; nmax/cm 2976, 2359, 1710, 1596, 1393, 1327, 1290, 1229
1
3
1
internal reference. C NMR spectra were obtained in CDCl at
3
and 1153; H NMR (700 MHz, CDCl ) d 1.28 (3H, d, J = 6, CH ),
3
H
3
either 126 or 176 MHz on Varian Inova-500 or Varian VNMRS
1.30 (3H, s, CH
3
), 1.31 (3H, s, CH
3
), 1.51 (1H, t, J = 13, CH
2
), 1.80
700 MHz spectrometers, respectively. All peaks are reported using
(1H, dd, J = 13 and 2.5, CH
2
3
), 2.25 (3H, d, J = 1, CH ), 3.70 (3H, s,
the residual protic solvent peak of CHCl (d = 77.00 ppm) as an
3
C
3
OCH ), 4.21–4.27 (1H, m, CH), 5.87 (1H, s, CH), 5.93 (1H, d,
1
1
1
3
internal reference. B NMR spectra were recorded at 128 MHz
on a Bruker Avance-400 spectrometer in ppm with respect to
J = 18, CH) and 6.92 (1H, d, J = 18, CH); C NMR (176 MHz,
CDCl ) d 13.5, 23.3, 28.3, 31.4, 46.1, 51.3, 65.2, 71.3, 121.0,
29–130, 149.2, 153.2, 167.8; B NMR (128 MHz, CDCl ) d 26
3
C
1
1
external BF
3
2
ÁOEt . IR spectra were recorded on a Perkin-Elmer
1
3
B
+
Paragon 1000 FT-IR Spectrometer. EI-MS analyses were performed
on an Agilent 6890N GC equipped with a 5973N MSD Performance
Turbo CI running an EI mode, and an Anatune Focus Autosampler/
liquid handler, using UHP helium as the carrier gas. ES-MS analyses
were performed on a Thermo-Electron Corp. Finnigan LTQ-FT mass
spectrometer, using the electrospray in positive ion mode (ES+)
to generate ions. Elemental analyses were obtained using an
Exeter Analytical CE-440 analyser. Melting points were obtained
using a Gallenkamp melting point apparatus.
(br s); m/z (ES) 252.1643 (M , C H BO , requires 252.1642).
13 21 4
237, 221, 196, 152, 137, 124 and 83.
Methyl (2E,4E)-5-iodo-3-methylpenta-2,4-dienoate 11. To a
dried Schlenk tube under a positive pressure of argon was
added a solution of the (2E,4E)-3-methyl-5-(4,4,6-trimethyl-
[1,3,2]-dioxaborinan-2-yl)-penta-2,4-dienoic acid methyl ester
(700 mg, 2.78 mmol), in dry THF (10 mL), and the tube cooled
to À78 1C in the absence of light. NaOMe (6.91 mL of a 0.5 M
solution in MeOH, 3.46 mmol) was added dropwise and the
mixture stirred for 30 min, followed by the addition of ICl
(
Z)-3-Iodobut-2-enoic acid methyl ester 7. Methyl tetrolate 6
(900 mg, 9.2 mmol), NaI (2.4 g, 16 mmol) and acetic acid (6 mL) (4.67 mL of a 1.0 M solution in DCM, 4.67 mmol) dropwise. The
were heated to reflux for 1.5 h, poured onto water (20 mL) and mixture was stirred for 1 h, warmed to room temperature, diluted
extracted with ether (2Â 30 mL). The combined organic extracts with Et O (80 mL), washed with 5% sodium metabisulphite
2
were washed with saturated NaHCO
3
(3Â 20 mL), 5% NaS
2
O
3
4
(30 mL), water (30 mL) and brine (30 mL). Drying (MgSO ) and
(20 mL) and brine (20 mL). Drying (MgSO ) and evaporation yielded evaporation gave the crude product as a orange oil. Purification
4
(Z)-3-iodobut-2-enoic acid methyl ester 7 as a clear oil (1.87 g, 90%). by silica gel chromatography (EtOAc : pet. ether, 1 : 9, a eluent),
All spectroscopic and analytical properties were identical to gave the product as a clear oil, which slowly crystallised as
3
9
those reported in the literature.
an off-white solid (602 mg, 86%); all spectral and analytical
3
9
(
E)-3-Iodobut-2-enoic acid methyl ester 8. A solution of properties were identical to those reported in the literature.
Z)-3-iodobut-2-enoic acid methyl ester 7 (4.38 g, 19.4 mmol) nmax/cm
and HI (0.25 g, 1.94 mmol) in benzene (40 mL) were heated to 1234, 1190 and 1153; H NMR (400 MHz, CDCl ) dH 2.24
À1
(
3070, 3013, 2943, 1708, 1612, 1433, 1386, 1359,
1
3
3
130 Mol. BioSyst., 2013, 9, 3124--3134
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Paper
3H, d, J = 1.2, CH
Molecular BioSystems
(
3
), 3.71 (3H, s, OCH
3
), 5.74 (1H, q, J = 0.8, CH), 6-ethynyl-1,1,4,4-tetramethyl-1,2,3,4-tetrahydronaphthalene 5 (0.40 g,
1
3
6.89 (1H, d, J = 15, CH), 7.10 (1H, d, J = 15, CH); C NMR 1.88 mmol) and Et
3
N (10 mL). The mixture was degassed using
(
126 MHz, CDCl ) d 13.6, 51.5, 84.8, 120.0, 148.5, 151.4, 167.2; the freeze–pump–thaw method (3Â), followed by the addition
3
+
C
m/z (EI) 252 (M ), 221, 193, 126 and 125.
2E,4E)-2-Methyl-7-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-
of Pd(OAc) (20 mg, 0.08 mmol), PPh (44 mg, 0.16 mmol)
2 3
(
and CuI (32 mg, 0.16 mmol). The mixture degassed using
naphthalen-2-yl)-hepta-2,4-dien-6-ynoic acid methyl ester. The the freeze–pump–thaw method (2Â) and stirred at room
dienyl iodide 11 (500 mg, 1.98 mmol), 6-ethynyl-1,1,4,4-tetra- temperature under argon. After 24 h, the reaction was diluted
methyl-1,2,3,4-tetrahydronaphthalene 5 (509 mg, 2.40 mmol) with Et
and triethylamine (10 mL) were added to a dried Schlenk tube (20 mL, 2Â), brine (20 mL), dried (MgSO
and the mixture degassed using the freeze–pump–thaw method give an orange oil. Purification by silica gel chromatography
2
O (80 mL), passed through Celite, washed with 5% HCl
4
) and evaporated to
(
3Â). Pd(OAc) (22 mg, 0.10 mmol), PPh (52 mg, 0.19 mmol) (EtOAc : petroleum ether, 5 : 95, as eluent) gave (E)-3-methyl-5-
2
3
and CuI (38 mg, 0.19 mmol), were then added and the mixture (5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-pent-2-en-
degassed by freeze–pump–thaw (3Â). The mixture was stirred at 4-ynoic acid methyl ester as a pale yellow oil (530 mg, 99%).
À1
room temperature for 5 h, diluted with EtOAc (80 mL), passed
n
max/cm 2957, 2195, 1715, 1612, 1433, 1342, 1269, 1198, 1180
1
through Celite, washed with 5% HCl (20 mL 2Â), brine (20 mL), and 1135; H NMR (700 MHz, CDCl
dried (MgSO ), 1.68 (4H, s, 2Â CH
) and concentrated. Purification by silica gel 1.28 (6H, s, 2Â CH
chromatography (pet. ether : EtOAc, 95 : 5, as eluent) gave (2E,4E)- CH ), 3.73 (3H, s, OCH ), 6.15 (1H, q, J = 1.5, CH), 7.21 (1H, dd,
-methyl-7-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)- J = 8 and 1.5, Ar), 7.27 (1H, d, J = 8, Ar), 7.41 (1H, d, J = 1.5, Ar);
C NMR (176 MHz, CDCl ) d 20.3, 31.9, 32.0, 34.5, 34.7, 35.1,
3 C
6%); nmax/cm 2956, 2192, 1715, 1608, 1490, 1434, 1358, 1232 35.1, 51.5, 90.5, 94.9, 119.4, 123.4, 127.0, 129.2, 130.5, 138.8,
3
) d 1.27 (6H, s, 2Â CH
3
),
4
3
2
), 2.40 (3H, d, J = 1.5,
3
3
2
1
3
hepta-2,4-dien-6-ynoic acid methyl ester as a yellow oil (510 mg,
À1
7
1
+
and 1154; H NMR 500 MHz, CDCl : d 1.27 (6H, s, 2Â CH ), 145.5, 146.8, 166.9; m/z (ES) 311.2011 (M + H , C H O ,
3
H
3
21 27 2
1
.28 (6H, s, 2Â CH
CH ), 3.73 (3H, s, OCH
CH), 6.70 (1H, d, J = 16, CH), 7.21 (1H, dd, J = 1.5 and 8, Ar), 7.26 thalen-2-yl)-pent-2-en-4-ynoic acid 9. (E)-3-Methyl-5-(5,5,8,8-tetra-
3
), 1.68 (4H, s, 2Â CH
2
), 2.31 (3H, d, J = 1.2, requires 311.2006). 279.
3
3
), 5.84 (1H, s, CH), 6.24 (1H, d, J = 16,
(E)-3-Methyl-5-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naph-
1
3
(
1H, d, J = 8, Ar), 7.41 (1H, d, J = 1.5, Ar); C NMR (126 MHz, methyl-5,6,7,8-tetrahydro-naphthalen-2-yl)-pent-2-en-4-ynoic acid
CDCl ) d 13.4, 31.9, 32.0, 34.5, 34.6, 35.1, 35.1, 51.5, 87.6, 96.0, methyl ester (85 mg, 0.274 mmol) was added to a stirred solution
14.7, 120.1, 120.4, 127.0, 128.9, 130.3, 143.7, 145.4, 146.3, of lithium hydroxide (46 mg, 1.10 mmol) in THF–H O (3 : 1,
3
C
1
1
3
2
+
51.4, 167.4; m/z (ES) 337.2164 (M + H , C H O , requires 20 mL) at room temperature. After 48 hours the reaction was
2
3
29 2
37.2162), 337, 305 and 215.
2E,4E)-2-Methyl-7-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro- extracted with ether (20 mL), dried (MgSO
naphthalen-2-yl)-hepta-2,4-dien-6-ynoic acid 12 (AH61). (2E,4E)- to give an off-white solid. Recrystallisation from acetonitrile
judged to be complete and acidified to pH 1 to 2 by 20% HCl,
(
4
) and concentrated
2
-Methyl-7-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-naphthalen-2-yl)- yielded the product as an off-white crystalline solid (25 mg,
À1
hepta-2,4-dien-6-ynoic acid methyl ester (0.51 g, 1.49 mmol), 31%). nmax/cm 2912, 2197, 1688, 1602, 1428, 1296, 1225 and
was dissolved in a THF water mixture (3 : 1, 20 mL) followed by 1145; H NMR (700 MHz, CDCl
1
3
) d 1.27 (6H, s, 2Â CH
3
),
the addition of LiOHÁH O (250 mg, 5.94 mmol). The mixture 1.28 (6H, s, 2Â CH ), 1.68 (4H, s, 2Â CH ), 2.41 (3H, d,
2
3
2
was stirred at room temperature for 48 h in the absence of light J = 1.5, CH ), 6.18 (1H, d, J = 1.5, CH), 7.22 (1H, dd, J = 8 and
3
1
3
after which the reaction was judged to be complete by tlc. The 1.5, Ar), 7.28 (1H, d, J = 8, Ar), 7.42 (1H, d, J = 1.5, Ar); C NMR
mixture was then acidified to pH 1 by the addition of HCl (176 MHz, CDCl ) d 20.6, 31.9, 32.0, 34.5, 34.7, 35.0, 35.1, 90.5,
3
C
(
20%), and extracted with diethyl ether (20 mL, 2Â). The sample 96.2, 119.3, 122.4, 127.1, 129.2, 130.6, 141.3, 145.5, 147.0, 169.0;
+
24 2
was evaporated to give the crude product as a yellow powder. m/z (ES) 296.1773 (M , C20H O , requires 296.1771), 279, 255,
This was re-crystallised from acetonitrile to yield the product as 84 and 43.
a yellow crystalline solid (394 mg, 82%); m.p. 205.1–206.7 1C;
nmax/cm 2500–3500, 2194, 1679, 1604, 1488, 1459, 1255 and yl)-hepta-2,4,6-trienoic acid methyl ester. To a dried Schlenk
(2E,4E,6E)-3-Methyl-7-(4,4,6-trimethyl-[1,2,3]-dioxaborinan-2-
À1
1
1
(
5
7
185; H NMR: (700 MHz, CDCl ) d 1.27 (6H, s, 2Â CH ), 1.28 tube under a positive pressure of argon was added Pd(OAc)2
3
H
3
6H, s, 2Â CH ), 1.68 (4H, s, 2Â CH ), 2.32 (3H, d, J = 1, CH ), (16.5 mg, 0.07 mmol), AgOAc (296 mg, 1.61 mmol), tri(o-tolyl)-
3
2
3
.87 (1H, s, CH), 6.29 (1H, d, J = 16, CH), 6.73 (1H, d, J = 16, CH), phosphine (45 mg, 0.148 mmol), methyl (2E,4E)-5-iodo-3-
.21 (1H, dd, J = 1.5 and 8, Ar), 7.27 (1H, d, J = 8, Ar), 7.41 (1H, d, methylpenta-2,4-dienoate (0.37 g, 1.47 mmol) and acetonitrile
1
3
3 C
J = 1.5, Ar); C NMR: (126 MHz, CDCl ) d 13.6, 31.9, 32.0, 34.4, (10 mL). The mixture was degassed using the freeze–pump–
3
1
4.6, 35.1, 35.1, 87.5, 96.6, 115.6, 119.9, 120.0, 127.0, 129.0, thaw method (2Â) followed by the addition of vinyl boronate 10
30.3, 143.5, 145.4, 146.4, 153.7, 172.0; m/z (ES) 321.1858 (265 mg, 1.72 mmol). The mixture was degassed again using
À
(M , C H O , requires 321.1860), 260, 186, 159 and 91; anal. the freeze–pump–thaw method before heating to 50 1C with
2
2
26 2
calcd for C H O : C, 81.95; H, 8.13. Found: C, 81.87; H, 8.10. vigorous stirring. After 22 h, the reaction was cooled to room
2
2
26 2
(
E)-3-Methyl-5-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-
naphthalen-2-yl)-pent-2-en-4-ynoic acid methyl ester. To a dried washed with 5% HCl (20 mL), water (40 mL) and brine (20 mL),
Schlenk flask under a positive pressure of argon was added dried (MgSO ) and evaporated. The product was purified by
E)-3-iodobut-2-enoic acid methyl ester 11 (0.36 g, 1.72 mmol), silica gel chromatography (EtOAc : pet. ether, 1 : 9, as eluent) to
temperature, diluted with Et O (60 mL), passed through Celite,
2
4
(
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Mol. BioSyst., 2013, 9, 3124--3134 3131

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Molecular BioSystems
Paper
give the product (2E,4E,6E)-3-methyl-7-(4,4,6-trimethyl-[1,2,3]- 7.19 (1H, dd, J = 8 and 1.5, Ar), 7.27 (1H, d, J = 8, Ar), 7.39 (1H, d,
1
3
dioxaborinan-2-yl)-hepta-2,4,6-trienoic acid methyl ester as a J = 1.5, Ar); C NMR (176 MHz, CDCl
3
) d
C
13.9, 31.9, 31.9,
À1
pale yellow oil (274 mg, 67%). nmax/cm 2973, 1711, 1614, 34.6, 34.6, 35.1, 35.1, 51.3, 88.2, 95.5, 98.8, 115.0, 120.2, 120.4,
391, 1303, 1237, and 1150; H NMR (700 MHz, CDCl ) d 1.28 127.0, 128.8, 130.1, 134.0, 137.5, 140.6, 145.3, 146.1, 167.5;
3H, d, J = 6, CH ), 1.31 (3H, s, CH ), 1.31 (3H, s, CH ), 1.51 (1H, m/z (ES) 363.2322 (M + H , C H O , requires 363.2319). 331,
1
1
3 H
+
(
3
3
3
25 31 2
t, J = 14, CH
2
), 1.80 (1H, dd, J = 14 and 3, CH
2
), 2.30 (3H, s, CH
3
), 289 and 215.
(2E,4E,6E)-3-Methyl-9-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-
CH), 5.81 (1H, s, CH), 6.36 (1H, d, J = 17, CH), 6.63 (1H, dd, naphthalen-2-yl)-nona-2,4,6-trien-8-ynoic acid 14. (2E,4E,6E)-3-
3
3.71 (3H, s, OCH ), 4.21–4.27 (1H, m, CH), 5.68 (1H, d, J = 17,
1
3
J = 17 and 11, CH) and 6.99 (1H, dd, J = 17 and 11, CH); C NMR Methyl-7-(4,4,6-trimethyl-[1,2,3]-dioxaborinan-2-yl)-hepta-2,4,6-
176 MHz, CDCl ) d 13.9, 23.3, 28.3, 31.4, 46.1, 51.2, 65.1, 71.1, trienoic acid methyl ester (120 mg, 0.331 mmol) was added to a
19.9, 130.0, 132.0, 136.6, 137.9, 146.0, 152.6, 167.5; m/z (ES) stirred solution of LiOH (42 mg, 0.993 mmol) in THF–H O (3 : 1,
(
3
C
1
2
1
2
+
78.1802 (M , C H BO , requires 278.1798), 190, 174, 162, 10 mL) at 4 1C. After the reaction was judged to be complete by
44, 136, 58, 52 and 44.
1
5
24
4
tlc, the reaction mixture was acidified to pH 1/2 by 20% HCl,
O (20 mL), dried (MgSO ) and evaporated to
(
2E,4E,6E)-7-Iodo-3-methyl-hepta-2,4,6-trienoic acid methyl extracted with Et
2
4
ester 13. To a dried Schlenk tube under a positive pressure of give an orange oil. Purification by silica gel chromatography
argon was added the (2E,4E,6E)-3-methyl-7-(4,4,6-trimethyl- (petroleum ether : EtOAc, 9 : 1, as eluent) gave the product as a
[
(
1,2,3]-dioxaborinan-2-yl)-hepta-2,4,6-trienoic acid methyl ester yellow solid (100 mg, 87%) and a 2.5 : 1 mixture of alkene
0.2 g, 0.72 mmol) in dry THF (5 mL). The mixture was cooled diastereoisomers. H NMR (700 MHz, CDCl ) d 1.26 (6H, s,
3 H
1
to À78 1C in the absence of light, followed by the addition of 2Â CH ), 1.27 (6H, s, 2Â CH ), 1.67 (4H, s, 2Â CH ), 2.32 (3H, s,
3
3
2
NaOMe (1.79 mL of a 0.5 M solution in MeOH, 0.895 mmol). CH ), 5.85 (1H, s, CH), 6.01 (1H, d, J = 14, CH), 6.36 (1H, d,
3
After 30 min, ICl (1.21 mL of a 0.5 M solution in CH Cl , J = 14, CH), 6.73 (2H, ddd, J = 14, 11 and 4, 2Â CH), 7.19 (1H, dd,
2
2
1
.21 mmol) was added to the mixture and stirred for 1 h. The J = 8 and 1.5, Ar), 7.28 (1H, d, J = 8, Ar), 7.39 (1H, d, J = 1.5, Ar);
1
3
reaction was warmed to room temperature, diluted with Et
2
O
3 C
C NMR (176 MHz, CDCl ) d 14.1, 31.9, 31.9, 32.0, 34.4, 34.6,
(
(
30 mL), washed with sodium metabisulphite (30 mL), water 35.1, 35.1, 88.1, 95.8, 99.0, 115.6, 119.4, 120.2, 126.9, 128.8,
30 mL), and brine (30 mL), dried (MgSO ) and evaporated to 130.2, 134.8, 137.2, 140.5, 145.4, 146.1, 154.4; m/z (ES) 347.2016
4
+
give an orange oil. Purification by silica gel chromatography (M À H, C24
27 2
H O , requires 347.2017).
(
EtOAc : pet. ether, 1 : 9, cooled to 0 1C) yielded the product as a
À1
Exposure to light
pale yellow oil (131 mg, 65%). nmax/cm 2946, 1710, 1609,
1
1
(
557, 1433, 1390, 1357, 1238, 1189 and 1152; H NMR Synthetic retinoid NMR samples were prepared and left to
500 MHz, CDCl ) d 2.31 (3H, d, J = 1, CH ), 3.73 (3H, s, stand in ordinary laboratory light, with additional irradiation
), 5.87 (1H, s, CH), 6.28 (1H, d, J = 15, CH), 6.51 (1H, ddd, from a standard fluorescent light ca. 30 cm above the sample.
3
H
3
OCH
3
J = 15, 11 and 5, CH), 6.62 (1H, d, J = 15, CH), and 7.16 (1H, ddd,
1
3
Biological analytical procedures
J = 15, 11 and 5, CH); C NMR (126 MHz, CDCl
3 C
) d 13.8, 51.4,
8
3.4, 120.7, 133.8, 136.3, 145.0, 151.8, 167.5; m/z (ES) 277.9799
Tissue culture. Human pluripotent TERA2.cl.SP12 embryonal
+
(M , C H IO , requires 277.9798), 247, 219, 127, 91, 65 and 50. carcinoma stem cells were maintained under standard labora-
9 11 2
3
1
(
2E,4E,6E)-3-Methyl-9-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro- tory conditions as previously described. In brief, cells were
naphthalen-2-yl)-nona-2,4,6-trien-8-ynoic acid methyl ester. The cultured in DMEM (Sigma) supplemented with 10% FCS (Gibco),
corresponding iodide (60 mg, 0.216 mmol), 6-ethynyl-1,1,4,4- 2 mM L-glutamine and 100 active units each of penicillin and
tetramethyl-1,2,3,4-tetrahydronaphthalene 5 (55 mg, 0.26 mmol) streptomycin (Gibco). Cultures were passaged using acid-washed
and Et
mixture degassed using the freeze–pump–thaw method (3Â). for counting, in which case a 0.25% trypsin EDTA (Cambrex)
Pd(OAc) (2.5 mg, 0.01 mmol), PPh (5.5 mg, 0.02 mmol) and solution was used. Cultures intended for flow cytometric and
3
N (2 mL) were added to a dried Schlenk tube and the glass beads (VWR) unless a single-cell suspension was required
2
3
CuI (4 mg, 0.02 mmol) were then added and the mixture RT-PCR analysis were set up in T25 flasks (Nunc) while 12-well
degassed using the freeze–pump–thaw method (3Â) and stirred plates (Nunc) were used for immunocytochemical studies and
at room temperature for 5 h. The reaction was diluted with cell viability/apoptopic analysis.
Et
2
O (60 mL), passed through Celite, washed with 5% HCl
Flow cytometry. Flow cytometry analysis was carried out
(
20 mL, 2Â), brine (20 mL), dried (MgSO
4
) and evaporated to on live cells using antibodies recognising cell surface markers.
give an orange oil. Purification by silica gel chromatography The expression of markers indicative of the stem cell SSEA-3
pet. ether : EtOAc, 95 : 5, as eluent) gave (2E,4E,6E)-3-methyl-7- (University of Iowa Hybridoma Bank) and TRA-1-60 (generous
(
(
4,4,6-trimethyl-[1,2,3]-dioxaborinan-2-yl)-hepta-2,4,6-trienoic acid gift from Prof. P. Andrews, University of Sheffield) or neural cell
À1
methyl ester as a pale yellow oil (50 mg, 64%). nmax/cm 2955, (A2B5, R&D Systems) phenotype was determined to indicate
2
1
181, 1709, 1604, 1435, 1358, 1240, and 1153; H NMR the status of cellular differentiation by TERA2.cl.SP12 cells.
(
1
(
700 MHz, CDCl ) d 1.27 (6H, s, 2Â CH ), 1.28 (6H, s, 2Â CH ), Suspensions of single EC cells of their differentiated derivatives
3
H
3
3
.67 (4H, s, 2Â CH
2
), 2.31 (3H, s, CH
3
), 3.71 (3H, s, OCH
3
), 5.82 were formed by the addition of 1 mL 0.25% trypsin–EDTA
1H, s, CH), 5.99 (1H, d, J = 15, CH), 6.34 (1H, d, J = 15, CH), 6.67 solution. The cell suspension was divided accordingly for
(1H, ddd, J = 15, 11 and 4, CH), 6.74 (1H, ddd, J = 15, 11 and 4, CH), flow cytometry and RT-PCR (see later) analysis accordingly.
3
132 Mol. BioSyst., 2013, 9, 3124--3134
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Paper
Molecular BioSystems
6
Cells were added to a 96-well plate (0.2 Â 10 cells per well) as a
suspension in wash buffer (0.1% BSA in PBS) for incubation
with primary (1 : 20 SSEA-3 and TRA-1-60, or 1 : 10 A2B5) and
FITC-conjugated secondary antibody (Cappell, 1 : 100) as pre-
viously described. Labelled cells were analysed in a Guave
EasyCytet Plus System (Millipore) flow cytometer. Thresholds
determining the numbers of positively expressing cells were set
against the negative control antibody P3X.
4 J. L. Napoli, Clin. Immunol. Immunopathol., 1996, 80, S52–S62.
5 S. A. Ross, P. J. McCaffery, U. C. Drager and L. M. De Luca,
Physiol. Rev., 2000, 80, 1021–1054.
6 G. Wald, Nature, 1968, 219, 800–802.
7 D. R. Soprano, P. Qin and K. J. Soprano, Annu. Rev. Nutr.,
2004, 24, 201–221.
8 S. J. Freemantle, K. H. Dragnev and E. Dmitrovsky, J. Natl.
Cancer Inst., 2006, 98, 426–427.
Gene expression analysis. Real time PCR was carried out on
cells immediately lysed after treatment with 0.25% trypsin–EDTA.
9 N. Lowe and R. Marks, Retinoids: a Clinicians Guide, London,
2nd edn, 1997.
Commercial RNA extraction kits and procedures (Qiagen) and 10 V. B. Christie, J. H. Barnard, A. S. Batsanov, C. E. Bridgens,
reverse transcription procedures (Applied Biosystems) were
followed. In brief, cells were lysed, homogenised using a 20-gauge
needle, and passed through an RNeasy spin column. DNase
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9 J. H. Barnard, J. C. Collings, A. Whiting, S. A. Przyborski and
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À1
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We thank the BBSRC and Reinnervate Ltd. for an industrial
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