Multigram synthesis of isobutyl-β- C -galactoside as a substitute of isopropylthiogalactoside for exogenous gene induction in mammalian cells

Article abstract of DOI:10.1021/jo2024569

Herein we report that isobutyl-β-C-galactoside (IBCG) is also a promising inducer of gene expression in mammalian cells and report a new synthetic route to the compound that should make obtaining the multigram quantities of material required for animal studies more feasible. A convenient synthesis of IBCG, an inducer of genes controlled by the lac operon system in bacterial cells, was achieved in 5 steps from galactose in 81% overall yield without any chromatographic separation steps. An optimized microwave-assisted reaction at high concentration was key to making the C-glycosidic linkage. A Wittig reaction on a per-O-silylated rather than per-O-acetylated or -benzylated substrate proved most effective in installing the final carbon atom.

Full text of DOI:10.1021/jo2024569

Article
pubs.acs.org/joc
Multigram Synthesis of Isobutyl-β-C-galactoside as a Substitute of
Isopropylthiogalactoside for Exogenous Gene Induction in
Mammalian Cells
Lin Liu,† Basma Abdel Motaal,‡ Marc Schmidt-Supprian,‡ and Nicola L. B. Pohl*,†
†Department of Chemistry, Department of Chemical and Biological Engineering, and the Plant Sciences Institute, Hach Hall, Iowa
State University, Ames, Iowa 50011-3111, United States
‡Molecular Immunology and Signal Transduction, Max Planck Institute of Biochemistry, D-82152 Martinsried/Planegg, Germany
S
* Supporting Information
ABSTRACT: Herein we report that isobutyl-β-C-galactoside (IBCG) is also a promising inducer of gene expression in
mammalian cells and report a new synthetic route to the compound that should make obtaining the multigram quantities of
material required for animal studies more feasible. A convenient synthesis of IBCG, an inducer of genes controlled by the lac
operon system in bacterial cells, was achieved in 5 steps from galactose in 81% overall yield without any chromatographic
separation steps. An optimized microwave-assisted reaction at high concentration was key to making the C-glycosidic linkage. A
Wittig reaction on a per-O-silylated rather than per-O-acetylated or -benzylated substrate proved most effective in installing the
final carbon atom.
system in mammalian cells ensures the exogenous genes are
expressed only when the inducer is added and tightly regulates
the gene transcription. When IPTG is used as the lac operon
inducer in animal models, the inducer is usually dissolved in
drinking water and fed to the animals. Since decomposition
products resulting from IPTG have an unpleasant thiol smell,
IPTG solutions have to be put into light-protected bottles and
carefully monitored.11,12 Even so, IPTG’s rapid clearance and
short half-life limit its usage and present a major drawback of
using this popular gene induction system in animal
systems.13−15
To circumvent this stability problem, our group has reported
the design and synthesis of a new lac inducer, isobutyl-β-C-
galactoside (IBCG, 1),16 as a C-glycoside analogue of IPTG.
Not only does this C-glycoside show at least equal gene
induction ability in bacterial systems compared to that of the S-
glycoside, but replacement of the S-glycosidic bond by a C-
glycosidic bond renders the resulting molecule much more
stable. Herein we report that IBCG is also a promising inducer
of gene expression in mammalian cells and report a new
synthetic route to the compound that should make obtaining
the multigram quantities of material required for animal studies
more feasible.
INTRODUCTION
■
Genetic engineering demands a tight regulation system to
control the expression of the introduced exogenous genes, and
this requirement is generally achieved via inducible gene
expression systems.1−3 Among the current available inducible
systems, lac operon-based systems are the most widely studied
and used.4,5 Isopropyl-β-D-thiogalactopyranoside (IPTG), a
lactose analogue, is routinely used as an inducer of the lac
operon in bacterial systems for in vitro studies to induce the
expression of exogenously introduced genes.4,6 This sugar
analogue binds to lac repressor to activate the gene tran-
scription machinery (Figure 1). Studies have also shown that
Previously, the synthesis of IBCG was achieved via either a
Lewis acid promoted reaction between galactose pentaacetate
and methallyltrimethylsilane or a Grignard reaction between
Figure 1. Lactose analogues such as allolactose, IPTG, and IBCG can
serve as inducers to bind the lac repressor and activate gene
transcription in lac operon regulated gene transcription.
this inducible lac operon/repressor system can be implemented
in mammalian cells and animal systems.7−10 Such an inducible
Received: November 29, 2011
Published: January 9, 2012
© 2012 American Chemical Society
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Scheme 1. Comparison of Heating Methods and Reaction Concentrations/Times for the Synthesis of β-C-Glycosidic Ketone 4
Figure 2. 1H NMR spectra in D2O of a 10.00 g reaction (2.4 M) under microwave heating conditions. Samples aliquots were removed at 30 min and
1, 2, 3, 4, 5, and 6 h.
bromoacetogalactose and excess isobutylmagnesium bromide.16
The former method employs a large excess of a relatively
expensive material (methallyltrimethylsilane, >$20/g) and
shows no α/β selectivity in the C-glycosidic bond formation
reaction. The latter method utilizes a Grignard approach, which
is highly exothermic and cumbersome to scale up. Both of the
methods require silica gel chromatography to purify the
product. To satisfy the need for larger amounts of IBCG
required for animal studies, we hoped to find a new route that
could offer a high overall yield on a multigram scale without
any chromatography steps.
particularly attractive as a way to quickly make β-C-glycosidic
ketones without prior protection of the carbohydrate hydroxyl
groups.19 A mixture of C-glycoside stereoisomers are formed in
the initial Knoevenagel condensation; however, extended
heating under basic conditions allow the equilibrium to shift
to the β-C-glycosidic pyranose ketone (Scheme 1).20 Many
recent studies utilized this strategy as a starting point to
synthesize β-C-glycoside analogues.21−27 Ideally, IBCG could
be obtained via a simple methylenation on the ketone by using
a Wittg-type reaction. However, even though the carbonyl
group in the β-C-glycosidic ketones obtained from natural
reducing sugars should provide an excellent opportunity to
perform C−C bond formation via a Wittig-type reaction to
obtain other β-C-glycoside analogues, this possibility remains
relatively unexplored. Most of the existing studies are based on
aldol reactions between the β-C-glycosidic ketone and an
aldehyde to form an α,β-unsaturated ketone. We found this
apparent absence of Wittig-type reaction on β-C-glycosidic
ketones quite intriguing and speculated that the base sensitivity
RESULTS AND DISCUSSION
■
Obviously key to any successful synthesis of IBCG is the
method for installation of the C-glycoside. Stereoselective
formation of C-glycosidic bonds have attracted significant
attentions in the recent years.17,18 Among methods for the
synthesis of β-C-glycosides, the one-step condensation between
free sugars in aqueous solutions and 2,4-pentanedione was
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Scheme 2. Wittig Reaction on Acyl-Protected Substrates
Scheme 3. Synthesis of IBCG via a Perbenzylated Ketone
of the ketone substrates might be a contributing factor. We
decided to pursue our IBCG synthesis through this route with
the hope of developing an efficient method for Wittig reactions
on the β-C-glycosidic ketones.
reported. The reaction was run at a concentration from 1.2 M
to as high as 4.8 M instead of the reported 0.15 M with similar
results. The water was removed from the reaction mixture
under reduced pressure, and then methanol/ethyl acetate was
added to separate the product from extra sodium bicarbonate.
The crude products still contained some sodium acetate but
could be either used directly in the following reaction or
purified by passing through a short silica gel plug followed by
recrystallization to give the desired β-C-glycosidic ketone 4 in
92% yield for a 5-g scale reaction.
With a method for the larger scale synthesis of the key
intermediate ketone 4 in hand, only a few steps should allow
elaboration of this ketone to the final compound 1. The
synthesis of β-C-glycoside 4 utilizes a well-studied property that
2-carbonylalkyl-C-glycopyranosides tend to undergo a retro-
Michael type addition initiated by the enolate formation and
followed by ring opening under mild basic conditions.32−35
However, this property that makes for a convenient synthesis of
β-C-glycoside ketones also makes the following transformations
under basic conditions troublesome. A straightforward way to
do the homologation is to use the Wittig reaction.36 No
precedents for such a Wittig reaction on substrates like
compound 4 have been reported. Even though the Wittig
reagent is relatively basic,37 we anticipated that there was a
reasonable chance to do the desired methylenation on the base-
sensitive ketone substrate if we could fine-tune the electronic
properties of the pyranose ring to make the homologation, and
not the enolate formation and ring-opening reaction, more
favorable. The Tebbe reagent provides a less basic alternative to
Wittig regents that could possibly give superior results on a
base-sensitive substrate38 but is less desirable in large-scale
reactions due to its high cost. Therefore, we decided to try the
First, an optimized synthesis of our desired intermediate
ketone 4 had to be developed. The synthesis of 4 has been
reported on a 500-mg scale at 0.15 M concentrations of
galactose after heating at 90 °C for 24 h.28 Unfortunately, the
long reaction time and dilute reaction conditions made the
reaction less desirable for multigram syntheses. We therefore
set out to probe the limits of this reaction. By careful
1
monitoring of the reaction by H NMR (Figure 2), we found
that formation of the C-glycosidic bond itself was a relatively
fast process; the mixture of isomers (mainly α-pyranose) was
then converted to the thermodynamically more stable β-
pyranose product 4 slowly upon extended heating. The
characteristic peaks of β-pyranose product 4 are two doublets
of doublets at δ 3.02 and 2.74 corresponding to the protons
from H-1′. The disappearance of peaks corresponding to H-1′
from the α-pyranose product around δ 2.92 indicates the shift
of the equilibrium has finished (Figure 2). To possibly
accelerate the reaction, microwave irradiation was attempted
instead of conventional heating.29−31 We found that the C-
glycosidic bond formation was finished under microwave
irradiation in only 30 min under reflux at 70−75 °C. The top
layer of the mixture containing the excess 2,4-pentanedione was
then discarded, and the reaction was heated in the microwave
reactor during which time the temperature of the reaction was
slowly raised to 100 °C as the excess THF evaporated. After 5.5
h, 1H NMR showed most of the mixture had been converted to
the desired β-C-glycoside 4 (Figure 2). We also found out that
the reaction could be run at much higher concentrations than
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Scheme 4. Revised Synthesis of IBCG Using TMS as a Protecting Group
Wittig reaction on the per-O-acetylated ketone 5a, since the
per-O-acetylation of ketone 4 was reported to give a high yield
easily28 and provides a readily available starting point for the
methylenation. The ketone 4 was per-acetylated using acetic
anhydride and pyridine to give per-O-acetylated compound
5a,28 which was then subjected to a Wittig reaction (Scheme 2).
However, no combination of varying base, temperature, or
order of addition was found in which the desired alkene 6a was
obtained in over 20% yield. The possible side products in this
reaction involved a retro-Michael addition under basic
conditions, followed by possible polymerization and decom-
position. The less base-sensitive benzoyl group was then
installed in place of acetyl groups, but the results were worse.
The protected sugar derivative 5b was obtained in only 30%
yield, and the Wittig reactions on 5b gave the desired product
in less than 5% yield. Apparently the Wittig reagent was too
basic for use with these peracetylated substrates. Non-basic
the per-benzyl substituted ketone 10 in 81% overall yield over 4
steps (Scheme 3).
The Wittig reaction on the per-benzyl substituted ketone 10
proceeded better than on the per-acetyl substituted ketone.
Using 2 equiv each of Wittig salt and t-BuOK, the alkene 11
was formed in 42% yield. When slightly excess base was used,
the yield of 11 dropped significantly. Hydrogenation of 11 gave
IBCG 1 in 94% yield. This route yields IBCG from galactose in
8 steps in 30% overall yield. However, this route is still not very
satisfactory, given the low yields and long step count.
To further improve the synthesis route, further fine-tuning of
the electronic effects was needed. Ideally, the Wittig substrate
would be even more electron-rich than the perbenzylated
substrate employed. Several examples of protected O-glycosides
using silyl groups as temporary protecting groups have been
reported.41−45 The per-O-TMS protected substrates are readily
prepared on large scale, and the ease of deprotection makes
them very convenient to use. However, the current studies
mainly focused on utilizing the improved solubility of the per-
O-silylated substrate in organic solvents and on the Lewis acid
catalyzed reactions of silyl protected hydroxyl groups. In the
latter case, the silyl groups were used as a proton surrogate.
Only a few studies used per-O-silylation as a method to change
the electronic properties of the parent compound. For example,
Gervay-Hague’s studies of glycosyl iodides42,46,47 showed that
the per-O-trimethlysilyl glycosyl iodides were more reactive
than per-O-benzyl donors in glycosylation reactions. We
proposed that by per-silylation we might be able to circumvent
the troublesome side reaction encountered in the Wittig
reaction by making the ketone substrate more electron-rich.
The per-TMS-silylation of 4 was tested under different
conditions, including using pyridine, Et3N/DMF, and Et3N/
CH2Cl2. Fortunately, it was found out that using crude 4
containing large amounts of NaOAc from the condensation did
not interfere with the per-silylation reaction. Since ketone 12
has good solubility in hexanes, it could be extracted from the
crude mixture directly to give pure product, leaving Et3N·HCl
and NaOAc as insoluble salts. The reaction in Et3N/CH2Cl2 is
slower than in DMF or pyridine, but the workup process is
easier. The Wittig reaction on 12 proceeded surprisingly well
and gave the methylenation product 13 in high yield. After
solvent removal, the crude mixture of the Wittig reaction
containing 13 and triphenylphosphine oxide was redissolved in
methanol/water and subjected to acidic silyl group cleavage
using Dowex 50X8 (H+ form). After filtration and solvent
removal, the crude product was redissolved in water and
extracted with EtOAc to remove triphenylphosphine oxide and
provide an aqueous solution of alkene 14. However, we
experienced problems in the hydrogenation of this alkene.
Unlike alkenes 6a or 11, the hydrogenation of 14 proceeded
very slowly; the reaction required more than 48 h to go to
39
methylenation conditions using TiCl4/Mg/CH2Cl2 on
substrate 5a were attempted, but the yield was also low
(∼20%). A Grignard reaction using methylmagnesium bromide
on 5a to install a methyl group also did not give satisfactory
results.
The electron-withdrawing acyl groups appeared to make the
retro-Michael addition more favorable, hence the side reactions
and low yields. Using more electron-donating groups such as
benzyl as the hydroxyl protecting groups might help. Per-O-
benzylated compound 10 (Scheme 3) has been made
previously from epimerization under basic conditions of the
corresponding α-ketone,33 which was obtained via oxymercura-
tion followed by oxidation of α-allyl-C-galactoside. Benzylation
of 4 seems to be a much more straightforward route to obtain
10. However, this perbenzylation reaction turned out to be
more problematic than expected. Direct benzylation of the
ketone 5a using NaH/BnBr gave complex mixtures, presumably
due to ring opening under basic conditions. Similar issues were
reported on the glucose ketone substrate.40 Using NaOH/BnBr
in THF with a phase transfer reagent did not give much
improvement. We therefore decided to protect the ketone first,
although this strategy would add undesirable additional steps to
the sequence. Using ethylene glycol, PTSA, and compound 4 in
a mixture of acetonitrile/benzene did not yield the ketal,
possibly due to the poor solubility of the initial ketone.
Interestingly, the glucose analogue could react under similar
conditions.40 The attempt at protecting ketone 4 in methanol
as a dimethyl ketal also did not proceed well.
Finally the per-acetylated ketone 5a was protected to form
ketal 7 using ethylene glycol and pyridinium p-toluenesulfonate
(PPTS) as a catalyst40 (using TsOH as catalyst led to the
decomposition of the substrate) in benzene in 3 h (Scheme 3).
Deacetylation, followed by benzylation and ketal removal, gave
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completion under 1000 psi of H2. We reasoned that even
though the H NMR of crude 14 seemed pure, trace amounts
protein (GFP) expressed from the original CAG promoter.
HeLa cells were made transgenic for these constructs by
cotransfection with a plasmid encoding SB100x,50 an enhanced
version of the Sleeping Beauty transposase. HeLa cells
expressing a fixed ration between Neptune and GFP were
purified by cell sorting. We then used SB100x-mediated
transposition to make the CAGop-Neptune/CAG-GFP HeLa
cells transgenic for LacI (Figure 3A). Expression of lacI resulted
in an over 40-fold repression of Neptune expression, while
leaving the GFP levels unaltered (data not shown). The
addition of the inducers IPTG or IBCG resulted in an increase
of Neptune expression over time. Our results showed that
IBCG was very well tolerated by HeLa cells at 1 mM
concentration and was comparable to IPTG in inducing gene
expression (Figure 3B).
In summary, a convenient route for the synthesis IBCG (1)
from galactose was developed that includes as key steps a
microwave-assisted synthesis of an intermediate β-C-glycoside
and a Wittig reaction on a per-O-silylated base-sensitive
substrate. Microwave-assisted conditions could significantly
reduce the reaction time for the β-C-glycoside formation and
also easily allows the reaction to be run at much higher
concentrations. The large yield differences of the Wittig
reaction on per-acetyl-, per-benzyl-, and per-silyl-protected
substrates further demonstrate the profound impact of
protecting groups. TMS groups can not only be used to
improve solubility of poly hydroxyl bearing compounds and
react as a proton surrogate under Lewis acid promoted
conditions, they can also change the electronic properties of
the substrate greatly. The Wittig reaction on the per-silyl
substituted β-C-glycosidic ketones provides a new and efficient
way of synthesizing novel β-C-glycoside analogues. In addition,
the developed route can produce material sufficiently pure for
biological studies without a single chromatographic separation.
Finally, IBCG has been shown capable of inducing lac operon
promoters for induction of gene expression not only in bacterial
systems but also in mammalian cells. This improved route
amenable to larger scale production of IBCG coupled with the
promising cell-based studies now sets the stage for testing of
this system in animal studies.
1
of phosphorus-containing compounds left due to the large
amount of Wittig salt used in the methylenation reaction might
be interfering with the hydrogenation reaction. Therefore, the
workup procedure for the Wittig reaction was revisited. Most of
the solvent was removed, and then hexane was added to
remove most of the triphenylphosphine oxide by filtration. The
resulting filtrate was concentrated, and the residue was
dissolved in methanol/water and subjected to Dowex 50X8
to provide 14. After EtOAc/water extraction, the hydro-
genation of 14 in ethanol took less than 1 h under high
pressure and could be done under atmospheric pressures of H2
1
in 8 h to give 1 as a white solid. The H NMR of crude 1
showed that it was sufficiently pure and no further purification
was needed. By using this modified route, we were able to
perform a larger scale synthesis of IBCG from 10.00 g of
galactose 2 (55.5 mmol), and obtained 9.90 g of 1 (45.4 mmol)
in 81% yield over 5 steps (95% average yield per step) without
any chromatography purification (Scheme 4). The large
difference in the Wittig reaction using a per-O-benzylated
substrate versus a per-O-silylated substrate shows the significant
affect a change in electronic properties of the substrate can
make in reaction yields.
Now that a route was available to readily provide the
multigram quantities of IBCG necessary to carry out animal
studies, the utility of the compound in inducing gene
expression in mammalian rather than bacterial cells needed to
be ascertained. Mammalian cells such as HeLa cells have less
diverse metabolic capabilities compared to bacterial systems,
and therefore decomposition of IPTG is less of an issue on the
time scale of the experiments. Such issues become problematic
when whole animal experiments are envisioned. However, the
permeability of IBCG compared to IPTG was unclear. In other
words, would IBCG reach the site of action to induce gene
expression? To test if IBCG will work as a lac operon inducer in
mammalian cells in the induction of protein expression, assays
of repressor activity utilizing the expression of fluorescent
proteins in HeLa cells were preformed (Figure 3). To this end
EXPERIMENTAL SECTION
■
General Experimental Methods. Reactions were performed
using flame-dried glassware under argon using anhydrous solvents
unless otherwise noted. Microwave-assisted reactions were performed
using a CEM Discover Microwave system. Thin layer chromatography
(TLC) was performed using glass-backed silica gel plates w/UV254.
Visualization of TLC plates was performed by UV light and 5% sulfuric
acid/ethanol. NMR spectra were recorded on a 400 MHz for 1H (100
1
MHz for 13C) spectrometer. H NMR and 13C NMR taken in CDCl3
spectra were referenced to the solvent peak at 7.260 ppm (1H) and
77.0 ppm (13C). Due to the severe overlap of 13C signals from
aromatic carbons in the range of 129−127 ppm in the 13C NMR of the
tetra-benzyl protected compounds 9 and 11, only clearly discernible
peaks from aromatic carbons on the benzyl groups are reported. The
Figure 3. (A) Scheme of promoters used to evaluate the induction
capabilities of IPTG and IBCG in HeLa cells. (B) Neptune expression
from the CAGop promoter was normalized to GFP expression and to
maximal Neptune expression in absence of LacI. The relative Neptune
expression after induction with IPTG or IBCG is shown. Fluorescence
intensities were determined by flow cytometry. Data shown are the
average of two independent experiments.
1
1
assignments of H NMR peaks were made primarily from 2D H−1H
COSY and edited 1H−13C HSQC spectra. High resolution mass
spectra (HRMS, ESI mode) were obtained using a Q-TOF LC−MS.
1-C-(β-D-Galactopyranosyl)-propan-2-one (4). To a round-
bottom flask were added D-galactopyranose 2 (4.00 g, 22.2 mmol),
NaHCO3 (7.40 g, 88.8 mmol), water (20 mL), and THF (10 mL). 2,4-
Pentanedione 3 (4.6 mL, 44 mmol, freshly distilled) was added; the
reaction started to turn light yellow. The flask was attached to a
condenser and put into a microwave reactor. The temperature of the
reaction was measured using the external sensor equipped in the
we cloned a construct placing the far-red fluorescent protein
Neptune behind the LacI-repressible Chicken −actin CMV
enhancer promoter sequences (CAGop)48 in between recog-
nition sites for the Sleeping Beauty transposase.49 As control, we
used a Sleeping Beauty transposon containing green fluorescent
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microwave reactor. The reaction was heated to reflux at 70−75 °C at
reaction was diluted with CH2Cl2, washed with water and saturated
NaHCO3 solution, and dried over Na2SO4. Silica gel chromatography
purification using hexanes/ethyl acetate 5:1 to 3:1 afforded the
product as a white solid (675 mg, 1.16 mmol, 89.2% for 4 steps). The
1H and 13CNMR of 10 spectra matches previously reported data.33
1
80 W with stirring. After 30 min, H NMR spectrum showed that all
the galactose had been consumed, and the top layer was separated and
discarded. The remaining solution was extracted with EtOAc (20 mL).
Then the undissolved solid was returned to the flask, and the aqueous
solution was heated at 75 °C at 80 W for another 30 min, gradually
2-(1-C-(2,3,4,6-Tetra-O-benzyl-β-D-galactopyranosyl) meth-
yl)-propene (11). To a flask containing PPh3CH3Br (830 mg, 2.32
mmol) and THF (5 mL) was added tBuOK (1 M in THF, 2.9 mL, 2.9
mmol) at 0 °C, and the reaction was stirred for 30 min. A solution of
10 (675 mg, 1.16 mmol) in THF (7 mL) was added dropwise into the
reaction, and the mixture was stirred for 8 h. A saturated NH4Cl
solution (10 mL) was added to quench the reaction, and the reaction
was extracted with EtOAc, washed with water, and dried over Na2SO4.
Solvents were removed under reduced pressure, and silica gel column
purification using hexanes/ethyl acetate 6:1 to 4:1 offered the product
11 (281 mg, 0.49 mmol, 42%). 1H NMR (400 MHz, CDCl3) δ 7.43−
7.23 (m, 20H, PhH), 4.96 (d, J = 11.2 Hz, 1H, -OCHPh), 4.95 (d, J =
11.6 Hz, 1H, -OCHPh), 4.77 (m, 2H, CCH2), 4.76 (d, J = 12.9 Hz,
1H, -OCHPh), 4.68 (d, J = 12.9 Hz, 1H, -OCHPh), 4.66 (d, J = 11.2
Hz, 1H, -OCHPh), 4.64 (d, J = 11.6 Hz, 1H, -OCHPh), 4.45(ABq, J =
11.8 Hz, 2H, -OCH2Ph), 3.99 (m, 1H, H-4), 3.67 (t, 1H, J = 8.8 Hz,
H-2), 3.63(dd, 1H, J = 2.5, 8.8 Hz, H-3), 3.59−3.48 (m, 3H, H-6a, H-
6b, H-5), 3.41 (dt, J = 1.6, 9.4 Hz, H-1), 2.57 (d, 1H, J = 14.6 Hz, H-
1′a), 2.25 (dd, 1H, J = 9.4, 14.6 Hz, H-1′b), 1.76 (s, 3H, CH3). 13C
NMR (101 MHz, CDCl3) δ 143.1, 138.8, 138.5, 138.4, 138.1, 128.42,
128.36, 128.19, 128.14, 128.02, 127.85, 127.68, 127.65, 127.62, 127.55,
127.53, 112.1, 85.0, 78.9, 78.7, 77.2, 75.4, 74.4, 73.8, 73.5, 72.3, 69.2,
39.8, 23.0. HRMS(ESI): calcd for C38H42NaO5 [M + Na]+ 601.2924,
found 601.2939.
1
heated to 90 °C for 3 h, and then to 100 °C for another 2 h. The H
NMR spectrum indicated that the reaction was finished, and the
yellow/orange reaction mixture was cooled and concentrated under
reduced pressure at room temperature. Methanol/ethyl acetate (1:1)
(100 mL) was added to the mixture to dissolve the product, and then
the remaining salt was removed by filtration. The solution was
concentrated to give a crude product of 4 containing NaOAc, which
could be used directly in the following experiments. The crude product
was passed through a short column using methanol/ethyl acetate 1:4
as eluting solvent, and recrystallization using methanol/ethyl acetate
gave 4 as white crystals (3.21 g, 14.5 mmol). The mother liquor was
concentrated and purified by silica gel column chromatography to give
additional 4 (1.02 g, 4.6 mmol). 1H NMR data matches data reported
in the literature.28
The reaction was also performed on a 10.00 g scale and on a 20.00 g
scale of galactose at concentrations of 2.4 and 4.8 M, respectively.
1-C-(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl)-propan-2-
one Ethylene Ketal (7). To a solution of 5a (500 mg, 1.30 mmol) in
benzene (15 mL) were added ethylene glycol (0.145 mL, 2.60 mmol)
and pyridinium p-toluenesulfonate (50 mg, 0.2 mmol). The reaction
was heated to reflux with a Dean−Stark apparatus. After 3 h, 1H NMR
indicated completion of the reaction. The solvent was removed under
reduced pressure. The resulting residue was dissolved in dichloro-
methane, washed with NaHCO3 (aq), and then dried over Na2SO4.
Solvents were removed under reduced pressure to provide 7 (550 mg,
1.27 mmol) as a white foam that was used without further purification.
1H NMR (400 MHz, CDCl3) δ 5.40 (dd, J = 1.2, 3.3 Hz, 1H, H-4),
Isobutyl-C-galactoside (1) by the benzyl ketone route. To a
solution of 11 (281 mg, 0.49 mmol) in methanol was added 10% Pd/
C (50 mg), and the reaction was stirred under H2 for 8 h. The reaction
was filtered and concentrated to give 1 as a hygroscopic white foam
1
(101 mg, 0.46 mmol, 94%). The H and 13C NMR data of 1 matches
5.05 (t, J = 9.7 Hz, 1H, H-2), 5.02 (dt, J = 3.3, 9.7 Hz, 1H, H-3), 4.13
(dd, J = 7.2, 11.3 Hz, 1H, H-6a), 4.05 (dd, J = 6.1, 11.3 Hz, 1H, H-6b),
3.98−3.82 (m, 5H, -OCH2CH2O-, H-5), 3.64 (dt, J = 1.6, 9,3 Hz, 1H,
H-1), 2.14 (s, 3H, OAc), 2.04 (s, 3H, OAc), 2.02 (s, 3H, OAc), 1.96
(s, 3H, OAc), 1.91 (dd, J = 8.5, 14.7 Hz, 1H, H-1′a), 1.75 (dd, J = 1.6,
14.7 Hz, 1H, H-1′b), 1.37 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3)
δ 170.2, 170.1, 169.9, 169.7, 108.4, 75.1, 74.0, 72.1, 68.9, 67.7, 64.4,
64.3, 61.8, 39.9, 24.5, 20.6, 20.53, 20.49, 20.42. HRMS(ESI): calcd for
C19H28NaO11 [M + Na]+ 455.1524, found 455.1517.
the reported.16
1-C-(2,3,4,6-Tetra-O-trimethylsilyl-β-D-galactopyranosyl)-
propan-2-one (12). Crude 4 (22.60 g) was synthesized from
galactose (10.00 g, 55.5 mmol), NaHCO3 (18.65 g, 222 mmol), and
2,4-pen-tanedione 3 (11.45 mL, 111 mmol, freshly distilled) in water
(50 mL) and THF (25 mL) according to the above procedure. The
crude product obtained from methanol/ethyl acetate extraction
contained ca. 10.40 g NaOAc as indicated by 1H NMR and used
directly.
1-C-(2,3,4,6-Tetra-O-benzyl-β-D-galactopyranosyl)-propan-
2-one Ethylene Ketal (9). Compound 7 (550 mg, 1.27 mmol) was
dissolved in CH3OH (15 mL), cooled to 0 °C, and treated with Na
(23 mg, 1.0 mmol). After 2 h, the solvent was removed under reduced
pressure to yield crude 8. The crude product was dissolved in DMF
(10 mL) and cooled to 0 °C. NaH (262 mg, 60%, 7.8 mmol) was
added, and the reaction was stirred for 30 min. Benzyl bromide (0.8
mL) was added, and the reaction was stirred at room temperature for 6
h before methanol (1 mL) was added. The mixture was extracted with
EtOAc, washed with water, and dried over Na2SO4. The solvents were
removed under reduced pressure to yield 9 as a syrup that was used
without further purification. 1H NMR (400 MHz, CDCl3) δ 7.47−7.29
(m, 20H, PhH), 5.05 (d, J = 11.2 Hz, 1H, -OCHPh), 5.01 (d, J = 11.8
Hz, 1H, -OCHPh), 4.82 (d, J = 11.8 Hz, 1H, -OCHPh), 4.74 (d, J =
11.8 Hz, 1H, -OCHPh), 4.73−4.67 (m, 2H, −CH2Ph), 4.58 (d, J =
11.9 Hz, 1H, -OCHPh), 4.53 (d, J = 11.9 Hz, 1H, -OCHPh), 4.06 (d, J
= 2.5 Hz,, H-4), 3.99−3.83 (m, 4H, -OCH2CH2O-), 3.75−3.65 (m,
2H, H-2, H-3), 3.64−3.59 (m, 3H, H-6a, H-6b, H-5), 3.52 (t, J = 9.0
Hz, 1H, H-1), 2.19 (d, J = 15.5 Hz, 1H,H-1′a), 1.87 (dd, J = 9.0, 15.5
Hz, 1H,H-1′b), 1.48 (s, 3H, CH3). 13C NMR (100 MHz, CDCl3) δ
138.8, 138.6, 138.4, 138.1, 128.6, 128.49, 128.45, 128.40, 128.37,
128.28, 127.98, 127.91, 127.78, 127.70, 127.67, 127.65, 127.60, 126.99,
109.4, 85.1, 78.6, 77.0, 76.87, 76.83, 73.8, 73.5, 72.4, 69.2, 65.3, 64.39,
64.35, 39.6, 24.7. HRMS(ESI): calcd for C39H44NaO7 [M + Na]+
647.2979, found 647.2987.
To a round-bottom flask containing crude 4 were added CH2Cl2
(100 mL) and Et3N (263 mL, 1.89 mol). The reaction was cooled to 0
°C, and freshly distilled chlorotrimethylsilane (48.3 mL, 380 mmol)
was added dropwise. The reaction was stirred at ambient temperature
for 16 h before the solvents were removed at reduced pressure.
Hexanes were added to the mixture, and the solution was filtered
through Celite, washed with hexanes, and concentrated to yield crude
12 as a yellow liquid that was used without further purification.
2-(1-C-β-D-Galactopyranosyl methyl)-propene (14). To a
round-bottom flask were added anhydrous PPh3CH3Br (29.80 g,
83.4 mmol) and t-BuOK (9.36 g, 83.4 mmol). The reaction was
cooled to 0 °C, and THF (200 mL) was added via cannula. The
resulting yellow solution was stirred at 0 °C for 5 min and warmed to
room temperature in 25 min. The solution was stirred at room
temperature for another 30 min and recooled to 0 °C. A solution of
crude 12 in THF (30 mL) was added dropwise to the reaction over 10
min. After stirring at 0 °C for another 10 min, the ice bath was
removed, and the reaction was stirred for 90 min. Acetone (5 mL) was
added, and the reaction mixture was partially concentrated to a slurry,
diluted with hexanes, and filtered. The filtrate was concentrated to
yield crude 13 as a yellow liquid that was used without purification.
The crude product 13 was dissolved in methanol/water (120 mL/
10 mL). Dowex 50X8 (H+ form, 10.00 g) was added, and the reaction
was stirred for 25 min, filtered, and concentrated under reduced
pressure. The resulting residue was suspended in water (250 mL) and
extracted with ethyl acetate (3 × 100 mL). The aqueous layer was
concentrated to give crude 14 as a pale yellow liquid that was used
1-C-(2,3,4,6-Tetra-O-benzyl-β-D-galactopyranosyl)-propan-
2-one (10). Crude benzyl ketal 9 was dissolved in CH2Cl2/TFA/H2O
(10:1:0.1, 10 mL total), and the reaction was stirred for 30 min. The
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The Journal of Organic Chemistry
Article
without further purification. 1H NMR (400 MHz, D2O) δ 4.70 (d, 2H,
J = 9.8 Hz, CCH2), 3.79 (d, 1H, J = 3.3 Hz, H-4), 3.58−3.48 (m,
2H, H-6ab), 3.48−3.41 (m, 2H, H-3, H-5), 3.31−3.26 (m, 2H, H-1,
H-2), 2.44 (d, J = 15.7 Hz, 1H, H-1′a), 2.03 (dd, J = 9.0, 15.7 Hz, H-
1′b), 1.61 (s, 3H, CH3). 13C NMR (100 MHz, D2O) δ 143.9, 111.9,
78.5, 77.6, 73.9, 71.0, 70.0, 61.2, 39.2, 21.6. HRMS(ESI): calcd for
C20H36NaO10 [2M + Na]+ 459.2201, found 459.2201.
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ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C NMR spectra of compounds 1, 4, 7, 9, 10, 11, and
14. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail: npohl@iastate.edu.
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■
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ACKNOWLEDGMENTS
■
N.L.B.P. acknowledges the Wilkinson Professorship in
Interdisciplinary Engineering. This work was supported in
part by the U.S. National Science Foundation (CHE-0911123).
We are also grateful to Bernard Binetruy for providing the
CAGop sequences and to Lajos Mates for the SB100x plasmid.
M.S. is supported by an Emmy Noether grant of the DFG.
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