Key words
isomannide - isosorbide - chiral amino alcohols - supported enzyme - lipase
Optically active amino alcohols are among the most versatile chemical structures that
can be employed as chiral inducers for asymmetric catalysis.[1] They are useful not only as chiral ligands, as in the enantioselective addition
of dialkylzinc reagents to carbonyl compounds,[2]
[3] but also as precursors of other chiral derivatives used as ligands in metal-catalyzed
enantioselective reactions[4,5] or as chiral organocatalysts.[6] Most chiral amino alcohols are derived from natural compounds, such as carbohydrates
or amino acids, which are readily available enantiopure sources and possess suitable
stereochemical features for good asymmetric induction. In particular, the manipulation
of the chiral pool available from biomass, which appears very attractive for the synthesis
of asymmetric auxiliaries[7] and organocatalysts,[8] could represent a simple and economical way to obtain chiral amino alcohols endowed
with interesting stereochemical features.[9] Among the biomass-based precursors suitable for the synthesis of amines and amino
alcohols, a great deal of attention has been addressed to (3R,3aR,6R,6aR)-hexahydrofuro[3,2-b]furan-3,6-diol and its (3R,3aR,6S,6aR) diastereoisomer, also known as isomannide and isosorbide, respectively. They are
renewable, inexpensive, and commercially available chiral compounds derived from the
dehydration of sorbitol and mannitol, respectively, which are waste products formed
during the processing of corn oil and byproducts from the starch industry.[10] Isomannide (1) and isosorbide (2) possess a vaulted structure due to the cis junction of the two tetrahydrofuran rings with two hydroxyl groups that in isomannide
are directed toward the inner of the chiral cavity (Figure [1]). Thanks to these stereochemical features, isomannide derivatives constitute real
chiral pockets, inside which enantioselective processes can take place with high effectiveness.[11] For these reasons most of the amino derivatives of isohexides show the isomannide
stereochemistry.[12]
[13]
[14]
Figure 1 Chemical structures of isomannide (1) and isosorbide (2)
In order to obtain the amino alcohol derivative 4 with the desired stereochemistry, a straightforward approach is represented by the
regioselective and stereospecific interconversion of the exo hydroxyl group of isosorbide to an amino group.[15] This transformation has been described and involves regioselective protection of
the endo hydroxyl, conversion of the exo hydroxyl into a benzenesulfonate group followed by its displacement by benzylamine;
the hydrogenolysis of the two benzyl groups gives the target amino alcohol (Scheme
[1]).[16]
Scheme 1 Comparison of synthetic routes to amino alcohol 4
The weak points of this protocol are the selective protection step, giving the endo benzyl ether 3 in 56% yield and the displacement of the benzenesulfonate group at high temperature,
causing the formation of an elimination byproduct.[17] Strategies to overcome these issues and develop a high-yielding synthetic protocol
require a highly selective protection for the endo hydroxyl group as well as a nucleophilic displacement of a good leaving group under
mild reaction conditions.
Lipase-catalyzed transesterification of isosorbide has been previously reported for
the regioselective synthesis of monoesters.[18]
[19]
[20]
[21]
[22]
[23]
[24] In general, the selectivity has been toward the endo hydroxyl group. Therefore, a biocatalyzed selective acetylation of the isosorbide
endo hydroxyl represents a high-yield protection step.[23]
[24] The transesterification has also been performed using immobilized enzymes, allowing
an easy recovery of the product. However, long reaction times were generally observed
to obtain complete conversion of isosorbide.[22]
[23]
Nucleophilic displacement can be performed using a stronger nucleophile than benzylamine,
which can react under mild conditions and can be easily converted into an amino group.
Sodium azide fulfills these requirements thanks to its high nucleophilicity and because
the obtained organic azides are easily reduced to the corresponding amines in high
yield.[25]
Herein we present a new straightforward synthetic protocol to access (3R,3aR,6R,6aR)-hexahydrofuro[3,2-b]furan-6-amino-3-ol (6-aminoisomannide), whose key points are the enzyme-catalyzed
regioselective acetylation of isosorbide (Scheme [1]) and the introduction of the azido group under mild reaction conditions. The synthesis
of the acetate 5 has been achieved under optimized conditions by using Amano Lipase PS which was immobilized
by adsorption onto a methacrylate resin. Moreover, the reusability of the biocatalyst
was investigated and the efficient biocatalytic step was evaluated on a preparative
scale.
Amano PS Lipase was immobilized onto octadecyl methacrylate resin ECR8806 according
to the supplier protocol.[26] Adsorption of lipases onto hydrophobic supports is a very easy immobilization method
that allows derivatives to be obtained that are more active and stable than other
immobilized lipases.[27] Indeed, by using this method the active site is usually unaffected by interaction
with the matrix, causing little or no conformational changes of the enzyme. The highly
hydrophobic surface of octadecyl methacrylate is particularly suitable for reversible
but strong adsorption of lipases and the immobilized derivatives show excellent performance
in applications in fully organic anhydrous solvents.[27]
As reported in Scheme [2], the first step of the synthesis was the regioselective enzymatic protection of
isosorbide 2 to give the monoacetate 5. Starting from a known protocol[24] an initial screening of the reaction conditions was made employing the supported
enzyme (see Table S1, Supporting Information).
Scheme 2 Synthesis of 6-aminoisomannide 4
The results show that, to obtain complete conversion, the use of anhydrous acetone
was crucial, due to the high activity of lipases in the hydrolytic reverse reaction.
Therefore, after immobilization the supported enzyme was carefully dried under vacuum
to avoid the presence of traces amount of water. The immobilization process led to
better results in terms of substrate conversion, maybe due to a purification of the
commercial enzyme during this process.
Analysis of the crude mixtures during the screening by means of 1H NMR spectroscopy never revealed the presence of the exo acetate, nor could it be detected by GC-FID analysis of reaction mixtures. In some
cases, the diacetate was obtained. Lowering the amount of vinyl acetate from 3.0 equivalents
to 2.0 equivalents allowed us to obtain the desired pure product, as confirmed by
GC-FID analysis (see Figure S3, Supporting information). The optimal amount of the
catalyst to convert isosorbide into 5 in a short time was found to be 0.7% w/w enzyme/substrate.
Therefore, as reported in Scheme [2], by using immobilized lipase with 2 equivalents of vinyl acetate in dry acetone
at room temperature the acetate 5 was obtained in quantitative yield and excellent selectivity (>99%, GC-FID) in very
short reaction time (1.5 h), which makes this protocol far superior to those reported
in the literature.[22]
[23] Under these optimized reaction conditions, the biotransformation was conducted
on a preparative scale (9.6 mmol of isosorbide).
The biocatalyst was also tested for its reuse. At the end of the reaction, the enzyme
was filtered, washed, and reused in consecutive reactions under the same experimental
conditions.
In each cycle, complete conversion of the substrate and high selectivity were obtained.
These results show that the immobilized lipase can be reused in subsequent runs without
loss in selectivity. However, a gradual drop in enzyme activity was observed but this
is not an issue as complete conversion of the substrate can still be achieved in reasonable
reaction times. Complete conversion of the substrate and high selectivity were observed
for the first two cycles after 3 h and 8 h, respectively. Notably, for large-scale
applications the enzyme may be desorbed after its inactivation and the support may
be reused.[26]
Once the 3-hydroxyl had been protected, the stereospecific interconversion of the
6-exo hydroxyl into 6-endo amine was achieved according to standard procedures (Scheme [2]).
By reacting acetate 5 with trifluoromethanesulfonic anhydride in the presence of pyridine at 0 °C the corresponding
triflate 6 was obtained in 90% yield. It is noteworthy that the reaction is clean, and the pure
product is obtained after simple workup (hydrolysis followed by liquid–liquid extraction),
without any further purification.
Nucleophilic displacement of the triflate group by sodium azide presented some initial
problems. Under standard reaction conditions, i.e., heating a 1:1.5 triflate–NaN3 mixture in DMF at 100 °C, the conversion of 6 was complete in short reaction times (2 h), but 45% of an elimination side product
was obtained. In order to improve the chemoselectivity of the process, the reaction
temperature was lowered to 50 °C and complete conversion of the substrate was achieved
in 5 h, but formation of a 40% yield of the elimination product was still observed.
At room temperature, triflate 6 reacted smoothly with NaN3 in DMF over a longer reaction time (30 h), but, unfortunately, a 40% yield of the
elimination side product was still observed. To drive the reaction wholly toward the
azide 7 an excess of sodium azide was used. These conditions markedly reduced the reaction
time (16 h) and product 7 was obtained in 73% yield after chromatographic purification.
The two final steps, reduction of the azido group and deprotection of the 3-hydroxy
group, were performed in one pot, employing lithium aluminum hydride as reducing agent
for both groups. The reaction was conducted at room temperature with a slight excess
of LiAlH4 in dry THF, and the conversion of substrate 7 was complete after 24 h. The workup procedure was crucial and it was performed with
a stoichiometric amount of water, in order to obtain oxides that could be readily
filtered off. Avoiding solvent extraction is mandatory to prevent loss of the amino
alcohol in the aqueous phase, due the water solubility of the compound. The pure amino
alcohol 4 was then obtained in quantitative yield after removal of the solvent.
Amino alcohol 4 can be used as precursor of other chiral derivatives, such as the hydroxyurea 8, obtained according to Scheme [3].
Scheme 3 Synthesis of hydroxyurea 8
As a proof of concept this derivative was used, as a chiral solvating agent, in the
enantiodiscrimination of the 3,5-dinitrobenzoylphenylglycine methylester. The hydroxyurea
8 produced splitting of various signals in the NMR spectrum of the racemic amino acid
derivative (Figure [2]): the baseline separation of the proton signals of the 3,5-dinitrobenzoyl group
therefore allows a ready determination of the enantiomeric composition of nonracemic
mixtures.
Figure 2 Selected regions of the 1H NMR spectrum (401 MHz, chloroform-d, 25 °C) of: a) 3,5-dinitrobenzoylphenylglycine methylester (c 30 mM); b) 1:1 mixture 8–3,5-dinitrobenzoylphenylglycine methylester (c 30 mM).
In conclusion, we have developed a straightforward and efficient synthesis of 6-aminoisomannide,
an amino alcohol with interesting structural features that make it a good candidate
as chiral auxiliary or as precursor of other chiral auxiliaries, such as hydroxyureas
or hydroxythioureas. The use of a supported enzyme for the acetylation step allowed
us to obtain a quantitative yield of the protected precursor with a completely regioselective
reaction, which was successfully used also on preparative scale. In this way, the
synthetic route, which starts from the biomass-based precursor isosorbide, allowed
us to obtain the amino alcohol 4 in 66% overall yield in four steps with only one chromatographic purification of
an intermediate. Starting from amino alcohol 4 chiral derivatives to be used in enantiodiscriminating processes can be readily obtained.
Further studies are in progress on this topic.
General Remarks
All the reactions involving sensitive compounds were carried out under dry argon,
in flame-dried glassware. Amano Lipase PS, from Burkholderia cepacian (≥30,000 U/g, pH = 7.0, 50 °C) was obtained from Sigma-Aldrich. If not noted otherwise,
reactants and reagents were commercially available and used as received from Alfa
Aesar, Purolite, Carlo Erba, TCI-Chemicals, and Sigma-Aldrich. Dichloromethane and
tetrahydrofuran were dried and degassed by an MB-SPS solvent purification system.
Analytical TLC was performed on precoated silica gel ALUGRAM Xtra G/UV254 plates.
Purifications were performed by flash chromatography on silica gel (40–63 μm). Melting
points were measured on a Reichert Thermovar Type 300429 Microscope. 1H NMR spectra were recorded in chloroform-d or in deuterium oxide on a Bruker 400 MHz NMR spectrometer. The following abbreviations
are used: br s = broad singlet, s = singlet, d = doublet, dd = double doublet, dt
= double triplet, t = triplet, q = quartet, m = multiplet. 13C NMR spectra were recorded at 101 MHz and 19F NMR spectra were recorded at 378 MHz. 1H NMR and 13C NMR chemical shifts (ppm) are referred to TMS as external standard. GC-FID analyses
were performed on a GC instrument with a split/splitless injector and FID detector
(Column: J&W HP-5ms GC Column, 30 m, 0.25 mm, 0.25 μm; method: 70 °C (hold 1min);
20 °C min–1 to 250 °C (hold 5 min); 20 °C min–1 to 280 °C (hold 10 min); t
R = 6.7 min (isosorbide); 8.0 min (5); 8.4 min (isosorbide diacetate)). UV-vis measurements were performed on a JASCO-V750
UV-vis spectrophotometer. Elemental analyses were obtained using an Elementar Vario
MICRO cube equipment. Optical rotations were measured in 1 dm cells at the sodium
D line, using an Anton Paar MCP 300 Polarimeter.
Immobilization of Lipase PS
Immobilization of Lipase PS
The immobilization was carried out following the protocol provided by Purolite: 5.48
g of Purolite octadecyl methacrylate resin ECR8806 was added to a solution of Amano
Lipase PS (6.16 mg in 41 mL of phosphate buffer 20 mM, pH = 7.0). The suspension was
gently stirred for 24 h at room temperature. After this time the support was filtered,
washed with phosphate buffer (20 mL, 2.9 mM, pH = 7.0), and dried under vacuum.
Enzymatic Assay
The activities of native and immobilized enzyme were determined by using a discontinuous
variant of the known assay.[28] To a solution of the native enzyme (0.5 mL, 50.0 μg mL–1 in phosphate buffer 100 mM, pH = 7.0) or to a dispersion of the immobilized enzyme
(0.54 mg in 0.5 mL of phosphate buffer 100 mM, pH = 7.0), 0.5 mL of p-nitrophenylpalmitate (14.3 mM solution in ethanol) were added, and the mixture was
incubated at 32 °C and 400 rpm for 5 min. The reaction was quenched by addition of
0.5 M Na2CO3 (0.5 mL) and centrifuged for 10 min at 6000 rpm. The supernatant was diluted with
distilled water and the amount of p-nitrophenol released was determined spectrophotometrically. One enzyme unit (U) was
defined as the amount of enzyme necessary to release 1 μmol of p-nitrophenol per minute under assay conditions.
(3R,3aR,6S,6aR)-Hexahydrofuro[3,2-b]furan-3-methylcarboxylate-6-ol (5)[24]
(3R,3aR,6S,6aR)-Hexahydrofuro[3,2-b]furan-3-methylcarboxylate-6-ol (5)[24]
In a two-necked round-bottom flask, under an argon atmosphere, vinyl acetate (1.8
mL, 19.5 mmol) and immobilized Amano Lipase PS (1.15 g, 23771 U) were added to a gently
stirred solution of isosorbide (1.41 g, 9.6 mmol) in dry acetone (19 mL). The mixture
was gently stirred at room temperature and progress of the reaction was monitored
by GC-FID. After 1.5 h complete conversion of the starting material was observed.
The reaction was terminated by filtering the immobilized enzyme, which was washed
with acetone (2 × 19 mL). The organic phases were combined, and the solvent was removed
under reduced pressure to afford the chemically pure product as a pale-yellow oil
(1.81 g, quantitative yield; > 99% purity as determined by GC-FID).
1H NMR (401 MHz, chloroform-d): δ = 5.13 (q, J = 5.4 Hz, 1 H), 4.83 (t, J = 4.8 Hz, 1 H), 4.39 (dt, J = 4.5, 0.9
Hz, 1 H), 4.35–4.28 (m, 1 H), 3.96–3.84 (m, 3 H), 3.75 (dd, J = 9.8, 5.3 Hz, 1 H),
2.45 (s, 1 H), 2.11 (s, 3 H).
13C NMR (101 MHz, chloroform-d): δ = 170.7, 88.3, 80.5, 76.3, 75.7, 74.3, 70.3, 20.8.
[α]D
20+105.7 (c 1.0, CHCl3); lit[29] [α]D
20 +107.7 (c 1.0, CHCl3).
Reusability of the Biocatalyst
Reusability of the Biocatalyst
Under an argon atmosphere, immobilized Amano Lipase PS (172.0 mg, 3559 U) and vinyl
acetate (390 μL, 4.2 mmol) were added to a solution of isosorbide in dry acetone (0.5
M, 2.8 mL). The reaction mixture was gently stirred at room temperature, and progress
of the reaction was monitored by GC-FID. The reaction was quenched when complete conversion
was achieved. The immobilized enzyme was filtered under argon, washed twice with dry
acetone (2.8 mL, 10 min of stirring at room temperature each time) and dried under
vacuum. Fresh batches of reaction medium containing isosorbide solution (0.5 M, 2.8
mL) and vinyl acetate (390 μL) were then added to the enzyme, and the mixture was
stirred at room temperature to start a new cycle.
(3R,3aR,6S,6aR)-Hexahydrofuro[3,2-b]furan-3-methylcarboxylate-6-trifluoromethanesulfonate (6)[12]
(3R,3aR,6S,6aR)-Hexahydrofuro[3,2-b]furan-3-methylcarboxylate-6-trifluoromethanesulfonate (6)[12]
Under an argon atmosphere, to a solution of 5 (1.18 g, 6.2 mmol) in dry CH2Cl2 (30 mL) and dry pyridine (600 μL, 7.5 mmol) at 0 °C, trifluoromethanesulfonic anhydride
(1.4 mL, 8.3 mmol) was slowly added over 5 min and the mixture was stirred at 0 °C.
The reaction was monitored by TLC analysis (hexane–ethyl acetate = 7:3 or CH2Cl2–acetone = 9 : 1). After 3 h the reaction was quenched with cold water (10 mL), the
phases were separated, and the organic phase was washed with H2O (10 mL) and dried over anhydrous Na2SO4. After filtration, the solvent was removed under reduced pressure to yield the pure
product as a pale-yellow oil (1.79 g, 90%).
Rf
= 0.44 (hexane–ethyl acetate = 7:3); 0.88 (CH2Cl2–acetone = 9:1).
1H NMR (401 MHz, chloroform-d): δ = 5.31 (d, J = 3.1 Hz, 1 H), 5.18 (q, J = 5.4 Hz, 1 H), 4.93 (t, J = 5.1 Hz,
1 H), 4.67 (d, J = 4.8 Hz, 1 H), 4.22 (d, J = 11.9 Hz, 1 H), 4.04 (dd, J = 11.9, 3.2
Hz, 1 H), 3.98 (dd, J = 10.0, 6.0 Hz, 1 H), 3.80 (dd, J = 10.0, 5.2 Hz, 1 H), 2.12
(s, 3 H).
13C NMR (101 MHz, chloroform-d): δ = 170.2, 118.6 (q, J = 318 Hz, –CF3), 89.4, 85.6, 81.0, 73.6, 73.0, 70.8, 20.7.
19F NMR (378 MHz, chloroform-d): δ = –74.85.
[α]D
20+81.6 (c 0.50, CHCl3); lit.[12]
[13] [α]D
20 +86.2 (c 0.52, CHCl3).
(3R,3aR,6R,6aR)-Hexahydrofuro[3,2-b]furan-6-azido-3-methylcarboxylate (7)
(3R,3aR,6R,6aR)-Hexahydrofuro[3,2-b]furan-6-azido-3-methylcarboxylate (7)
To a solution of 6 (486.9 mg, 1.5 mmol) in N,N-dimethylformamide (15 mL), sodium azide (598.7 mg, 9.2 mmol) was added, and the mixture
was stirred at room temperature. The reaction was monitored by TLC analysis (hexane–acetone
= 8:2). After 16 h the reaction was stopped. Diethyl ether (30 mL) was added to the
mixture, and the precipitated solid was filtered off. The filtrate was concentrated
under reduced pressure, and the crude product was dissolved in diethyl ether (30 mL)
and washed with brine–water (1:1, 15 mL). The aqueous phase was extracted with diethyl
ether (3 × 30 mL), the organic phases were combined, and dried over anhydrous Na2SO4. After filtration, the solvent was removed under reduced pressure to give the crude
product as a yellow oil that was purified by flash chromatography (silica gel, hexane–acetone
= 8:2) to give the pure product as a yellow oil (233.1 mg, 73%).
Rf
= 0.23 (hexane–acetone = 8:2)
1H NMR (401 MHz, chloroform-d): δ = 5.13 (q, J = 6.0 Hz, 1 H), 4.73 (t, J = 5.6 Hz, 1 H), 4.62 (t, J = 4.9 Hz,
1 H), 4.09–4.00 (m, 2 H), 3.90 (dd, J = 9.7, 6.1 Hz, 1 H), 3.84 (ddd, J = 8.9, 6.9,
5.1 Hz, 1 H), 3.71 (t, J = 8.7 Hz, 1 H), 2.12 (s, 3 H).
13C NMR (101 MHz, chloroform-d): δ = 170.5, 82.7, 81.3, 74.1, 71.2, 70.2, 62.1, 20.7.
Elem. Anal.: calcd for C8H11N3O4: C, 45.07; H, 5.20; N, 19.71; O, 30.02. Found: C, 44.85; H, 5.32; N, 19.88.
[α]D
20 +219.7 (c 0.61, CHCl3).
(3R,3aR,6R,6aR)-Hexaydrofuro[3,2-b]furan-6-amino-3-ol (4)[16]
(3R,3aR,6R,6aR)-Hexaydrofuro[3,2-b]furan-6-amino-3-ol (4)[16]
Under an argon atmosphere, to a solution of 7 (177.4 mg, 0.83 mmol) in dry THF (14 mL), LiAlH4 (84.8 mg, 2.2 mmol) was slowly added at 0 °C. The heterogeneous mixture was stirred
for 5 min at 0 °C and then it was allowed to warm slowly to room temperature. After
24 h, the mixture was cooled to 0 °C and water (190 μL) was slowly added. The solids
were filtered off and washed with diethyl ether (3 × 5 mL), and the solvent was removed
under reduced pressure to give the pure product as a white solid (120.0 mg, quantitative
yield).
1H NMR (401 MHz, deuterium oxide): δ = 4.62 (t, J = 4.8 Hz, 1 H), 4.49 (t, J = 4.6
Hz, 1 H), 4.48–4.40 (m, 1 H), 4.16–4.02 (m, 2 H), 3.64–3.53 (m, 2 H), 3.47–3.35 (m,
1 H).
13C NMR (101 MHz, deuterium oxide): δ = 82.9, 82.3, 72.9, 72.3, 71.9, 54.7.
[α]D
25 +126.9 (c 0.22, MeOH); lit.[16] [α]D
25 +110.9 (c 0.5, MeOH).
Mp 115–117 °C; lit.[16] mp 115 °C.
(3R,3aR,6R,6aR)-Hexaydrofuro[3,2-b]furan-6-[(3,5-dimethylphenyl)carbamoyl]amino-3-ol (8)
(3R,3aR,6R,6aR)-Hexaydrofuro[3,2-b]furan-6-[(3,5-dimethylphenyl)carbamoyl]amino-3-ol (8)
Under an argon atmosphere, to a solution of (3R,3aR,6R,6aR)-hexahydrofuro[3,2-b]furan-6-amino-3-ol 4 (89.8 mg, 0.62 mmol) in dry THF (20 mL), 3,5-dimethylphenyl isocyanate (70 μL, 0.50
mmol) was slowly added. The solution was stirred at room temperature, and the reaction
was monitored by TLC analysis (CH2Cl2–acetone = 9 : 1). After 24 h, the solvent was removed under reduced pressure, and
the residue was dissolved in CH2Cl2 (35 mL). The organic solution was washed with H2O (3 × 10 mL) and dried over anhydrous Na2SO4. After filtration, the solvent was removed under reduced pressure to give the crude
product (195.7 mg) as a white solid. This was purified using a Biotage Isolera Cromatograph
(CH2Cl2–acetone) to give the pure product as a white solid (105.2 mg, 72%).
1H NMR (401 MHz, chloroform-d): δ = 6.90 (s, 2 H), 6.73 (s, 2 H), 5.64 (br s, 1 H), 4.58 (t, J = 5.0 Hz, 1 H),
4.55–4.46 (m, 2 H), 4.31 (q, J = 5.4 Hz, 1 H), 4.28–4.23 (m, 1 H), 3.88 (dd, J = 9.6,
5.5 Hz, 1 H), 3.64 (dd, J = 9.6, 5.2 Hz, 1 H), 3.46 (t, J = 8.5 Hz, 1 H), 2.27 (s,
6 H).
13C NMR (101 MHz, chloroform-d): δ = 155.5, 139.1, 138.0, 125.9, 118.7, 82.1, 81.7, 74.7, 72.6, 72.4, 53.5, 21.3.
Elem. Anal.: calcd for C15H20N2O4: C, 61.63; H, 6.90; N, 9.58; O, 21.89. Found: C, 61.81; H, 6.85; N, 9.65.
[α]D
25 +67.4 (c 0.51, CHCl3).
Mp 158–160 °C.
