Exploitation of a Multienzymatic Stereoselective Cascade Process in the Synthesis of 2-Methyl-3-Substituted Tetrahydrofuran Precursors

Article abstract of DOI:10.1021/acs.joc.6b02927

Enantiopure 2-methyl-3-substituted tetrahydrofurans are key precursors of several biologically active products (drugs, flavors, and agrochemicals). Thus, a stereocontrolled and efficient methodology for the obtainment of these synthons is highly desirable. We exploited a two-step multienzymatic stereoselective cascade reduction of α-bromo-α,β-unsaturated ketones to give the corresponding bromohydrins in good yields, with high ee and de values. The cascade process is catalyzed by an ene-reductase and an alcohol dehydrogenase. Further manipulations of these bromohydrins, by two diastereodivergent routes, allowed the preparation of the tetrahydrofuran synthons. One route is based on a lipase catalyzed cleavage of the protecting group. The second route is characterized by a camphor sulfonic acid mediated isomerization of a β-hydroxyepoxide to give the tetrahydrofuran-2-ol. Finally, the synthesis of the most odorous and pleasant stereoisomer of the roasted meat aroma, i.e., (2S,3R)-2-methyl-3-thioacetate tetrahydrofuran, is reported as well.

Full text of DOI:10.1021/acs.joc.6b02927

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Article
Exploitation of a Multienzymatic Stereoselective Cascade Process in
the Synthesis of 2-Methyl-3-Substituted Tetrahydrofurans Precursors
Elisabetta Brenna, Michele Crotti, Francesco Gilberto
Gatti, Ludovico Marinoni, Daniela Monti, and Sara Quaiato
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b02927 • Publication Date (Web): 17 Jan 2017
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The Journal of Organic Chemistry
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Exploitation of a Multienzymatic Stereoselective Cascade
Process in the Synthesis of 2-Methyl-3-Substituted Tetrahy-
drofuran Precursors
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Elisabetta Brenna,†,§ Michele Crotti,† Francesco G. Gatti,*,†,§ Ludovico Marinoni, Daniela Monti,‡
and Sara Quaiato†
†Dipartimento di Chimica, Materiali ed Ingegneria Chimica ”G. Natta”, Politecnico di Milano, P.zza Leonardo da Vinci 32, 20133
Milano, Italy
‡Istituto di Chimica del Riconoscimento Molecolare C.N.R. Via Mario Bianco, 9, 20131, Milano, Italy
§The Protein Factory, Politecnico di Milano and Università degli Studi dell'Insubria, Via Mancinelli 7, 20131 Milano, Italy
KEYWORDS: Cascade multienzymatic process; stereoselective synthesis; ene-reductases; alcohol dehydrogenases; li-
pases; cross-metathesis; bromohydrin.
ABSTRACT: Enantiopure 2-methyl-3-substituted tetrahydrofurans are key precursors of several biologically active prod-
ucts (drugs, flavors and agrochemicals). Thus, a stereocontrolled and efficient methodology for the obtainment of these
synthons is highly desirable. We exploited a two-steps multienzymatic stereoselective cascade reduction of α-bromo-α,β-
unsaturated ketones to give the corresponding bromohydrins in good yields, with high ee and de values. The cascade pro-
cess is catalyzed by an ene-reductase and an alcohol dehydrogenase. Further manipulations of these bromohydrins, by
two diastereodivergent routes, allowed the preparation of the tetrahydrofuran synthons. One route is based on a lipase
catalyzed cleavage of the protecting group. The second route is characterized by a camphor sulfonic acid mediated isom-
erization of a β-hydroxyepoxide to give the tetrahydrofuran-2-ol. Finally, the synthesis of the most odorous and pleasant
stereoisomer of the roasted meat aroma, i.e. (2S,3R)-2-methyl-3-thioacetate tetrahydrofuran, is reported as well.
diastereoisomer of 2 was prepared by Sharpless asymmet-
INTRODUCTION
ric dihydroxylation (AD-mix-β), but not with a very high
stereoselectivity (ee values ranged between 62% and
Chiral tetrahydrofurans are important building blocks
of organic chemistry, indeed, in the last decades a consid-
80%). Then, the two enantiomers of trans-2 were ob-
erable attention has been devoted to the development of
tained by Mitsunobu inversion of C(3) stereocentres of
efficient and stereocontrolled methodologies for the syn-
the cis enantiomers, conserving most of their original op-
thesis of 2,5-disubstituted tetrahydrofuranic derivates,1
key structural fragments of many natural products. How-
ever, less attention has been dedicated to the 2,3-
disubstituted tetrahydrofurans, which however are largely
employed in the preparation of many products of indus-
trial relevance such as aromas,2 drugs3 and agrochemicals4
(Figure 1).
tical purity.2
Thus, the development of efficient strategies for the
stereoselective synthesis of the key synthons 3-bromo-2-
methyl tetrahydrofuran 1 and 3-hydroxy-2-methyl tetra-
hydrofuran 2 (Figure 1), is highly desirable. For example,
these synthons may find a valuable application in the
preparation of the most odorous and pleasant stereoiso-
mer of the roasted meat aroma, i.e. (2S,3R)-3.
To knowledge of the authors no enantioselective syn-
theses of the very useful bromoderivative5 1 have ever
been reported; whereas, each enantiomer of the cis-
Figure 1. Selected examples of biologically active prod-
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ucts bearing a 2-methyl-3-substituted tetrahydrofuran
moiety.
1
2
3
4
RESULTS AND DISCUSSION
5
6
7
8
Figure 2 shows the retrosynthetic analysis of 1 and 2. It
relies on the stereospecific reduction of the (Z)-
bromoketone I to give the corresponding bromohydrin II;
the latter might be converted by regioselective tosylation
of the primary alcohol into III, which is then transformed
into 1 by ring closure. Diastereo- and/or enantiocomple-
mentary asymmetric catalytic transformations often re-
quire completely new and time consuming set-up of the
experimental conditions, moreover it is not always so triv-
ial to prepare the enantioform of the catalyst. Thus, in our
synthetic strategy we show that starting from the same
bromohydrin II, it is possible to obtain 2, diastereocom-
plementary synthon of 1, by means of a ring-expansion of
epoxide IV.
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Scheme 1. ADH and ER multienzymatic system for the syn-
thesis of optically enriched alcohols: a) sequential enzyme
addition; b) cascade procedure.
The products are usually obtained in good yields, with
excellent enantioselectivities, and for the ketones, with
good diastereoselectivities and high optical purity. In ad-
dition, we found that the ADHs, quite often, are not able
to reduce the carbonyl group of α,β-unsaturated ketones.
This uncommon chemoselectivity for a reducing agent,10
makes the combination of ADHs with ERs particularly
advantageous in a cascade process. In fact, possible spon-
taneous product racemizations, side-product formations
and intermediate product inhibition effects can be miti-
gated by this approach.9,b-d
Figure 2. Retrosynthesis of the diastereocomplementary
synthons 1 and 2.
According to our retrosynthetic plan, especially for the
crucial stereoselective transformations, a biocatalytic ap-
proach6 seems to be the best choice, indeed it is well
known that the bromo substituent is extremely labile to
the metal-catalyzed reduction, making this extremely
valuable catalysis unfeasible with our strategy.
Scheme 2. Synthesis of α-bromo-α,β-unsaturated ketones
4a-d.
The synthesis of the starting materials 4a-d is shown in
Scheme 2. First, the α,β-unsaturated ketones 3a-d with
orthogonal protective groups of the terminal alcohol (ace-
tyl and benzoate esters vs benzyl and triethylsilyl ethers)
were easily prepared in a good yield and with an excellent
diastereoselectivity (E/Z, >99:1) by cross-metathesis11: we
used an excess of the cheap methylvinyl ketone with re-
spect to the O-protected allylic alcohol, in presence of the
2nd generation of Grubbs catalyst12,13 (see SI for the prepa-
ration of alcohols). Then, the ketones were transformed
into the (Z)-α-bromoderivative (4a-d) by a one-pot two
steps procedure: i) stereospecific addition of bromine at
low temperature (-50 °C), ii) followed by in situ elimina-
tion with NEt3 affording the products in fairly good yields
(60-70%) and with a good de (Z/E, >97:3).14 Remarkably,
compound 4b was isolated by crystallization in hex-
ane/Et2O (9:1, -30°C), whereas the other bromoderivatives
In the last few years several one-pot multienzymatic
systems7,8 have been investigated. In relation to the
chemoselectivity of the enzymes involved, the latter can
be added to the reaction mixture either step by step or
simultaneously.
In particular, systems based on the combination of an
enzyme with ene-reductase (ER) activity with a second
one with alcohol dehydrogenase (ADH) activity, have
proven to be extremely efficient9 (Scheme 1). Especially,
these systems are well suited for the stereoselective re-
duction of both functionalities of prochiral α,β-
unsaturated ketones/aldehydes: the C=C double bond by
ERs and the CO carbonyl group by ADHs.
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(4a and 4c-d) required column chromatography purifica-
tion.
Thus, the C=C double bond reduction of the substrates
4a-d was tested with a set of different recombinant ERs
belonging to the Old Yellow Enzymes family15 (OYE1-3),
using for the NADPH cofactor regeneration a glucose de-
hydrogenase16 (GDH) and glucose as sacrificial co-
substrate. The results and the experimental conditions are
summarized in Table 1.
shown that OYE3 was the ER with the highest catalytic
1
2
3
4
5
6
7
8
activity (entry 5 vs 4 and 6), but a significant decrease of
conversions was observed. A good compromise between
conversion and enzyme consumption was reached by us-
ing 65 ꢀg/mL of OYE3 (entry 7), similar results were
achieved with 4a-b (data not shown). The stereochemical
configuration of the products (vide infra) was assigned
(S), in agreement with a flipped binding mode of the sub-
strate into the catalytic site of the protein.18 Unfortunately
any attempt to determinate the ee by chiral GC or HPLC
was unsuccessful.
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Table 1. ER catalyzed reduction of (Z)-4a-d.a
However, at the end of each biotransformation together
to the α-bromoketone product (5a-c) and to the unreact-
ing (E) diastereoisomer19 ((E)-4a-c) a small amount of α-
hydroxyketone, i.e. 6a-c (detected by GC-MS, Table 1),
was always present. This result can be related to the fact
that, even in such very mild experimental conditions it is
not uncommon to have side reactions for halocarbonyl
derivatives. For instance Faber et al. have shown that
2,3,3-trihaloesters, products of the ER catalyzed reduction
of trihaloderivatives of the methyl acrylic ester, undergo
to complete and spontaneous elimination of HX at neu-
tral pH and at 30 °C.20 In our case, we hypothesized that
as soon the product is formed during the biotransfor-
mation, a nucleophilic displacement of the brom0 sub-
stituent21 with water could occur to give the correspond-
ing hydroxyl ketone. This hypothesis was confirmed by
comparing the GC retention time of a sample of 6c on
purpose synthetized (see SI). The product/side-product
ratio was around 1:10, independently of the protective
group and to the type of ER used. Thus, even if it is well
known that the ERs of OYE family exhibit their highest
activity at neutral pH,22 we repeated the reductions at
lower pH values with the aim of minimizing the hydroly-
sis, since these reactions are usually carried out in pres-
ence of inorganic bases. By performing the reactions at
pH=5.2 and pH=4.0 (entries 9 and 10, respectively), a
gradual increase of the 5c/6c ratio was observed, but this
fact was accompanied by a corresponding decrease of the
conversions. Eventually, the best results on preparative
scale (≈ 1 mmol) in terms of conversion, enzyme con-
sumption and side-product formation were achieved at
pH=6.0 (entry 11). Under these experimental conditions,
both 5b and 5c could be isolated after column chromatog-
raphy purification in a good yield (around 85%). In the
case of the O-Ac protected ketone the yield was lower
(around 53%), because the higher hydrophilicity and vola-
tility of 5a made its isolation more difficult and therefore
not quantitative.
Entry
R
ER
pH
Conv.[b]
Ratio[b]
5/6
(%)
1
2
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Ac
Bz
OYE1
OYE2
OYE3
OYE1
OYE2
OYE3
OYE3
OYE2
OYE3
OYE3
OYE3
OYE3
OYE3
OYE3
OYE3
OYE3
7.0
7.0
7.0
7.0
7.0
7.0
7.0
6.0
5.2
4.0
6.0
6.0
6.0
6.0
6.0
6.0
>99
>99
88:12
90:10
87:13
88:12
90:10
87:13
85:15
94:6
94:6
96:4
93:7
94:6
96:4
_
3
>99
4
20 [c]
16 [c]
5
6
7
24 [c]
>99 [d]
>99 [d]
88 [d]
73 [d]
85 [e],[f]
95
8
9
10
11
12
13
>99
[g]
14 SiEt3
_
15
16
Ac
Bz
53 [d],[f]
86 [d],[f]
93:7
96:4
(a) Typical experimental conditions of the ER screening on
1 mL scale: substrate conc. 5 mM, 1 % DMSO co-solvent, ER:
100 ꢀg/mL, GDH 5U, 0.1 mM NADP+, 20 mM glucose, pH=7
phosphate buffer 50 mM, 30 °C, reaction time: 1 day, 180 rpm;
(b) By GC-MS; (c) ER: 12 ꢀg/mL; (d) ER: 65 ꢀg/mL; (e) Yield
after column chromatography; (f) On a preparative scale: 1
mmol of substrate; (g) No conversion.
First of all, substrates 4a-c are very well accepted by all
tested ERs, since the conversions using the standard
amount of enzyme (≈100 ꢀg/mL for a 5 mM concentration
of substrate) were quantitative (entries 2-12-13) inde-
pendently of the ER used (for instance entries 1-3 for 4a).
An exception was ketone 4d that did not react at all (en-
try 14), this result being likely due to the presence of the
more sterically demanding -OSiEt3 protective group.17
Then, in view of a reaction scale-up we optimized the
amount of ER to be added, using 4c as model compound.
By lowering the amount of enzyme to 12 ꢀg/mL, it was
Next, we focused our attention on the ADH catalyzed
reduction of the carbonyl group, which for methyl ke-
tones, quite often proceeds with a very high stereoselec-
tivity.6 A panel of commercially available ADHs was
screened, while for the NAD(P)H cofactors regeneration
we used the same system employed in the biotransfor-
mations catalyzed by the ERs.
As most of the screened ADHs did not reduce the car-
bonyl group of the α,β-unsaturated ketones 4a-c (see Ta-
ble S1 of SI), this very high chemoselectivity8a,9b-c opens
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the possibility to couple the two biocatalyzed reactions in duced by the same ER but affording the products with a
much higher stereoselectivity (ees >99);23 the stability of
these ketones at the typical biotransformation conditions
is likely due to the lower acidity of the proton in α to the
carbonyl group relative to the corresponding proton of
the α-bromo ketones. Thus, by coupling the OYE3 cata-
lyzed reductive step with that of an ADH in a cascade
process, in principle, it should be possible to minimize
the racemization by converting the ketone, as soon it is
formed, into the more stable alcohol. Moreover, in this
case the application of a cascade process was facilitated
by the fact that the selected ADHs (EVO030 and EVO270)
were completely chemoselective towards the saturated
ketones, thus avoiding the detrimental formation of the
allylic alcohol side-product.
1
2
3
4
5
6
7
8
a one-pot cascade process. The ADHs stereoselectivity
was preliminarily tested on the model compound (3S)-5c.
Conversion and diastereoselectivity are summarized in
Table 2. Three ADHs showing pro (S) stereoselectivity
were identified (entries 1-3), but the diastereomeric excess
of the syn-bromohydrin, i.e. (2S,3S)-7c, were not optimal,
since in the best case we obtained a modest de=38% with
EVO030. At this stage, because we did not know the opti-
cal purity of (S)-5c, we were not able to attribute these
low de values neither to a low stereoselectivity of the
OYE3 catalyzed step nor to the ADH mediated reduction
of the CO group. With the pro (R) ADHs (entries 4-7) the
diastereoselectivity was higher, but still not satisfactory,
indeed the best result was achieved with EVO270 (entry
4, de=72%). Finally, we tested the most performing pro (S)
and pro (R) ADH, i.e. EVO030 and EVO270 respectively,
with the other two substrates 5a-b (entries 8-11, Table 2),
the results in terms of selectivity and conversion being
consistent with those obtained with 5c.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
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60
Table 3. Multienzymatic cascade reduction of (Z)-4b-c.a
Table 2. ADHs catalyzed reduction of (3S)-5a-c.a
Entry
R
ADH
ADH/OYE3 Conv.[b] Syn [c] Anti [c]
(%)
(%)
(%)
1
Bn EVO030
Bn EVO030
Bn EVO030
Bn EVO030
Bn EVO270
Bn EVO270
Bn EVO270
Bn EVO270
Bz EVO030
1:1
3:1
96.3
93.3
88
98
99
98
0.3
0.5
0.5
1
12
2
2
3
4
5
6
7
8
9
Entry
R
ADH
Conv.[b]
(%)
Syn [c]
(%)
Anti [c]
(%)
6:1
99.6
89.1 [d]
1
3:1
2
1
2
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Ac
Bz
Bz
Bz
CPADH
DRADH
EVO030
EVO270
EVO420
KRED
50.1
60.7
62.9
68.9
14.4
22.6
20.8
17.8
39.3
37.1
2:1
97.2
99.7
99.5
99.5
99
1
66.9
99.3
93.3
99.6
99.1
1:1
99.3
3
31.1
0.5:1
0.5:1
3:1
99.3
90.3 [d]
4
5
85.6
77.4
79.2
82.1
99.1
99
99
1
6
7
10 Bz EVO030
11 Bz EVO270
3:1
87.4 [d]
90.3 [d]
1
PLADH
EVO030
EVO030
EVO270
EVO420
97.2
99.3
98.6
98.2
93.7
0.5:1
99
8
9
10
11
61.9
65.9
15.8
38.1
(a) Typical experimental conditions of the OYE3+ADH
screening on 1 mL scale: substrate conc. 5 mM, 1 % DMSO
co-solvent, OYE3: 65 ꢀg/mL, ADH: see the Table, GDH 5U,
0.1 mM NAD++0.1 mM NADP+, 20 mM glucose, pH= 7 phos-
phate buffer 50 mM, 30 °C, reaction time: 12 hr, 180 rpm; (b)
By GC-MS; (c) By GC-MS: the bromohydrins syn and anti
were transformed into the O-silyl derivatives by adding N-
methyl-N-(trimethylsilyl) trifluoroacetamide 12 hr before of
GC-MS analysis; (d) The reaction was repeated on a prepara-
tive scale (2 mmol).
34.1
84.2
79.6
20.4
(a) Typical experimental conditions of the ADH screening
on 1 mL scale: substrate conc. 5 mM, 1 % DMSO co-solvent,
ADH: 200 ꢀg/mL, GDH 5U, 0.1 mM NAD++0.1 mM NADP+,
20 mM glucose, pH=7 phosphate buffer 50 mM, 30 °C, reac-
tion time: 12 h, 180 rpm; (b) By GC-MS; (c) By GC-MS, since
the bromohydrins syn and anti are not well separated, were
transformed into the O-silyl derivatives by adding N-methyl-
N-(trimethylsilyl)trifluoroacetamide 12 hr before of GC analy-
sis.
In the study of coupled reactions, we focused our atten-
tion on substrates (E)-4b-c, those with the orthogonal
protective groups and best conversions. The multienzy-
matic cascade processes were first carried out on the
screening scale, as described before; conversion and dia-
stereoselectivity are reported in Table 3. The main param-
eter to be optimized was the ADH/OYE3 ratio. First, the
cascade reduction of 4c with OYE3 and EVO030 in a 1:1
ratio afforded the syn-7c (entry 1) with a much higher dia-
Even if the preliminary results were not encouraging,
we thought that the low de values might be ascribed
mainly to a partial racemization of the α-bromoketone
5a-b rather than to a low stereoselectivity of OYE3, since
similar α-alkyl substituted methylenones have been re-
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the most pleasant and characterizing odor between all
stereoselectivity with respect to the sequential procedure
1
2
3
4
5
6
7
(de=76% vs de=38%). Subsequently, by increasing the ra-
tio to 3:1 and then to 6:1, the de value further increased up
to 98% (entries 2-3). These results overall confirmed that
OYE3 reduces these substrates with a high enantioselec-
tivity, but the optical purity of the products is partially
lost by means of a racemization occurring during the bio-
transformation.24
possible stereoisomers of 3. In contrast, both enantiomers
of cis-3 diastereoisomer impart to the food an unpleasant
sulfur note.
8
9
Then, we repeated the cascade process with the enantio
complementary ADH, i.e. EVO270. The 1:1 enzymes ratio was
already good since we obtained the anti-7c with an excellent
de=99% (entry 6), the same outcome being achieved even
when further reducing the respective amount of EVO270
(entry 7). Thus, with the optimized experimental conditions,
we scaled-up the reactions (around 2 mmol of starting mate-
rial) affording the bromohydrins syn-7b, anti-7b, syn-7c and
anti-7c after column chromatography purification in quite
good yields (over 80%) and with almost the same selectivity
observed on the analytical scale (entries 4, 8 and 10-11). Final-
ly, together to a lower extent of product racemization, we
observed that also the formation of the side product 6b-c
diminished as well to almost negligible amounts.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Lastly, in order to accomplish our synthetic plan, the
deprotection protocol of (3S,4S)-6b was investigated, the
classical approach by alkaline hydrolysis being unfeasible
for the presence of the bromohydryn functionality. Even-
tually, the enzymatic hydrolysis of the benzoate25 ester
was performed at neutral pH by adding the lipase from
Candida rugosa to the reaction mixture at the end of the
cascade process26 catalyzed by OYE3+EVO030. Thanks to
this further enzymatic step, it was possible to obtain the
diol (3S,4S)-8 in an overall yield of 77% (Scheme 3) with-
out any column purification.
Scheme 4. Synthesis of (2S,3S)-3 and (2S,3R)-3.
Thus, with (2S,3S)-7c and (3S,4S)-8 in our hands we
prepared the stereoisomers (2S,3S)-3 and (2S,3R)-3 of the
roasted meat flavor (Scheme 4). First, the bromohydrin
(2S,3S)-7c was converted into the epoxide (2R,3S)-9 with
NaH in THF, with the same procedure but starting from
anti-7c the epoxide (2R,3R)-9 was obtained as well. Then
the OBn protective group of (2R,3S)-9 was removed by
hydrogenolysis with Pd/C affording the epoxyalcohol
(2R,3S)-10. The latter, was rearranged in presence of a
substoichiometric amount of camphorsulfonic acid (CSA)
to give the less strained tetrahydrofuranol (2S,3R)-2, how-
ever this compound was not isolated due to his high vola-
tility, but it was promptly converted into the mesyl deriv-
ative (2S,3R)-11 in an overall yield of 64%. The rearrange-
ment of β-hydroxy epoxides to tetrahydrofuran-2-ols pro-
ceeds with inversion of stereochemistry at C(3) stereocen-
tre of the epoxide ring; to our knowledge just few cata-
lysts have been studied (MgI2 and BF3·OEt2)28,29 compared
to the extensive investigations that have been carried out
for the rearrangement of γ-hydroxy epoxide homo-
logues.30 Thus, we studied this rearrangement on a model
compound (see Table S2 of SI); between all reagents test-
ed the CSA gave the best results. Finally, the mesyl deriva-
tive 11 was transformed into (2S,3S)-3 by SN2 with AcSK in
DMF,2b with an excellent ee=99.6% (by chiral GC), which
Scheme 3. Coupling of OYE3+EVO030 cascade with the hy-
drolysis step catalyzed by the lipase from Candida rugosa in
the one-pot synthesis of (2S,3S)-8 from 4b.
Stereoselective synthesis of the roasted meat aro-
ma. The 2-methyl-tetrahydrofuran-3-thioacetate 3 is one
of the main aromas of the roasted meat, and it is pro-
duced in the racemic form during cooking process by a
Maillard reaction. Quite often chiral fragrances and fla-
vors exhibit different odor properties in function of the
relative and absolute stereochemical configuration.27 Re-
cently, an organoleptic study2b has identified (2S,3R)-3 as
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Page 6 of 11
where required the solvents were dried over molecular sieves
confirmed the high stereoselectivity of the cascade pro-
cess.
Concerning the second stereoisomer, i.e. (2S,3R)-3, its
1
(4 Å). H and 13C NMR spectra were recorded on 400 or 500
1
2
MHz spectrometer at room temperature, using TMS as an
1
3
4
5
6
7
8
9
internal standard for H and CDCl3 for 13C; chemical shifts δ
synthesis was more straightforward. The diol (3S,4S)-8
was regioselectively tosylated in pyridine at 0 °C and the
tosyl intermediate cyclized in situ affording the bromo
derivative (2S,3S)-1 in an overall yield of 54% and with an
excellent de>97% (by GC-MS, see SI). The almost com-
plete conservation of the high diastereomeric excess of
the starting material syn-8 into the final product shows
that the tosylation28 of the primary alcohol proceeds with
a remarkable regioselectivity. Then, the bromoderivative
2 was converted into (2S,3R)-3 modifying the procedure
adopted for the SN2 of 11. Indeed, accidentally during the
bulb to bulb distillation of (2S,3S)-1 at high temperature
(110 °C, 15 mmHg) we found that the product isomerized31
are expressed in ppm relative to the reference. High-
resolution MS spectra were recorded with a Q-TOF mass
spectrometer, equipped with an ESI source. The GC-MS
analyses of all compounds were performed on a column with
a low polarity stationary phase (30 m x 0.25 mm x 0.25 ꢀm).
Program temperature: 60 °C (1 min)/6 °C min−1/150 °C (1
min)/12 °C min−1/280 °C (5 min). For the very volatile com-
pounds 2 and 10 was used a different method: 38 °C (9 min) /
3 °C min–1 / 90 °C (1 min) / 6 °C min–1 / 180 °C (1 min) / 15 °C
min–1 / 280 °C (5 min). The enantiomeric excess values (ee) of
compounds 3 were determined by chiral GC analysis (column
DactButilSililBeta CDX) with a program temperature: 60 °C /
1 °C min-1/95 °C/90 °C min-1 / 220 °C (2 min). TLC analyses
were performed on pre-coated silica gel 60 F254 plates, and
spots were visualized either by UV light (254 nm) or by
spraying with phosphomolybdic acid reagent. All chromato-
graphic separations were carried out on silica gel columns
(230−400 mesh). Optical rotations were determined on a dig-
ital automatic polarimeter at 589 nm (sodium D line) and are
given at rt in °cm3 g–1 dm–1.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
to the trans diastereoisomer (cis/trans 1:1 by H NMR, see
SI) and it decomposed as well. Thus, in order to avoid
these side reactions during the nucleophilic substitution
with AcSK, it is crucial to carry out the reaction at tem-
perature below 100 °C in DMSO and in presence of diben-
zo-18 crown 6.
The O-protected allylic alcohols (OAc, OBz, OBn and OSiEt3,
respectively) for the cross metathesis were prepared follow-
ing reported procedures.33
CONCLUSION
Enzymes and Strains. OYE1 from Saccharomyces pastoria-
nus, OYE2-3 from Saccharomyces cerevisiae and GDH from
Bacillus megaterium were overexpressed in E. coli BL21 (DE3)
strains harboring a specific plasmid, according to standard
molecular biology techniques as described in ref. [16]. Pro-
tein concentrations were determined according to Bradford
test, using bovine serum albumine (BSA) as a standard.
CPADH from Candida parapsilosis was purchased from Julich
Chiral Solutions GmbH (now CODEXIS). PLADH from Par-
vibaculum lavamentivorans, DRADH from Deinococcus radi-
odurans, KRED (ketoreductase) from an unspecified source
and lipase from Candida rugosa were purchased from Sigma-
Aldrich. EVO420, EVO270 and EVO030 from an unspecified
source were purchased from Evocatal GmbH and used with-
out further purifications.
The integration of at least two or more transformations
in a multienzymatic cascade process is particularly ap-
pealing, since the enzymes, typically operating at very
similar conditions, are intrinsically more compatible to
this strategy than other type of catalysis. In this work we
demonstrated that the one-pot multienzymatic cascade
process, encompassing four different enzymes (ER, ADH,
GDH and lipase) compares better in terms of stereoselec-
tivity and side-products formation with respect to the
one-pot enzyme sequential addition procedure.
Besides, one of the main drawbacks of the ERs based
catalysis is the lack of wild-type enzymes with opposite
stereoselectivity, and even if the C=C reduction is stereo-
specific, it is not always possible to prepare a regioisomer
or diastereoisomer of the starting material that once re-
duced gives the opposite enantiomer (substrate engineer-
ing9g strategy). The generation of ER variants showing
opposite stereoselectivity by protein engineering using
either a (semi)-rational32a,b or a directed evolution32c ap-
proach has permitted in some cases to overcome this lim-
itation. In this case we by-passed this problem by devising
a diastereodivergent synthesis of synthons 1 and 2. Thus,
since differently from the ERs there is large availability of
ADHs with either a pro (S) or pro (R) enantioselectivity,
by means of our synthetic route is possible to have full
access to all stereoisomers of any 2-methyl-3-substituted
tetrahydrofuran. In this regard we prepared the most
pleasant roasted meat aroma (2S,3R)-2-methyl-3-
thioacetate tetrahydrofuran in an overall yield of 17% and
with a diastereoselectivity of 96%.
General Procedure for the Preparation of Compounds
3a-d via Cross-Metathesis. To a well stirred solution of O-
protected allylic alcohol (17 mmol), freshly distilled methyl
vinyl ketone (4.8 g, 68 mmol) and CuI (96 mg, 0.51 mmol) in
anhydrous and degassed Et2O (85 mL) at 35 °C under a N2
atmosphere was added the 2nd gen. Grubbs’ catalyst (144 mg,
0.17 mmol). After 24 hr the reaction was directly concentrat-
ed under vacuum and purified by column chromatography
(eluent: hexane/AcOEt, 9:1) giving the product as a liquid/oil.
(E)-4-Oxopent-2-en-1-yl acetate (3a):10 liquid, 1.64 g, yield
1
68%; tr=9.43 min 99% purity by GC; H NMR (400 MHz,
CDCl3)
δ 6.76 (dt, J=4.7 and 16.1 Hz, 1H), 6.26 (dt, J=1.9 and
16.1 Hz, 1H), 4.77 (dd, J=1.9 and 4.3 Hz, 2H), 2.29 (s, 3H), 2.13
(s, 3H); 13C NMR (100 MHz, CDCl3)
δ 197.7, 170.3, 139.8, 131.0,
62.6, 27.3, 20.7; GC-MS: m/z (%) 100 (M+-42, 40), 83 (50), 71
(20), 43 (100).
(E)-4-Oxopent-2-en-1-yl benzoate (3b): oil, 2.53 g, yield
1
73%; tr=22.86 min 99% purity by GC; H NMR (400 MHz,
Experimental Section
General Remarks. Chemicals and solvents were purchased
CDCl3)
δ 8.09 (dt, J=1.4 and 7.1 Hz, 2H), 7.60 (m, 1H), 7.47 (t,
J=7.8 Hz, 2H), 6.88 (dt, J=4.6 and 16.1 Hz, 1H), 6.36 (dt, J=1.9
and 16.1 Hz, 1H), 5.02 (dd, J=1.9 and 4.6 Hz, 2H), 2.30 (s, 3H);
from suppliers and used without further purification, while
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The Journal of Organic Chemistry
13C NMR (100 MHz, CDCl3)
δ 197.7, 165.9, 140.0, 133.4, 131.0,
cording to Table 1) containing glucose (4 equiv. with respect
to 4a-d), NADP+ (0.1 mM), GDH (5 U mL−1), and an OYE1-3
(12÷100 μg mL−1, according to Table 1). The mixture was
stirred for 24 h in an orbital shaker (180 rpm, 30 °C). The so-
lution was extracted with EtOAc (3 x 0.5 mL), centrifuged
(15000 g, 1.5 min). The combined organic phase was dried
over Na2SO4 and analyzed by GC-MS, in the following we re-
port the GC-MS of 6a-c. Conversions are reported in Table 1.
3-Hydroxy-4-oxopentyl acetate (6a): tr=11.12 min, GC-MS:
m/z (%) 142 (M+-18, 3), 118 (3), 43 (100).
129.7, 129.5, 128.53, 63.1, 27.3; GC-MS: m/z (%) 203 (M+-1, 1),
159 (2), 144 (2), 122 (2), 105 (100).
1
2
3
4
5
6
7
8
(E)-5-(Benzyloxy)-pent-3-en-2-one (3c):34 oil, 2.65 g, yield
82%; tr = 19.76 min 99% purity by GC; 1H NMR (400 MHz,
CDCl3)
δ 7.35 (m, 5H), 6.79 (dt, J=4.6, 9.1 and 16.2 Hz, 1H),
6.34 (dt, J = 1.6, 3.3 and 13.2 Hz, 1H), 4.58 (s, 2H), 4.20 (dd,
J=1.8 and 4.5 Hz, 2H), 2.26 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ
197.9, 142.9, 137.8, 130.4, 128.5, 127.9, 127.7, 73.0, 68.9, 27.2;
GC-MS: m/z (%) 190 (M+, 1), 160 (5), 145 (10).
9
3-Hydroxy-4-oxopentyl benzoate (6b): tr=22.00 min, GC-
MS: m/z (%) 205 (M+-18, 3), 163 (3), 122 (80).
(E)-5-((Triethylsilyl)oxy)pent-3-en-2-one (3d): liquid, 2.90
g, yield 80%; tr=15.69 min 98% purity by GC; 1H NMR (400
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5-(Benzyloxy)-3-hydroxypentan-2-one (6c): tr=20.59 min,
GC-MS: m/z (%) 190 (M+-18, 3), 162 (19), 122 (80).
MHz, CDCl3)
and 15.8 Hz, 1H), 4.37 (dd, J=2.1 and 3.7 Hz, 2H), 2.27 (s, 3H),
0.97 (t, J=8.0, 9H), 0.62 (m, 6H); 13C NMR (100 MHz, CDCl3)
δ 6.83 (dt, J=3.7 and 15.8 Hz, 1H), 6.35 (dt, J=2.1
General Procedure for the Preparation of α-Bromo Ke-
tone (S)-5a-c with OYE3. The substrate 4a-c (1 mmol) dis-
solved in DMSO (1 mL, final 5 mM substrate, 0.5% DMSO
cosolvent) was added to a KPi buffer solution (200 mL, 50
mM, pH 6.00) containing glucose (4 equiv with respect to
4a-c), NADP+(0.1 mM), GDH (5 U mL−1), and OYE3 (65 μg
mL−1). The mixture was stirred for 24 h at room temperature.
The solution was extracted with EtOAc (3 x 50 mL), the or-
ganic phase was dried over Na2SO4, concentrated under re-
duced pressure and the crude material was submitted to col-
umn chromatography purification (eluent: gradient hex-
ane/AcOEt from 9:1).
δ
198.4, 146.1, 128.9, 61. 9, 27.3, 6.7, 4.4; GC-MS: m/z (%) 214
(M+, 3), 185 (75), 157 (40).
General Procedure for the Preparation of Compounds
4a-d. To a well stirred solution of 3a-d (9.6 mmol) in CH2Cl2
(50 mL) at -50 °C was added drop-wise a solution of Br2 (1.54
g, 9.7 mmol) in CH2Cl2 (15 mL) over 1 h under a N2 atmos-
phere. After complete consumption (checked by TLC) of the
reactant some drops of 1-hexene were added to remove the
excess of Br2. To the reaction mixture at -30°C was added
dropwise NEt3 (0.99 g, 9.8 mmol) and then left to reach 0 °C.
The mixture was washed with HCl (0.2 M, 50 mL) and brine
(sat., 50 mL), dried over Na2SO4 and concentrated under re-
duce pressure. The crude material of products of 4a and 4c-d
were subjected to column chromatography purification (gra-
dient: hexane/AcOEt from 9:1), whereas 4b was crystalized in
hexane/Et2O (9:1; -30°C).
(S)-3-Bromo-4-oxopentyl acetate ((S)-5a): 118 mg, yield
56%; tr=12.45 min 97% purity by GC; [α]D = -1.14° (c 1.40,
CH2Cl2); 1H NMR (400 MHz, CDCl3)
δ
4.39 (dd, J=5.9 and 8.4
Hz, 1H), 4.20 (m, 2H), 2.39 (m, 1H), 2.39 (s, 3H), 2.20 (m, 1H),
2.05 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ 200.9, 170.7, 61.6,
49.9, 32.4, 26.7, 20.8; GC-MS: m/z (%) 182 (M+-41, 20), 180
(20), 164 (25), 162 (25),138 (4), 136 (4); HRMS (ESI) calcd for
C7H11BrNaO3+ [M+Na]+ 244.9784, found 244.9793.
(Z)-3-Bromo-4-oxopent-2-en-1-yl acetate (4a): yellow oil,
1
1.29 g, yield 61%; tr=13.94 min 97% purity by GC; H NMR
(400 MHz, CDCl3)
δ 7.19 (t, J=4.8 Hz, 1H), 4.86 (d, J=5.2 Hz,
2H), 2.48 (s, 3H), 2.13 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ
(S)-3-Bromo-4-oxopentyl benzoate ((S)-5b): 285 mg, yield
86%; tr=22.89 min 99% purity by GC; [α]D = +1.34° (c 0.56,
190.9, 170.5, 139.3, 126.9, 64.0, 26.2, 20.7; GC-MS: m/z (%) 180
(M+-41, 25), 178 (25), 163 (35), 161 (35),149 (30), 151 (30).
(Z)-3-Bromo-4-oxopent-2-en-1-yl benzoate (4b): yellow–
1
CH2Cl2); H NMR (400 MHz, CDCl3)
δ 8.02 (m, 2H), 7.58 (m,
1H), 7.45 (m, 2H), 4.47 (m, 3H), 2.56 (m, 1H), 2.41 (s, 3H), 2.35
(m, 1H); 13C NMR (101 MHz, CDCl3)
δ 200.9, 166.4, 133.2, 129.8,
white solid, 2.03 g, yield 75%; tr=22.86 min 98% purity by
129.6, 128.5, 62.1, 49. 9, 32.6, 26.8; GC-MS: m/z (%) 244 (M+-
41, 5), 242 (5), 205 (5), 163 (15); HRMS (ESI) calcd for
C12H13BrNaO3+ [M+Na]+ 306.9940, found 306.9948.
1
GC; H NMR (400 MHz, CDCl3)
δ 8.07 (dt, J=1.4 and 7.1 Hz,
2H), 7.60 (m, 1H), 7.47 (m, 2H), 7.32 (t, J=5.2 Hz, 1H), 5.12 (d,
J=4.8 Hz, 2H), 2.50 (s, 3H); 13C NMR (101 MHz, CDCl3)
δ
(S)-5-(Benzyloxy)-3-bromopentan-2-one ((S)-5c): 230 mg,
yield 85%; tr = 21.38 min 99% purity by GC; [α]D = -114° (c
191.00, 166.1, 139.4, 133.5, 129.8, 129.3, 128.5, 127.01, 64.5, 26.2;
GC-MS: m/z (%) 283 (M+-1, 1), 203 (10), 179 (1), 161 (15), 105
(100).
1.02, CH2Cl2); 1H NMR (400 MHz, CDCl3)
δ
7.32 (m, 5H), 4.56
(m, 1H), 4.49 (s, 2H), 3.61 (m, 2H), 2.39 (s, 1H), 2.35 (m, 4H),
2.15 (m, 1H); 13C NMR (125 MHz, CDCl3)
201.4, 138.0, 129.4,
(Z)-5-(Benzyloxy)-3-bromopent-3-en-2-one (4c): yellow
1
δ
oil, 1.56 g, yield 60%; tr = 22.36 min 99% purity by GC; H
127.7, 127.7, 73.2, 67.0, 51.0, 33.8, 26.5; GC-MS: m/z (%) 244
(1), 163 (2), 131 (55), 91 (99); HRMS (ESI) calcd for
C12H15BrNaO2+ [M+Na]+ 293.0148, found 293.0152.
NMR (400 MHz, CDCl3)
δ 7.34 (m, 5H), 7.23 (dd, J = 4.9, 9.8
Hz, 1H), 4.56 (s, 2H), 4.33 (d, J = 4.9, 2H), 2.41 (s, 3H); 13C
NMR (100 MHz, CDCl3)
δ 190.6, 143.0, 137.5, 128.5, 128.0,
127.9, 126.0, 73.3, 70.4, 25.8; GC-MS: m/z (%) 269 (M++1, 1),
252 (M+-18, 2), 162 (4), 91 (99).
General Procedure for the Screening-Scale of the ADH
Mediated Reduction. The substrate 5a-c (5 µmol) dissolved
in DMSO (10 µL, final 5 mM substrate, 1% DMSO cosolvent)
was added to a KPi buffer solution (0.99 mL, 50 mM, pH 7.0)
containing glucose (4 equiv. with respect to 5a-c), NAD+ and
NADP+ (0.1 mM each), GDH (5 U mL−1), and an ADH (200 μg
mL−1, according to Table 2). The mixture was stirred for 12 h
in an orbital shaker (180 rpm, 30 °C). The work-up was the
same used for the OYEs screening. One day before of the GC-
MS analysis the sample was derivatized by addition of N-
(Z)-3-Bromo-5-((triethylsilyl)oxy)pent-3-en-2-one (4d):
yellow oil, 1.13 g, yield 40%; tr=19.24 min 99% purity by GC;
1H NMR (400 MHz, CDCl3)
δ
7.26 (d, J = 4.6, 1H), 4.47 (d, J =
4.6 Hz, 1H), 2.47 (s, 3H), 0.98 (t, J = 8.1, 9H), 0.65 (q, J = 8.1,
6H); 13C NMR (100 MHz, CDCl3)
190.9, 146.5, 124.4, 63.7,
δ
25.9, 6.5, 4.3; GC-MS: m/z (%) 292 (M+, 1), 265 (40), 237 (20).
General Procedure for the Screening of the OYE Medi-
ated Reduction. The substrate 4a-d (5 µmol) dissolved in
DMSO (10 µL, final 5 mM substrate, 1% DMSO cosolvent)
was added to a KPi buffer solution (0.99 mL, 50 mM, pH ac-
methyl-N-(trimethylsilyl)trifluoroacetamide.
Conversions
and diastereoselectivities are reported in Table 2.
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General Procedure for the Screening of the OYE3+ADHs
(3S,4S)-3-bromopentane-1,4-diol ((3S,4S)-8). To the reac-
1
2
3
4
5
6
7
8
Cascade Reduction. The substrate 4b-c (5 µmol) dissolved
in DMSO (10 µL, final 5 mM substrate, 1% DMSO cosolvent)
was added to a KPi buffer solution (0.99 mL, 50 mM, pH 7.0)
containing glucose (4 equiv with respect to 4b-c), NAD+ and
NADP+ (0.1 mM each), GDH (5 U mL−1), OYE3 (65 μg mL−1)
and an ADH (32÷390 μg mL−1, according to the ratio indicat-
ed in Table 3). The work-up and the GC-MS analysis are the
same used for ADHs screening. Conversions and diastereose-
lectivities are reported in Table 3.
General Procedure for the Preparation of Bromohydrin
7b-c by OYE3+ADHs Cascade Reduction. The substrate
4b-c (2 mmol) dissolved in DMSO (1 mL, final 10 mM sub-
strate, 0.5% DMSO cosolvent) was added to a KPi buffer solu-
tion (200 mL, 50 mM, pH 7) containing glucose (8 mmol),
NAD+ or NADP+ (0.1 mM according to ADH dependence),
GDH (5 U mL−1, 2000 U), ADH (see Table 3) and OYE3 (65 μg
mL−1). The mixture was stirred for 1 day at rt. The reaction
mixture was extracted with EtOAc (3x50 mL), the organic
phase was dried over Na2SO4, concentrated under reduced
pressure and submitted to column chromatography purifica-
tion.
tion mixture of the cascade process (OYE3+EVO030) for sub-
strate 4c was added lipase from Candida rugosa (30 mg), the
neutral pH was maintained with a pH controlled dosing
pump (1 M NaOH). After completeness of hydrolysis the so-
lution was washed with Et2O/hexane (1:1, 2x30 mL) and then
the aqueous phase was extracted with EtOAc (150 mL) in a
continuous liquid-liquid extractor apparatus, occasionally
the pH was adjusted to the neutrality. After 12 hr the organic
phase was dried over Na2SO4, concentrated under reduced
pressure to give the crude material of sufficient purity for the
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
1
next step. Overall yield 78%; oil, 281 mg, 94% purity by H-
1
NMR; [α]D = -23.1 ° (c 0.9, CH2Cl2); H NMR (400 MHz,
CDCl3)
2H), 2.15 (m, 2H), 1.31 (d, J=6.3 Hz, 3H); 13C NMR (101 MHz,
CDCl3) 70.2, 61.6, 60.2, 38.0, 21.1.
δ 4.26 (m, 1H), 3.89 (m, 1H), 3.81 (m, 2H), 2.92 (bs,
δ
General Procedure for the Ring-Closure of Bromohy-
drins 7c. To an ice cooled solution of 7c (1.0 mmol) in dry
THF (5 mL) was added NaH (oil suspension 60 %, 3.4 mmol
of NaH). The reaction mixture was left stirring at 0 °C for 2
hr; then, it was added Et2O (20 mL) and NH4Cl (sat., 30 mL).
The organic layer was washed several times with brine (sat., 3
x 20 mL) and NH4Cl (sat., 20 mL); after that, it was dried
over Na2SO4 and concentrated under reduced pressure. The
crude material was submitted to column chromatography
purification (eluent: gradient hexane/AcOEt, 99:1 → 7:3) to
give the epoxide as a colorless oil.
(3S,4S)-3-Bromo-4-hydroxypentyl benzoate (syn-7b): oil,
501 mg, yield=87.4%; [α]D = -52.9 ° (c 1.4, CH2Cl2); tr=23.63
min 95% purity by GC; de=98% by GC; 1H NMR (400 MHz,
CDCl3)
δ 8.03 (m, 2H), 7.57 (m, 1H), 7.44 (m, 2H), 4.66 (m,
1H), 4.48 (m, 1H), 4.19 (dt, J=4.1 and 10.1 Hz, 1H), 3.83 (bs, 1H),
2.38 (m, 2H), 2.15 (bs, 1H), 1.34 (d, J=6.2 Hz, 3H); 13C NMR
(2R,3S)-2-(2-(benzyloxy) ethyl)-3-methyloxirane ((2R,3S)-
(100 MHz, CDCl3)
δ
166.4, 133.1, 130.0, 129.6, 128.4, 70.5, 62.9,
9): yield 87%; 168 mg; tr = 18.69 min 99% purity by GC; [α]D
61.3, 34.7, 21.4; GC-MS: m/z (%) 244 (M+-43, 3), 242 (3), 189
1
= -11.0° (c 0.8, CH2Cl2); H NMR (500 MHz, CDCl3)
δ 7.32 (m,
(40), 163 (35); HRMS (ESI) calcd for C12H15BrNaO3 [M+Na]+
+
5H), 4.54 (s, 2H), 3.66 (dd, J = 5.8, 7.1 Hz, 2H), 3.06 (m, 2H),
1.88 (m, 2H), 1.28 (d, J = 5.5 Hz, 3H); 13C NMR (125 MHz,
309.0097, found 309.0102.
(3S,4R)-3-Bromo-4-hydroxypentyl benzoate (anti-7b): oil,
520 mg, yield=90%; [α]D = -50.8 ° (c 1.3, CH2Cl2); tr=23.78 min
CDCl3)
GC-MS: m/z (%) 191 (M+ -1, 1), 174 (M+ -18, 2), 159 (15), 91 (99).
(2R,3R)-2-(2-(Benzyloxy) ethyl)-3-methyloxirane
δ 138.5, 128.5, 127.7, 73.2, 67.7, 54.8, 52.6, 28.5, 13.5;
1
1
95% purity by GC; de=99% by H-NMR; H NMR (400 MHz,
CDCl3) 7.95 (d, J=7.2 Hz, 2H), 7.49 (t, J=7.2 Hz, 2H), 7.36 (t,
δ
((2R,3R)-9): yield 80%; 154 mg; tr = 18.48 min 99% purity by
1
J=7.2 Hz, 2H), 4.55 (m, 1H), 4.38 (m, 1H), 4.21 (dt, J=4.1 and
10.1 Hz, 1H), 3.85 (m, 1H), 2.85 (bs, 1H), 2.53 (s, 3H), 2.32 (m,
1H), 2.10 (m, 1H), 1.24 (d, J=6.2 Hz, 3H); 13C NMR (100 MHz,
GC; [α]D = 28.4° (c 0.84, CH2Cl2); H NMR (500 MHz, CDCl3)
δ
7.34 (m, 5H), 4.53 (s, 2H), 3.61 (m, 2H), 2.79 (m, 2H), 1.91
(m, 1H), 1.77 (m, 1H), 1.31 (d, J = 4.9 Hz, 3H); 13C NMR (125
CDCl3)
δ 166.4, 133.1, 130.0, 129.6, 128.4, 70.2, 62.6, 60.5, 32.8,
MHz, CDCl3)
δ 138.5, 128.5, 127.7, 73.2, 67.2, 57.5, 54.8, 32.7,
19.4; GC-MS: m/z (%) 244 (M+-43, 3), 242 (3), 189 (40), 163
17.8; GC-MS: m/z (%) 191 (M+ -1, 1), 174 (M+ -18, 2), 159 (15), 91
(99).
(35); HRMS (ESI) calcd for C12H15BrNaO3 [M+Na]+ 309.0097,
+
found 309.0105.
2-((2R,3S)-3-Methyloxiran-2-yl) ethanol ((2R,3S)-10). To a
stirred solution of (2R,3S)-9 (211 mg, 1.1 mmol) in Et2O (5 mL)
was added Pd/C (5% wt, 10 mg) under an H2 atmosphere and
it was left to stir at rt until complete absorption of H2. Thus,
the reaction mixture was filtered on a celite pad and concen-
trated slowly under reduced pressure. The crude material
was submitted to column chromatography purification (elu-
ent: gradient pentane/Et2O, 99:1 → 1:1) to give (2R,3S)-9 as a
colorless liquid. Yield 94%; 105 mg; tr = 16.26 min 99% purity
by GC; [α]D = +24.5° (c 1, CH2Cl2); 1H NMR (400 MHz, CDCl3)
(2S,3S)-5-(Benzyloxy)-3-bromopentan-2-ol (syn-7c): oil,
487 mg, yield=89%; tr = 22.12 min 99% purity by GC; [α]D = -
1
37.1° (c 1.6, CH2Cl2); de=98% by GC; H NMR (400 MHz,
CDCl3)
δ 7.34 (m, 5H), 4.53 (s, 2H), 4.25 (m, 1H), 3.68 (m,
3H), 2.29 (m, 3H), 1.30 (d, J = 6.2 Hz, 3H); 13C NMR (100 MHz,
CDCl3)
δ 138.0, 128.5, 127.8, 73.3, 70.0, 67.8, 62.3, 35.8, 21.4;
GC-MS: m/z (%) 272 (M+ -1, 1), 175 (5), 107 (45), 91 (99);
HRMS (ESI) calcd for C12H17BrNaO2 [M+Na]+ 295.0304,
+
found 295.0322.
(2R,3S)-5-(Benzyloxy)-3-bromopentan-2-ol (anti-7c): oil,
493 mg, yield=90.3%; tr = 22.42 min 99% purity by GC;
δ
3.87 (m, 2H), 3.08 (m, 2H), 1.78 (m, 3H), 1.29 (d, J = 5.4 Hz,
3H); 13C NMR (100 MHz, CDCl3)
δ 60.6, 55.0, 52.3, 30.3, 13.3;
1
GC-MS: m/z (%) 101 (M+ -1, 5), 71 (80), 57 (30), 45 (99).
(2S,3R)-2-Metyltetrahydrofuran-3-yl methanesulfonate
((2S,3R)-11).
de=98% by GC; [α]D = -29.0 ° (c 1.0, CH2Cl2); H NMR (400
MHz, CDCl3)
δ 7.34 (m, 5H), 4.54 (s, 2H), 4.31 (q, J = 5.7, 11.2
Hz, 1H), 3.92 (q, J = 5.5 and 11.1 Hz, 1H), 3.74 (m, 1H), 3.65 (m,
1H) 2.60 (d, J = 5.6 Hz, 1H), 2.15 (q, J = 5.6, 11.6 Hz, 2H), 1.30
To a stirred solution of CSA (47 mg, 0.2 mmol) in dry CH2Cl2
(2 mL) was added dropwisely the epoxyalcohol 10 (102 mg,
1.0 mmol) in dry CH2Cl2 (2 mL) at 28 °C. The reaction was
completed after 6 days. Due to the high volatility of the tet-
rahydrofuran (2S,3R)-2, the reaction mixture was used with-
out any work-up for the next step; tr = 11.76 min, purity 75%
(d, J = 6.3 Hz, 3H); 13C NMR (100 MHz, CDCl3)
δ 137.9, 128.5,
127.7, 73.4, 70.5, 67.5, 61.4, 34.0, 19.6; GC-MS: m/z (%) 272
(M+ -1, 1), 175 (5), 107 (45), 91 (99); HRMS (ESI) calcd for
C12H17BrNaO2+ [M+Na]+ 295.0304, found 295.0318.
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by GC; GC-MS: m/z (%) 102 (M+, 1), 87 (M+-15, 3), 69 (2), 57
trated under reduced pressure. The crude material was sub-
1
2
3
4
5
6
7
8
(99); we did not isolate it, since it is a very volatile product.
To the ice cooled stirred solution of 2 was added NEt3 (202
mg, 2.0 mmol), methanesulfonyl chloride (149 mg, 1.3 mmol)
and it was left stirring overnight at rt. After 1 day, to the reac-
tion mixture was added water (100 mg), then it was concen-
trated under reduced pressure and the crude material was
submitted to column chromatography purification (eluent:
gradient hexane/AcOEt, 8:2 → 1:1) to give the mesylate as a
mitted to column chromatography purification (eluent: gra-
dient hexane/AcOEt, 9:1→7:3) to give the product as a yellow
oil. Yield 49%; 47 mg; tr = 10.07 min 98% purity by GC;
de=97% by GC-MS, ee>99 % by chiral GC, tr 17.17 min; [α]D
-25.4 ° (c 0.78, CH2Cl2) Lett. [α]D = -20.5 ° (c 4.49, CHCl3, ref.
2a); 1H NMR (400 MHz, CDCl3)
3.97 (m, 1H), 3.79 (m, 2H),
3.53 (m, 1H), 2.48 (m, 1H), 2.34 (s, 3H), 1.86 (m, 1H), 1.28 (d, J
=
δ
= 6.1 Hz, 3H); 13C NMR (100 MHz, CDCl3)
δ 195.3, 78.0, 66.7,
46.7, 33.3, 30.6, 19.2; GC-MS: m/z (%) 159 (M+ -1, 1), 145 (M+ -
15, 1), 116 (10), 103 (4), 84 (80).
yellow oil. Yield 64%; 115 mg; tr = 13.15 min 99% purity by GC;
9
1
[α]D = -34.1° (c 0.92, CH2Cl2); H NMR (400 MHz, CDCl3)
δ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
4.81 (m, 1H), 4.11 (m, 1H), 4.01 (m, 1H), 3.89 (m, 1H), 3.02 (d, J
ASSOCIATED CONTENT
Supporting Information. All H and 13C NMR spectra, the
study of the epoxide/tetrahydrofuran isomerization on a
model compound and the chiral GC of compound 1. In addi-
tion the screening on the chemoselectivity of ADHs. This
material is available free of charge via the Internet at
http://pubs.acs.org.
= 1.4 Hz, 3H), 2.27 (m, 1H), 2.16 (m, 1H), 1.25 (dd, J = 1.9, 6.5
Hz, 3H); 13C NMR (100 MHz, CDCl3)
δ 85.1, 79,8, 66.4, 38.6,
1
32.6, 18.5; GC-MS: m/z (%) 165 (M+ -15, 5), 136 (5), 101 (50), 84
(50).
(2S,3S)-2-Methyltetrahydrofuran-3-thiol acetate ((2S,3S)-
3). A stirred solution of AcSK (171 mg, 1.5 mmol) and 12 (90
mg, 0.5 mmol) in dry DMF (2 mL) under a N2 atmosphere
was heated at reflux for 6 hr; then, it was added Et2O (5 mL).
The reaction mixture was filtered on a celite pad; the celite
was washed several times with Et2O (3x5 mL). Then, the
combined organic phase was washed with brine (sat., 5 mL x
3), dried over Na2SO4 and concentrated under reduced pres-
sure. The crude material was submitted to column chroma-
tography purification (eluent: gradient hexane/AcOEt,
9:1→7:3) to give the product as a yellow oil. Yield 70%; 56
mg; tr = 10.97 min 99% purity by GC; de>99.9 by GC,
ee=99.6% by chiral GC, tr 20.95 min; [α]D = -21.4 ° (c 0.78,
CH2Cl2) vs Lett. [α]D = -8.09 ° (c 4.37, CHCl3, ref. 2a); 1H NMR
AUTHOR INFORMATION
Corresponding Author
* E-mail: francesco.gatti@polimi.it F.G.G.
Author Contributions
The manuscript was written through contributions of all au-
thors. All authors have given approval to the final version of
the manuscript.
ACKNOWLEDGMENT
(400 MHz, CDCl3)
δ 4.13 (m, 1H), 4.04 (m, 1H), 3.91 (dt, J =
We acknowledge Dr. Davide Tessaro and Dr. Davive De Se-
meis for the very fruitful discussion and support on the set-
up of the Lipase catalysed hydrolysis. We thank Dr. Marina
Montagna of PA Aromatics Flavours Srl for providing a sam-
ple of (±)-3. This work was supported by Fondazione Cariplo,
grant n. 2014-0568, INBOX project.
8.3, 16.5, 6.2 Hz, 1H), 3.74 (dt, J = 8.4, 16.9, 6.2 Hz, 1H), 2.47
(m, 1H), 2.34 (s, 3H), 1.94 (m, 1H), 1.20 (d, J = 6.2 Hz, 3H); 13C
NMR (100 MHz, CDCl3)
δ 195.4, 76.5, 66.0, 46.3, 33.6, 30.6,
16.9; GC-MS: m/z (%) 159 (M+ -1, 1), 145 (M+ -15, 3), 116 (10),
103 (4), 84 (80).
(2S,3S)-3-Bromo-2-methyltetrahydrofuran ((2S,3S)-1). To
an ice–cold and well stirred solution of 8 (300 mg, 1.6 mmol)
in pyridine (0.8 mL) was added portion wise tosyl chlorode
(430 mg) under a N2 atmosphere. After complete consump-
tion of 8 (checked by TLC, AcOEt/hexane 6:4) the reaction
was left at rt for 1 h. Then, the ice-cooled reaction mixture
was diluted with Et2O/pentane (1:1, 5 mL). After trituration of
the gummy solid the supernatant liquid was separated, and
and washed with HCl (1 M, 3x5 mL). The organic phase was
dried over Na2SO4 and then concentrated under reduced
pressure to give a yellow liquid of sufficient purity for the
next step. Yield 54%; 143 mg; a sample was distilled with bulb
to bulb apparatus (50 °C, 1 mmHg); tr = 5.81 min 94% purity
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19
We have seen already in the past that α-substituted α,β-
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20
Tasnádi, G.; Winkler, C. K.; Clay, D.; Hall, M.; Faber, K. Catal.
Sci. Technol. 2012, 2, 1548-1552.
21
For a computational study on the hydrolysis of α-halo-ketone
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2011, 76, 8294–8299.
22
Chaparro-Riggers, J. F.; Rogers, T. A.; Vazquez-Figueroa, E.;
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Parmeggiani, F.; Sacchetti, A. Org. Biomol. Chem. 2013, 11, 2988-
2996.
24
We left a sample of 4c at rt for 2 weeks, the reduction with
EVO030 gave a 1:1 mixture of syn and anti diastereoisomers (see
SI).
25 Esmurziev, A.; Sundby, E.; Hoff, B. H. Eur. J. Org. Chem. 2009,
1592–1597.
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