On the selectivity of oxynitrilases towards α-oxygenated aldehydes

Article abstract of DOI:10.1016/S0040-4020(01)00054-0

Different α-alkoxy and α,β-di-alkoxy substituted aldehydes have been submitted to the catalytic action of the oxynitrilases from almond (PaHNL) or from Hevea brasiliensis (HbHNL), in order to explore the possibility of using these enzymes for the preparation of complex cyanohydrins. The selectivity of both enzymes towards these compounds was found to be largely dependent on the substitutents, being low with the aldehydes carrying the sterically more demanding phenyl substituent. Contrary to the chemical addition of HCN, which always occurs with a slight preference for the formation of the anti diastereoisomers, the enzymatic cyanuration - occurring with a facial preference, Si or Re according to the biocatalyst used - gave a mixture of cyanohydrins that, depending on the starting enantiomeric aldehyde, can be enriched in the syn diastereoisomers.

Full text of DOI:10.1016/S0040-4020(01)00054-0

TETRAHEDRON
Pergamon
Tetrahedron 57 (2001) 2213±2220
On the selectivity of oxynitrilases towards
a-oxygenated aldehydesq
Paola Bianchi,a Gabriella Roda,a,b Sergio Riva,a,p Bruno Danieli,b
Antonina Zabelinskaja-Mackovac and Herfried Grienglc
aIstituto di Biocatalisi e Riconoscimento Molecolare, C.N.R., via Mario Bianco 9, I-20131 Milano, Italy
Dipartimento di Chimica Organica ed Industriale, Centro C.N.R. di Studio per le Sostanze Organiche Naturali, Universita di Milano,
b
Á
via Venezian 21, I-20133 Milano, Italy
cInstitute of Organic Chemistry, Technical University Graz, Stremayrgasse 16, A-8010 Graz, Austria
Received 28 September 2000; revised 13 December 2000; accepted 4 January 2001
AbstractÐDifferent a-alkoxy and a,b-di-alkoxy substituted aldehydes have been submitted to the catalytic action of the oxynitrilases from
almond (PaHNL) or from Hevea brasiliensis (HbHNL), in order to explore the possibility of using these enzymes for the preparation of
complex cyanohydrins. The selectivity of both enzymes towards these compounds was found to be largely dependent on the substitutents,
being low with the aldehydes carrying the sterically more demanding phenyl substituent. Contrary to the chemical addition of HCN, which
always occurs with a slight preference for the formation of the anti diastereoisomers, the enzymatic cyanurationÐoccurring with a facial
preference, Si or Re according to the biocatalyst usedÐgave a mixture of cyanohydrins that, depending on the starting enantiomeric
aldehyde, can be enriched in the syn diastereoisomers. q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
reaction outcomes obtained with another oxynitrilase with
opposite stereospeci®city, namely the enzyme from Hevea
brasiliensis (HbHNL)5 and compared with the data recently
reported in the literature for chemical6 and enzymatic7 trans-
formations of similar substrates.
Exploitation of oxynitrilases for the stereoselective syn-
thesis of (R)- and (S)-cyanohydrins from aldehydes and
ketones is a well-assessed methodology nowadays.2 As a
part of our general interest in enzymatic catalysis in organic
solvents,3 we have been studying the performances of these
biocatalysts towards several different carbonylic substrates
and, speci®cally, focusing attention on the in¯uence of a
stereocenter already present in the molecule on the selec-
tivity displayed by these enzymes.1,4
2. Results and discussion
2.1. Synthesis of the aldehydes 3±9
Most of the aldehydes have been prepared from the corre-
sponding alcohol using the Swern procedure.8 Speci®cally,
4 and 5 were obtained from the commercially available
racemic alcohols, while the precursor of 9 was obtained
by condensation of glycerol and cyclohexanone.9
In this respect, very recently, we have reported on the in¯u-
ence of simple a- and b-alkoxy substituents on the stereo-
selectivity displayed by almond oxynitrilase (PaHNL)
towards a set of aldehydes (i.e. racemic 1±3).1 As an
extension of this previous work, we report here on the
results obtained with the racemic a-alkoxy aldehydes 4
(monocyclic analog of 3) and 5, and with the a,b-di-alkoxy
aldehydes 6±8 (enantiomerically pure) and 9 (racemic)
(Fig. 1). The diastereoselectivity of HCN addition to these
compounds catalyzed by PaHNL has been matched with the
Aldehyde (R)-6 was obtained from mannitol,10 and its enan-
tiomer (S)-6 was prepared from a commercially available
ascorbic acid derivative.11
Aldehyde (2R,3S)-7 was prepared from d-threonine accord-
ing to Scheme 1A, and its enantiomer (2S,3R)-7 was simi-
larly obtained from l-threonine.
q Oxynitrilases in organic synthesis, Part 4.1
Finally, the enantiomeric compounds (2R,3S)-8 and
(2S,3R)-8 were prepared via Sharpless hydroxylation of
methyl cinnamate in the presence of (DHQ)2PHAL and
(DHQD)2PHAL, respectively (Scheme 1B).12
Keywords: almond oxynitrilase (PaHNL); Hevea brasiliensis oxynitrilase
(HbHNL); cyanohydrins; aldehyde oxynitrilases.
Corresponding author. Tel.: 139-02-285-000-00; fax: 139-02-285-000-
36; e-mail: rivas@ico.mi.cnr.it
p
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(01)00054-0

2214
P. Bianchi et al. / Tetrahedron 57 (2001) 2213±2220
Figure 1.
2.2. Stereochemical correlations
enantiomers of the aldehydes 6±8) have been analyzed
either by chiral HPLC or, after acylation, by chiral GC,
obtaining base-line separated peaks.
The aldehydes 3±9 have been chemically transformed into
the corresponding cyanohydrins (Xa±d). The four dia-
stereoisomers (or the two epimers obtained from the pure
The absolute con®guration of the stereogenic center of the
Scheme 1.

P. Bianchi et al. / Tetrahedron 57 (2001) 2213±2220
2215
analyzed by chiral chromatographic techniques. The combi-
nation of the whole of this information allowed us to
evaluate the stereochemical outcome of the enzymatic
reactions.
Scheme 2. Acetylation of racemic cyanohydrins catalyzed by lipase PS.
2.3. Almond oxynitrilase-catalyzed synthesis of cyano-
hydrins
cyanohydrins was determined by exploiting the well-known
selectivity of lipase PS in the acylation of these compounds
(Scheme 2).13 However, it should be noted that, at variance
to the usual esteri®cation of (2S)-cyanohydrins (Rˆalkyl or
aryl), formally, the (2R)-stereoisomers are acylated in the
presence of a-oxygenated substituents (RˆCHR0OR00), due
to the CIP priority rules.
In analogy with our previous investigations, acetone cyano-
hydrin was used as HCN donor for the PaHNL-catalyzed
synthesis of cyanohydrins in iso-propyl ether, as shown for
the aldehyde 9 (Scheme 4).16 Table 1 reports the results
obtained with the aldehydes 4±9 (`2' refers to the cyano-
hydrin stereogenic carbon and `3' refers to its a-carbon, as
indicated in compound 9a). No transformation was
observed in the absence of the enzyme.
Then, to complete the correlation between the absolute
con®gurations of the products and their own chiral chro-
matographic peaks, preliminary resolution of the synthetic
precursors of the aldehydes 414 and 515 was accomplished
(Scheme 3). Additionally, the (R)-alcohol, precursor of (D)-
9, was commercially available.
The data of Table 1 deserve some comments. Concerning
substrate reactivity, all the compounds were quantitatively
converted to the corresponding cyanohydrins, except (R)-6.
This aldehyde proved to be quite unsuitable for the PaHNL
active site and, after several attempts, we only observed a
11% conversion at best. Diastereomeric excesses of the
products were below 70%, being particularly low with the
aldehydes 8 and 9 carrying the more sterically demanding
The enantiomerically enriched aldehydes 4, 5 and 9 were
then chemically transformed into the corresponding cyano-
hydrins, and the diastereoisomeric mixtures, enriched in the
products with a de®ned absolute con®guration at C-3,
Scheme 3.
Scheme 4.

2216
P. Bianchi et al. / Tetrahedron 57 (2001) 2213±2220
Table 1. PaHNL-catalyzed transformation of aldehydes 4±9 using acetone cyanohydrin as cyanide source
Substrate
Cyanohydrins
% Diastereomeric compositiona
2S,3R (Xa)
2R,3R (Xb)
d.e. 3R
2S,3S (Xc)
2R,3S (Xd)
d.e. 3S
4
4a±d
5a±d
6a,b
6c,d
7a,b
7c,d
8a,b
8c,d
9a±d
41.6
37.9
41.6
13.2
8.4
58.4
51.8
63.7
16.8b
30.6
39.7
14.5
14.0
35.7
47.9
5
(R)-6
(S)-6
(2R,3S)-7
(2S,3R)-7
(2R,3S)-8
(2S,3R)-8
9
73.8
55.1
26.2
44.9
47.6
10.2
25.6
49.5
23.1
74.4
50.5
27.3
48.8b
1.0b
45.3
29.4
54.7a
20.2
9.4
18.5
8.3b
a
In the presence of a-oxygenated substituents, due to the C.I.P. priority rules, formally the (2S)-cyanohydrins are the `natural' products produced by PaHNL.
Inversion of the `natural' expected selectivity.
b
substituents. Additionally, we observed a peculiar inversion
6,7 we repeated these biotransformations using PaHNL
adsorbed on celite R-630w and HCN as cyanurating agent.
Additionally, the reactions were also performed with the
(S)-speci®c oxynitrilase from Hevea brasiliensis (HbHNL)
to compare the catalytic outcomes of two unrelated
enzymes. The results are reported in Table 2, together
with the diastereomeric composition of the corresponding
cyanohydrins obtained by chemical cyanuration of 4±9. (In
fact, we observed that the spontaneous addition of the
chemical HCN to these compounds was not negligible any
of the natural selectivity towards one of the enantiomers of
the dioxolane type substrates 6±9. The 3:1 ratio obtained
with (2R,3S)-7 in favor of the `wrong' diastereoisomer is, by
far, the highest d.e. value (48.8%) reported up to now,
related to the inversion of the natural selectivity of this
enzyme.
In order to directly compare our results with some data
reported in the literature for the aldehydes (R)-6 and (S)-
Table 2. Comparison of the performances of PaHNL and HbHNL with the aldehydes 4±9
Substrate
Method
Product
% Diastereomeric compositiona
2S,3R (Xa)
2R,3R (Xb)
d.e. 3R
2S,3S (Xc)
2R,3S (Xd)
d.e. 3S
4
A
B
C
4a±d
5a±d
6a,b
6c,d
7a,b
7c,d
8a,b
8c,d
9a±d
47.6
5.4
22.7
4.6
45.9
27.6
82.4
78.9
9.7
42.6
3.9
27.5
5.2
44.9
22.2
78.2
84.0
10.5
5
A
B
C
33.7
17.1
22.4
16.2
32.9
27.2
35.1
31.6
9.5
35.6
18.9
27.3
14.5
31.1
23.1
42.1
24.4
8.3
(R)-6
A
B
C
47.1
34.9
42.9
52.9
65.1
57.1
5.8b
30.2
14.2
(S)-6
A
B
C
66.4
29.8
42.2
33.6
70.2
57.8
32.8
40.4
15.6
(2R,3S)-7
(2S,3R)-7
(2R,3S)-8
(2S,3R)-8
9
A
B
C
34.2
48.1
47.2
65.8
51.9
52.8
31.6b
3.8
5.6
A
B
C
52.6
35.1
49.1
47.4
64.9
50.9
5.2
29.8
1.8
A
B
C
48.2
52.7
49.2
51.8
47.3
50.8
3.6b
5.4
1.6
A
B
C
49.3
49.9
52.4
50.7
50.1
47.6
1.4b
0.2
4.8
A
B
C
21.8
16.9
21.6
28.3
33.0
28.7
13.0b
32.3
14.1
30.3
18.3
28.2
19.6
31.8
21.6
21.4
26.9
13.3
A: PaHNL, HCN; B: HbHNL, HCN; C: HCN.
a
In the presence of a-oxygenated substituents, due to the C.I.P. priority rules, formally the (2S)-cyanohydrins are the `natural' products produced by PaHNL
and the (2R)-cyanohydrins are the `natural' products produced by HbHNL.
Inversion of the `natural' expected selectivity.
b

P. Bianchi et al. / Tetrahedron 57 (2001) 2213±2220
2217
longer after several hours of incubation under these reaction
conditions).
brasiliensis (HbHNL) was obtained from Roche diag-
nostics. Lipase PS from Pseudomonas cepacia was
purchased from Amano. Celitew R-630 was from Fluka.
Acetone cyanohydrin and other reagents were from Aldrich.
A,0.6 M HCN solution in methyl tert-butyl ether was
prepared according to Brussee.19 HPLC analyses were
performed using a Chiralcel OD column (from DIACEL)
and a Jasco 880/PU instrument equipped with a Jasco 875
UV/VIS detector (reading was done at 254 nm). GC
analyses were performed using a Chrompack capillary
column fused silica gel coated with CP-cyclodex B236 M
and a Hewlett Packard 5890 series II instrument. FT-IR
spectra were recorded using a Jasco 610 instrument
As expected, PaHNL and HbHNL gave complementary
reaction outcomes with most of the substrates. However,
at variance to PaHNL, HbHNL did not show any inversion
of its natural selectivity with the enantiomers of aldehydes
6±9.
Aldehyde (R)-6 con®rmed to be a poor substrate for PaHNL
and the d.e. (5.8%) was even worse than the one obtained
with acetone cyanohydrin (16.8%, Table 1): the conversion
was quantitative, but we cannot exclude that it was partly
due to spontaneous chemical cyanuration. The results
obtained by the action with HbHNL on this aldehyde were
slightly better in terms of d.e.
1
equipped with a DTGS detector. H NMR spectra were
recorded on a Bruker AC-300 at 300 MHz using CDCl3 as
a solvent.
The data in Table 2 clearly shows that the selectivity of
HbHNL towards these compounds was also generally low,
particularly with the set of aldehydes 6±9, that never gave
d.e. higher than 50%. However, it deserves to be empha-
sized that, while the chemical addition of HCN to
compounds 3±9 always occurred with a slight preference
for the formation of the anti diastereoisomers, the enzymatic
cyanurationÐoccurring with a facial preference, Si or Re
according to the biocatalyst usedÐgave mixtures of cyano-
hydrins that, depending on the starting enantiomeric
aldehyde, can be enriched in the syn diastereoisomers
(Table 2).
3.2. Synthesis of aldehydes
3.2.1. Racemic 4. This compound was prepared from the
corresponding alcohol using the Swern procedure8 and
controlling the reaction outcome by TLC (eluent: hexane/
AcOEtˆ7:3). At 2608C, a solution of DMSO (3.7 ml,
51.7 mmol) in CH2Cl2 (70 ml) was added dropwise to a
2 M solution of oxalyl chloride (15.5 ml, 31.0 mmol) in
CH2Cl2. The mixture was stirred for 20 min and then a
solution of tetrahydropyran-2-methanol (3 g, 25.8 mmol)
in CH2Cl2 was added dropwise. The mixture was stirred
for 10 min and then Et3N (18 ml, 129.1 mmol) was slowly
added. The reaction mixture was warmed up to room
temperature, stirred for 30 min and H2O was added. When
the mixture became clear, it was extracted with CH2Cl2, the
solvent was evaporated and the product was puri®ed by ¯ash
chromatography (eluent: hexane/AcOEtˆ8:2) followed by
bulb-to-bulb distillation (808C, 20 mmHg) to give the title
compound 4 (1.3 g, 44%); [Found: C, 59.8; H, 7.9. C5H8O2
requires C, 60.00; H, 8.00%]. FT-IR, nmax(CHCl3): 2989,
A possible synthetic application of the cyanuration of these
compounds is the so-called Kiliani±Fischer one-carbon
homologation of carbohydrates. Using this well established
methodology,17 HCN addition to the starting carbo-
hydratesÐfollowed by acid-catalyzed nitrile hydrolysis,
lactonization and reduction to re-establish the aldehydic
groupÐallows the formation of epimeric one-carbon
elongated sugars. However, despite the fact that the starting
molecules (formally a-hydroxy aldehydes) are enantio-
merically pure, poor asymmetric induction has always
been observed in this chemical formation of cyanohydrins
and therefore new and more selective methodologies would
be synthetically quite useful. Unfortunately, the reaction
outcomes obtained with the a,b-di-alkoxy aldehydes
described in this paper do not support the idea of a more
ef®cient (in term of diastereoselectivity) Kiliani±Fischer
sugar homologation using a chemo-enzymatic approach.
2867, 1730, 1216, 1074 cm21. H NMR, dˆ9.60 (brs, 1H,
1
CHO); 4.05 (td, 1H, J1ˆ4 Hz, J2ˆ12 Hz, H-6eq); 3.80 (dd,
1H, J1ˆ3 Hz, J2ˆ10 Hz, H-2); 3.52 (dt, 1H, J1ˆ12 Hz,
J2ˆ4 Hz, H-6ax).
3.2.2. Racemic 5. This compound was prepared similarly
from the corresponding alcohol. The product was puri®ed
by ¯ash chromatography (eluent: hexane/AcOEtˆ7:3)
followed by bulb-to-bulb distillation (508C, 20 mmHg) to
give the title compound 5 (1.23 g, 42%); [Found: C, 62.9; H,
8.6. C6H10O2 requires C, 63.16; H, 8.77%]. FT-IR,
n
max(CHCl3): 2944, 2861, 1741, 1217, 1094 cm21 1H
.
Finally, the signi®cant inversion of the natural diastereo-
selectivity of PaHNL with the aldehyde (2R,3S)-7 (the
same effect observed with (R)-6 and (2R,3S)-8) as well as
the similar result previously obtained with 2-naphthyl-acet-
aldehyde,1 deserve to be better investigated at a molecular
level, as it will be possible as soon as the three-dimensional
structure of PaHNL will be elucidated.
NMR, dˆ9.65 (brs, 1H, CHO), 4.26 (dt, 1H, J1ˆ2 Hz,
J2ˆ8 Hz, H-5a), 3.90±3.70 (m, 2H, H-2 and H-5b).
3.2.3. 2,3-O-Isopropylidene-d-glyceraldehyde ((R)-6).
This compound was prepared from d-mannitol according
to a published procedure.10 (A) Under inert atmosphere,
the catalyst stannous chloride (15 mg) was added to a
solution of d-mannitol (10 g, 55 mmol) in 2,2-dimethoxy-
propane (16 ml) and dimethoxyethane (24 ml). The mixture
was re¯uxed until the starting material disappeared (TLC:
CHCl3/MeOHˆ10:0.2), then it was cooled to room
temperature and pyridine (20 ml) was added. The solvent
was evaporated and the white solid was dissolved in
CH2Cl2 (100 ml) by heating at the boiling point. The
3. Experimental
3.1. Materials and methods
Prunus amygdalusoxynitrilase (PaHNL) was isolated from
grounded almonds.18 The oxynitrilase from Hevea

2218
P. Bianchi et al. / Tetrahedron 57 (2001) 2213±2220
mixture was ®ltered, CH2Cl2 was evaporated and the solid
residue was crystallized from diisobutyl ether to give pure
mannitol diacetonide. Yield 7.15 g (50%). (b) A saturated
solution of NaHCO3 (1 ml) was added to a solution of the
previously obtained mannitol diacetonide in CH2Cl2
(20 ml). The mixture was cooled at 08C and sodium
metaperiodate (3.29 g) was slowly added (30 min), keeping
the temperature under 358C (TLC: CHCl3/MeOHˆ10:0.2).
After 4 h, magnesium sulphate (1 g) was added and after
10 min, the mixture was ®ltered. The solvent was evapo-
rated and the residue was puri®ed by bulb-to-bulb distil-
lation (708C, 20 mmHg). FT-IR, nmax(CHCl3): 2945, 2870,
1723, 1423, 1215 cm21. 1H NMR: dˆ9.70 (d, 1H, Jˆ3 Hz,
CHO); 4.40 (dt, 1H, J1ˆ3 Hz, J2ˆ7.8 Hz, H-2); 4.25±4.00
(2dd, each 1H, J1ˆ8 Hz, J2ˆ10 Hz, H-3a and H-3b); 1.50
(s, 3H, CH3); 1.40 (s, 3H, CH3).
rated to give the alcohol that was puri®ed by bulb-to-bulb
distillation (1158C, 20 mmHg). Yield 3 g (92%). (D) The
alcohol was oxidized using the Swern protocol.8 The
product was puri®ed by ¯ash chromatography (eluent:
hexane/AcOEtˆ75:25) and followed by bulb-to-bulb distil-
lation (1008C, 20 mmHg) to give the title compound
((2R,3S)-7) (1.35 g, 46%); [Found: C, 58.1; H, 8.2.
C7H12O3 requires C, 58.33; H, 8.33%]; FT-IR: nmax(CHCl3):
2990, 2895, 1721, 1454, 1382, 1252, 1098 cm21. 1H NMR,
dˆ9.65 (d, 1H, Jˆ3 Hz, CHO); 4.11 (dq, 1H, J1ˆ8 Hz, J2ˆ
6 Hz, H-3); 3.81 (dd, 1H, J1ˆ8 Hz, J2ˆ3 Hz, H-2); 1.49 (s,
3H, CH3±C); 1.41 (s, 3H, CH3±C); 1.39 (d, 3H, Jˆ6 Hz,
CH3±CH).
3.2.6. 4-Deoxy-2,3-O-isopropylidene-d-threose ((2S,3R)-
7). This compound was prepared similarly from l-threonine.
3.2.4. 2,3-O-Isopropylidene-l-glyceraldehyde ((S)-6).
This compound was prepared from 5,6-O-isopropylidene-
l-gulono-1,4-d-lactone according to a published pro-
cedure.11 At 08C, a 3 M NaOH solution was added to a
sodium metaperiodate solution (9.8 g in 23 ml of H2O)
keeping the temperature under 58C until pH 5.5 was
reached. The mixture was warmed up to room temperature
and 5,6-O-isopropylidene-l-gulono-1,4-d-lactone (5 g,
22.9 mmol) was added. During the reaction, the pH was
kept at 5.5 by adding 15% w/v Na2CO3. When the reaction
was ®nished (TLC: hexane/AcOEtˆ7:3), the mixture was
saturated with NaCl (12 g) and ®ltered. The water solution
was extracted with CH2Cl2. The extracts were dried with
Na2SO4 and the solvent was evaporated to give the aldehyde
that was puri®ed by bulb-to-bulb distillation (708C,
20 mmHg). 1H NMR, dˆ9.70 (d, 1H, Jˆ3 Hz, CHO);
4.40 (dt, 1H, J1ˆ3 Hz, J2ˆ8 Hz, H-2); 4.25-4.00 (2dd,
each 1H, J1ˆ8 Hz, J2ˆ10 Hz, H-3a and H-3b); 1.50 (s,
3H, CH3); 1.40 (s, 3H, CH3).
3.2.7. (3S)-3-Phenyl-2,3-O-isopropylidene-d-glyceralde-
hyde ((2R,3S)-8). This compound was prepared from
methyl cinnamate. (A) To N-methylmorpholino-N-oxide
(NMNO) at 60% (40 ml), a solution of cinnamate (5 g,
30.86 mmol) in tert-butanol (200 ml) was added,
followed by the addition of (DHQ)2(PHAL) (62 mg) and
K2OsO4(OH)4 (127 mg); (TLC, hexane/AcOEtˆ7:3).
After 28 h, a solution of sodium sulphite in 80 ml of water
was added, the reaction mixture was stirred for 1 h and the
water was evaporated. The residue was extracted with
AcOEt and washed with 1N HCl and then with brine. The
organic phase was evaporated and the solid recrystallized
from AcOEt. Yield 4.23 g (70%). (B) 2,2-Dimethoxy-
propane (5.3 ml, 0.44 mol) and catalytic p-toluenesulfonic
acid were added to the residue in CH2Cl2 (13 ml; TLC:
hexane/AcOEtˆ7:3). When the reaction was ®nished, the
solvent was evaporated and the residue reduced to the
alcohol (1 M LiAlH4 in THF, yield 3.3 g, 73%), then
oxidized to the aldehyde using the Swern protocol8 and
®nally puri®ed by ¯ash chromatography (eluent: CHCl3/
MeOHˆ99:1) followed by bulb-to-bulb distillation
(1258C, 5 mmHg) to give the title compound ((2R,3S)-8)
(1.8 g, 55%); [Found: C, 69.6; H, 6.6. C12H14O3 requires
C, 69.90; H, 6.80%]; FT-IR, nmax(CHCl3): 2996, 2942,
3.2.5. 4-Deoxy-2,3-O-isopropylidene-l-threose ((2R,3S)-
7). This compound was prepared from d-threonine. (A) At
258C, a solution of NaNO2 (3.13 g, 0.045 mol) in H2O
(6.6 ml) and a solution of 95±97% H2SO4 (1.28 ml,
0.023 mol) in H2O (3.3 ml) were added, slowly and simul-
taneously, to a solution of d-threonine (5 g, 0.042 mol) in
H2O (50 ml) keeping the temperature under 58C. The reac-
tion mixture was warmed to room temperature and stirred
overnight. The solvent was evaporated and the residue was
dissolved in 50 ml of EtOH. The solid residue was ®ltered
through celite and the solvent was evaporated. The residue
was esteri®ed with MeOH (40 ml) saturated with gaseous
HCl. (B) 2,2-Dimethoxypropane (5.04 ml, 0.042 mol) and
catalytic p-toluenesulfonic acid were added to the methyl
ester dissolved in acetone (17 ml) and the reaction mixture
was allowed to stand for 4 h at room temperature (TLC:
hexane/AcOEtˆ7:3). The product was puri®ed by ¯ash
chromatography (eluent: hexane/AcOEtˆ7:3). Total yield
3.9 g (53%). (C) Under inert atmosphere at 08C, a solution
of the acetonide ester (3.9 g, 22.41 mmol) in THF (60 ml)
was carefully dropped into a 1.0 M solution of LiAlH4 in
THF (23 ml). After 10 min, the mixture was warmed up to
room temperature. (TLC: hexane/AcOEtˆ7:3). The excess
of LiAlH4 was destroyed by adding AcOEt, then 1.5 ml of
H2O was added and the salts were ®ltered through celite.
The solution was dried (Na2SO4) and the solvent was evapo-
1
2902, 1718, 1461, 1385, 1238 cm21. H NMR, dˆ9.82 (d,
1H, Jˆ2 Hz, CHO); 7.30±7.50 (m, 5H, Ph); 5.09 (d, 1H,
Jˆ8 Hz, H-3); 4.21 (dd, 1H, J1ˆ8 Hz, J2ˆ2 Hz, H-2); 1.45
(s, 3H, CH3); 1.42 (s, 3H, CH3).
3.2.8. (3R)-3-Phenyl-2,3-O-isopropylidene-l-glyceralde-
hyde (2S,3R)-8. This compound was prepared similarly
using the enantiomeric catalyst (DHQD)2PHAL.
3.2.9. Racemic 2,3-O-cyclohexylidene-d,l-glyceralde-
hyde 9. This compound was prepared by condensation of
glycerol and cyclohexanone (¯ash chromatography, eluent:
hexane/AcOEtˆ7:3),9 followed by oxidation according to
the Swern procedure,8 and ®nal puri®cation by ¯ash
chromatography (eluent: CHCl3/MeOHˆ99:1, yield of the
Swern oxidation 2 g, 67%), followed by bulb-to-bulb distil-
lation (1008C, 5 mmHg) to give the title compound 9;
[Found: C, 63.2; H, 8.1. C9H14O3 requires C, 69.90; H,
6.80%]. FT-IR, nmax(CHCl3): 2943, 2859, 1739, 1217,
1098 cm21
.
1H NMR, dˆ9.71 (d, 1H, Jˆ2 Hz, CHO);
4.0±3.6 (m, 3H, H-2 and CH2-3); 1.80±1.30 (m, 10H, cyclo-
hexyl moiety).

P. Bianchi et al. / Tetrahedron 57 (2001) 2213±2220
2219
3.3. General procedure for the chemical synthesis of
cyanohydrins (Method C of Table 2)
1.37 (2d, 1.5H each, Jˆ6 Hz, CH3±CH). Compounds 7c,d.
GC-analyses (as acetates), Tiˆ508C; tiˆ1 min; rateˆ58C/
min; Tfˆ2008C; ret. times (min): 7c, 23.705; 7d, 24.470.
1H NMR: dˆ4.52 (d, 1H, Jˆ5 Hz, H-2); 4.14 (m, 1H,
H-3); 3.75 (m, 1H, H-4); 1.45 and 1.43 (2s, 3H, CH3);
1.38 and 1.37 (2d, 1.5H each, Jˆ6 Hz, CH3±CH).
To a solution of 20 mg of aldehyde in 1 ml of 80% v/v
AcOH, NaCN (3 equiv.) dissolved in 1 ml of water was
added dropwise at 08C. When the reaction was over, water
was added and the mixture was extracted with ethyl ether.
The organic phase was washed with NaHCO3, dried with
Na2SO4 and evaporated. The cyanohydrins mixtures (two
epimers or four diastereoisomers) were puri®ed by ¯ash
chromatography on silica using a mixture of hexane/
AcOEt (7:3 for 4a±d, 5a±d, and 6a±d; 8:2 for 7a±d, 8a±
d, and 9a±d) as the eluent.
3.3.5. Compounds 8a,b. Found: C, 66.7; H, 6.4. C13H15O3N
requires C, 66.95; H, 6.44%. HPLC analysis, eluent:
hexane/iPrOHˆ96:4; ¯ow rateˆ0.5 ml/min; ret. time
(min): 8a, 57.31; 8b, 33.58. FT-IR, nmax(CHCl3): 3620±
3120 (br), 2993, 2944, 2907, 2252, 1461, 1385,
1
1238 cm21. H NMR: dˆ7.30±7.45 (m, 5H, Ph); 4.98 and
4.86 (2d, 0.5 H each, Jˆ8 Hz, H-4); 4.51 and 4.48 (d, 0.5 H
each, Jˆ3 Hz, H-2); 4.05 and 4.02 (2dd, 0.5 H each, J1ˆ
9 Hz, J2ˆ4 Hz, H-3); 1.66 and 1.64 (2s, 1.5 H each, CH3±
C); 1.52 and 1.50 (2s, 1.5 H each, CH3±C). Compounds
8c,d. HPLC analysis, eluent: hexane/iPrOHˆ96:4; ¯ow
3.3.1. Compounds 4a±d. Found: C, 56.4; H, 6.9. C6H9O2N
requires C, 56.69; H, 7.09%. GC-analysis (as acetates),
Tiˆ508C; tiˆ5 min; rateˆ38C/min; Tfˆ2008C; ret. times
(min): 4a, 44.160; 4b, 43.272; 4c, 43.108; 4d, 44.410. FT-
IR, nmax(CHCl3): 3650±3100 (br), 2984, 2875, 2250, 1219,
1
rateˆ0.5 ml/min; ret. time (min): 8c, 31.31; 8d, 28.46. H
1
1075 cm21. H NMR (for carbon number see Scheme 4):
NMR: dˆ7.30±7.45 (m, 5H, Ph); 4.98 and 4.86 (2d, 0.5 H
each, Jˆ8 Hz, H-4); 4.51 and 4.48 (d, 0.5 H each, Jˆ3 Hz,
H-2); 4.05 and 4.02 (2dd, 0.5 H each, J1ˆ9 Hz, J2ˆ4 Hz,
H-3); 1.66 and 1.64 (2s, 1.5 H each, CH3±C); 1.52 and 1.50
(2s, 1.5 H each, CH3±C).
dˆ4.34 and 4.29 (2d, 0.5H each, Jˆ6 Hz, CH(CN)OH);
4.10 (m, 1H, H-6eq); 3.50 (m, 2H, H-2 and H-6ax).
3.3.2. Compounds 5a±d. Found: C, 59.4; H, 7.6. C7H11O2N
requires C, 59.57; H, 7.80%. GC-analysis (as acetates),
Tiˆ1008C; tiˆ5 min; rateˆ0.38C/min; Tfˆ2008C; ret.
times (min): 5a, 56.115; 5b, 49.025; 5c, 46.377; 5d,
57.629. FT-IR, nmax(CHCl3): 3640±3120 (br), 2945, 2862,
3.3.6. Compounds 9a±d. Found: C, 66.4; H, 8.2.
C10H15O2N requires C, 66.30; H, 8.29%. GC-analysis (as
butanoates), Tiˆ1008C; tiˆ5 min; rateˆ18C/min; Tfˆ
2008C; ret. times (min): 9a, 93.257; 9b, 89.885; 9c,
89.377; 9d, 92.840. FT-IR: nmax(CHCl3): 3600±3080 (br),
2940, 2860, 2252, 1217, 1098 cm21. 1H NMR: dˆ4.44 and
4.42 (2d, 0.5 H each, Jˆ4 Hz, H-2); 4.31 (m, 1H, H-3); 4.14
(m, 1H, H-4a); 3.99 (m, 1H, H-4b).
1
2251, 1447, 1222, 1086 cm21. H NMR, dˆ4.43 and 4.41
(2d, 0.5 H each, Jˆ4 Hz, CH(CN)OH); 4.18 and 3.95 (m,
3H, H-2 and CH2-5).
3.3.3. Compounds 6a,b. Found: C, 53.6; H, 6.9. C7H11O3N
requires C, 53.50; H, 7.01%. GC-analysis (as acetates),
Tiˆ508C; tiˆ1 min; rateˆ58C/min; Tfˆ2008C; ret. times
(min): 6a, 25.521; 6b, 24.655. FT-IR, nmax(CHCl3): 3600±
3100 (br), 2943, 2896, 2251, 1423, 1215 cm21. 1H NMR, as
acetates: diastereoisomer 2R,3R, dˆ5.44 (d, 1H, Jˆ6 Hz,
H-2); 4.35 (m, 1H, H-3); 4.18 (dd, 1H, J1ˆ10 Hz, J2ˆ7 Hz,
H-4a); 4.05 (dd, 1H, J1ˆ10 Hz, J2ˆ5 Hz, H-4b); 2.16 (s,
3H, CH3±CO); 1.5 (s, 3H, CH3); 1.38 (s, 3H, CH3); dia-
stereoisomer 2S,3R, dˆ5.30 (d, 1H, Jˆ6 Hz, H-2); 4.39
(m, 1H, H-3); 4.15 (dd, 1H, J1ˆ9 Hz, J2ˆ6 Hz, H-4a);
3.92 (dd, 1H, J1ˆ9 Hz, J2ˆ4 Hz, H-4b); 2.17 (s, 3H,
CH3±CO); 1.5 (s, 3H, CH3); 1.4 (s, 3H, CH3). Compounds
6c,d. GC-analysis (as acetates), Tiˆ508C; tiˆ1 min;
rateˆ58C/min; Tfˆ2008C; ret. times (min): 6c, 24.392; 6d,
3.4. General procedures for the enzymatic synthesis of
the cyanohydrins
3.4.1. Using acetone cyanohydrin (Table 1). To a solution
of 250 mg of aldehyde in 10 ml of isopropyl ether con-
taining 1.3 equiv. of acetone cyanohydrin, PaHNL (,1500
units) dissolved in 500 ml of 0.1 M citrate buffer pH 5.5, was
added and the biphasic system shaken at room temperature
for 5 days. At the end of the reaction, the two phases were
separated. The aqueous phase was extracted with isopropyl
ether, the organic phases were dried with Na2SO4 and
evaporated. The cyanohydrins were puri®ed by ¯ash
chromatography.
1
25.520. H NMR, as acetates: diastereoisomer 2S,3S, dˆ
5.44 (d, 1H, Jˆ6 Hz, H-2); 4.35 (m, 1H, H-3); 4.18 (dd,
1H, J1ˆ10 Hz, J2ˆ7 Hz, H-4a); 4.05 (dd, 1H, J1ˆ10 Hz,
J2ˆ5 Hz, H-4b); 2.16 (s, 3H, CH3±CO); 1.5 (s, 3H, CH3);
1.38 (s, 3H, CH3); diastereoisomer 2R,3S, dˆ5.30 (d, 1H,
Jˆ6 Hz, H-2); 4.39 (m, 1H, H-3); 4.15 (dd, 1H, J1ˆ9 Hz,
J2ˆ6 Hz, H-4a); 3.92 (dd, 1H, J1ˆ9 Hz, J2ˆ4 Hz, H-4b);
2.17 (s, 3H, CH3±CO); 1.5 (s, 3H, CH3); 1.4 (s, 3H, CH3).
3.4.2. Using HCN (Methods A and B of Table 2). Method
A. PaHNL (,400 units), dissolved in 100 ml of 0.1 M citrate
buffer pH 5.5, was dropped homogeneously on celite
(300 mg). The catalyst was added to isopropyl ether
containing 250 mg of aldehyde and 2 equiv. of HCN, and
the reaction was shaken at room temperature at 150 rpm for
3 days. At the end of the reaction, the mixture was ®ltered,
celite was washed with isopropyl ether and the organic
phases were evaporated. The cyanohydrins were puri®ed
by ¯ash chromatography.
3.3.4. Compounds 7a,b. Found: C, 56.0; H, 7.4. C8H13O3N
requires C, 56.14; H, 7.60%. GC-analysis (as acetates),
Tiˆ508C; tiˆ1 min; rateˆ58C/min; Tfˆ2008C; ret. times
(min): 7a, 24.031; 7b, 24.780. FT-IR: nmax(CHCl3) 3630±
Method B. HbHNL (,6360 units in 1.2 ml of citrate buffer
pH 4.5) was added to a solution of 250 mg of aldehyde in
1.5 ml of isopropyl ether. Neat HCN (5 equiv.) was added at
08C, and the reaction was stirred at 158C until the complete
3076 (br), 2992, 2904, 2250, 1454, 1382, 1252, 1098 cm21
.
1H NMR: dˆ4.52 (d, 1H, Jˆ5 Hz, H-2); 4.14 (m, 1H, H-3);
3.75 (m, 1H, H-4); 1.45 and 1.43 (2s, 3H, CH3); 1.38 and

2220
P. Bianchi et al. / Tetrahedron 57 (2001) 2213±2220
conversion of the substrate was reached. Celite was added to
absorb the enzyme and the aqueous phase; the organic phase
was dried with Na2SO4 and evaporated. The cyanohydrins
were puri®ed by ¯ash chromatography.
Austrian treaty on scienti®c and technological cooperation
are gratefully acknowledged. Work in Graz was also
®nanced from the special research program `Biocatalysis'
of the Austrian Research Fund.
3.5. General procedure for the acylation with lipase PS
References
Approximately 30 mg/ml of cyanohydrin were dissolved in
methyl tert-butylether containing 10% v/v vinyl acetate.
Lipase PS immobilised on celite13 (20 mg/ml) was added,
and the suspension was shaken at room temperature until
about 50% of conversion was reached. The enzyme was
®ltered, the solvent was evaporated and the products were
puri®ed by ¯ash chromatography.
1. For Part 3, see: Roda, G.; Riva, S.; Danieli, B. Tetrahedron:
Asymmetry 1999, 10, 3939±3949.
2. (a) Griengl, H.; Schwab, H.; Fechter, M. TIBTECH 2000, 18,
252±256. (b) Schmidt, M.; Griengl, H. Top. Curr. Chem.
1999, 200, 194±225. (c) Effenberger, F. Chimia 1999, 53,
3±10. (d) Griengl, H.; Hickel, A.; Johnson, D. V.; Kratky,
C.; Schmidt, M.; Schwab, H. J. Chem. Soc., Chem. Commun.
1997, 1933±1940.
3.6. Stereochemical correlations
3. (a) Carrea, G.; Riva, S. Angew. Chem., Int. Ed. Engl. 2000, 39,
4000±4028. (b) Riva, S. In Enzymatic reactions in organic
media; Koskinen, A. P. G., Klibanov, A. M., Eds.; Blackie
A and P: London, 1995; pp 140±169. (c) Carrea, G.; Ottolina,
S.; Riva, S. Trends Biotechnol. 1995, 13, 63±70. (d) Danieli,
B.; Riva, S. Pure Appl. Chem. 1994, 66, 2215±2218.
4. (a) Danieli, B.; Frattini, S.; Roda, G.; Carrea, G.; Riva, S.
J. Mol. Catal. B: Enzymatic 1998, 5, 223±228. (b) Danieli,
B.; Barra, C.; Carrea, S.; Riva, S. Tetrahedron: Asymmetry
1996, 7, 1675±1682.
3.6.1. (S)-4. This compound was prepared by oxidation of
(S)-tetrahydropyran-2-methanol according to a published
procedure.14 This alcohol was obtained via kinetic reso-
lution of the racemic butanoyl ester catalyzed by porcine
pancreatic lipase (Scheme 3). The enzyme was added to a
solution of the racemic ester (2 ml) in phosphate buffer pH
7.2. The mixture was shaken at room temperature (TLC:
hexane/AcOEtˆ8:2), and after 6 h, extracted with AcOEt.
The organic phase was dried with Na2SO4 and evaporated.
The product was puri®ed by ¯ash chromatography (eluent:
hexane/AcOEtˆ9:1, then 8:2, then 1:1).
5. Schmidt, M.; Herve, S.; Klempier, N.; Griengl, H.
Tetrahedron 1996, 52, 7833±7840 (and references therein).
6. Ward, D. E.; Hrapchak, M. J.; Sales, M. Org. Lett. 2000, 2,
57±60.
3.6.2. (S)-5. This compound was prepared by oxidation of
(S)-tetrahydrofuran-2-methanol. This racemic alcohol was
resolved into its enantiomers by crystallizing the half esters
formed with O,O0-diacetyl-(2R,3R)-tartaric acid according
to a published procedure (Scheme 3).15 To a stirred mixture
of racemic alcohol (2.37 g, 23.2 mmol) and pyridine
(185 mg, 2.3 mmol), (2R,3R)-diacetyltartaric anhydride
(5 g, 23.1 mmol) was added. Stirring was continued for
20 min at room temperature, then 0.5 ml of ethyl acetate
was added and the temperature was raised to 80±858C.
After 1.5 h heating, 3 ml of ethyl acetate was added, and
the mixture was allowed to cool to room temperature, then
kept for 10 h at 58C. The crystals were ®ltered, washed with
7. Roos, J.; Effenberger, F. Tetrahedron: Asymmetry 1999, 10,
2817±2828.
8. Mancuso, A. J.; Swern, D. Synthesis 1981, 165±185.
9. Renoll, M.; Newman, M. S. Org. Synth. 1955, 3, 502±504.
10. Schmid, R.; Bryant, J. D. Org. Synth. 1993, 72, 6±12.
11. Hubschwerlen, C.; Specklin, J.-L.; Higelin, J. Org. Synth.
1993, 72, 1±5.
12. Wang, Z.-M.; Kolb, H. C.; Sharpless, K. B. J. Org. Chem.
1994, 59, 5104±5105.
13. Inagaki, M.; Hiratake, J.; Nishioka, T.; Oda, J. J. Org. Chem.
1992, 57, 5643±5649.
14. Kwatelay Quartey, E. G.; Hustad, J. A.; Faber, K.; Anthonsen,
T. Enzyme Microb. Technol. 1996, 19, 361±366.
ethyl acetate and dried. [a]25 ˆ213.3 (c 1.2, CHCl3;
D
È
15. Mravik, A.; Bocskei, Z.; Keszei, S.; Elekes, F.; Fogassy, E.
lit.:2 13.6). The solid was slowly added to 25 ml of a stirred
solution of NaOH 1 M and allowed to stand for 18 h at room
temperature (TLC: hexane/AcOEtˆ7:3). The aqueous solu-
tion was extracted with CH2Cl2; Na2SO4 (2 g) was dissolved
in the aqueous phase, and this was extracted again with
CH2Cl2. The organic phases were dried with Na2SO4 and
evaporated.
Tetrahedron: Asymmetry 1996, 7, 1477±1484.
16. Ognyanov, F.; Datcheva, V. K.; Kyler, K. S. J. Am. Chem. Soc.
1991, 113, 6992±6996.
17. (a) Kiliani, H. Ber. Dtsch. Chem. Ges. 1885, 18, 3066±3072.
(b) Fischer, E. Ber. Dtsch. Chem. Ges. 1889, 22, 2204±2205.
18. Chiari, M.; Gelain, A.; Riva, S.; Tura, D. Electrophoresis
1997, 18, 2050±2054.
19. (a) Zandbergen, P.; van der Linden, J.; Brussee, J.; van der
Gen, A. In Preparative biotransformation: Whole cells and
isolated enzymes; Roberts, S. M., Ed.; Wiley: New York,
1995 (Module 4.5.1). (b) Zandbergen, P.; van der Linden, J.;
Brussee, J.; van der Gen, A. Synth. Commun. 1991, 21, 1387±
1395.
3.6.3. (R)-9. This compound was obtained by oxidation of
the corresponding commercially available alcohol.
Acknowledgements
Grants obtained within the framework of the Italian±