Enzymatic kinetic resolution of 1,3-dioxolan-4-one and 1,3-oxathiolan-5-one derivatives: Synthesis of the key intermediate in the industrial synthesis of the nucleoside reverse transcriptase inhibitor Amdoxovir

Article abstract of DOI:10.1002/adsc.200303184

The resolution of racemic 1,3-dioxolan-4-one and 1,3-oxathiolan-5-one derivatives such as (4-oxo-1,3-dioxolan-2-yl)methyl 2-methylpropanoate (2) by enzymatic solvolysis was investigated. The resolution of 2, a precursor for the synthesis of the nucleoside reverse transcriptase inhibitor Amdoxovir, was optimized in terms of solvent/nucleophile, reaction conditions, and enzyme. The use of lipase from Candida antarctica B (CALB) and methanol as nucleophile and solvent resulted in an effective resolution and the product (R)-2 could be easily isolated. Products of substrate decomposition can be isolated and reused for the synthesis of racemic 2. The broad range of application for this enzymatic resolution was demonstrated by the resolution of further substrates with different substitution patterns. This process gives a new and unprecedented access to enantiopure 1,3-dioxolan-4-ones and 1,3-oxathiolan-5-ones.

Full text of DOI:10.1002/adsc.200303184

FULL PAPERS
Enzymatic Kinetic Resolution of 1,3-Dioxolan-4-one and
1,3-Oxathiolan-5-one Derivatives: Synthesis of the Key
Intermediate in the Industrial Synthesis of the Nucleoside
Reverse Transcriptase Inhibitor AMDOXOVIR
Alfred Popp,a,* Andrea Gilch,a Anne-Laure Mersier,a Hermann Petersen,b
Jodoca Rockinger-Mechlem,a J¸rgen Stohrera
a
Consortium f¸r elektrochemische Industrie GmbH, Zielstattstrasse 20, 81379 M¸nchen, Germany
Fax: (þ49)-89-74844-242, e-mail: alfred.popp@wacker.com
b
Wacker-Chemie GmbH, Johannes-Hess-Strasse 24, 84489 Burghausen, Germany
Received: October 21, 2003; Accepted: March 26, 2004
Abstract: The resolution of racemic 1,3-dioxolan-4- ucts of substrate decomposition can be isolated and
one and 1,3-oxathiolan-5-one derivatives such as (4- reused for the synthesis of racemic 2. The broad range
oxo-1,3-dioxolan-2-yl)methyl
2-methylpropanoate of application for this enzymatic resolution was dem-
(2) by enzymatic solvolysis was investigated. The res- onstrated by the resolution of further substrates with
olution of 2, a precursor for the synthesis of the nu- different substitution patterns. This process gives a
cleoside reverse transcriptase inhibitor Amdoxovir, new and unprecedented access to enantiopure 1,3-di-
was optimized in terms of solvent/nucleophile, reac- oxolan-4-ones and 1,3-oxathiolan-5-ones.
tion conditions, and enzyme. The use of lipase from
Candida antarctica B (CALB) and methanol as nucle-
ophile and solvent resulted in an effective resolution Keywords: enzyme catalysis; kinetic resolution; lipas-
and the product (R)-2 could be easily isolated. Prod- es; solvolysis
Introduction
Enantiopure compounds are used as starting materials
and intermediates in the synthesis of agrochemicals
and pharmaceuticals. Many of these compounds are cur-
rently prepared and marketed as racemates or mixtures
Scheme 1. Enantiopure dioxolan-4-one and oxathiolan-5-one
derivatives.
Due to its chemical nature this structure is not prone
of diastereomers. However, in many cases, the desired to resolution by crystallization of diastereomeric salts.
physiological effect is brought about by only one enan- To date, no direct resolution of the mentioned racemates
tiomer/diastereomer. The other isomer is, in the most fa- has been reported. Only the indirect route via enzymatic
vorable case, inactive, but it also may counteract the de- cleavage of hydrolyzable groups in the side chain in the
6]
sired effect or even be toxic. Besides from stereoselec- 2-position of the ring has been described.[1,3
tive processes, the resolution of racemates is of great im- Amdoxovir (1) {(ꢀ)-(2R,4R)-2-amino-[2-(hydroxy-
portance for the preparation of compounds of high methyl)-1,3-dioxolan-4-yl]adenine also known as (ꢀ)-
enantiopurity. b-d-2,6-diaminopurine dioxolane or DAPD}, originally
Especially enantiopure dioxolan-4-one (X¼O) and developed at the University of Georgia,[7] Emory Uni-
oxathiolan-5-one derivatives (X¼S, Scheme 1) are of versity[8] and Triangle Pharmaceuticals Inc.,[9 11] is an ex-
great interest because they are intermediates in the perimental nucleoside reverse transcriptase inhibitor
synthesis of compounds active in anti-viral therapy. (NRTI)[12] now owned by Gilead Sciences, Inc.[13] As
For example, 2-(hydroxymethyl)-1,3-dioxolan-4-one Amdoxovir is currently being evaluated in phase II, it
(Scheme 1: X¼O, R ¼H, TBDPS) is a key intermediate was necessary to develop an economic and reliable in-
in the synthesis of Dioxolane-T,[1,2] whereas 2-(hydroxy- dustrial large-scale process[14] in order to fulfill the mate-
methyl)-1,3-oxathiolan-5-ones (Scheme 1: X¼S , R¼ rial requirements for clinical trials. In this process, ac-
alkyl, silyl, acyl) can be used as building blocks for the cording to retrosynthetic studies the most important
preparation of the oxathiolanyl-nucleoside Covira- key intermediate is the enantiopure 1,3-dioxolan-4-
¾ [2 4]
cil .
one (R)-2 (Scheme 2).
682
¹ 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
DOI: 10.1002/adsc.200303184
Adv. Synth. Catal. 2004, 346, 682 690

Enzymatic Kinetic Resolution of 1,3-Dioxolan-4-one and 1,3-Oxathiolan-5-one
FULL PAPERS
Enzymatic Solvolysis of (R,S)-2
Hydrolysis
One of the most common methods for resolving race-
mates of esters and similar compounds is the enzyme-
catalyzed aqueous hydrolysis.[22 24] The major drawback
of this method is the insufficient solubility of most sub-
strates in aqueous solutions, but this can be avoided by
adding organic solvents. In order to identify an appropri-
ate enzyme, we firstly used a two-phase system [0.1 M
phosphate puffer at pH 7 and tert-butyl methyl ether
(TBME)] and 30 commercially available lipases and es-
Scheme 2. Retrosynthetic identification of the key building
block.
Results and Discussion
Resolution strategies have always played a central role terases.[25]
in the preparation of optically active compounds despite
Since, during a hydrolysis, both ester and lactone moi-
the factthat the maximum theoretical yield is 50% based eties of (R,S)-2 can be cleaved several degradation
on the racemic starting material. Considering this fact, products are possible (Scheme 4).
the basis of an economically successful resolution proc-
The hydroxymethyldioxolanone 4 and iso-butyric acid
ess is a cheap racemate. Even if the unwanted enantiom- 5 are formed after hydrolysis of the ester group in the
er (distomer[15]) can be returned to the production proc- side-chain whereas 6 and 7 are fragments of the cleavage
ess (e.g., after racemization) this involves additional of the dioxolanone ring. We could identify all these
work and occupancy of reactors. In our case the racemic products during our screening of the various lipases (ex-
starting material (R,S)-2 can be obtained according to cept for the stereochemistry of the byproduct 4), but in
known protocols.[16]
most cases a rapid and uncontrollable hydrolysis of the
Starting from chloroacetaldehyde dimethyl acetal and side-chain ester moiety took place. An enrichment of
potassium butyrate, a nucleophilic substitution leads to one enantiomer in the remaining substrate was observed
the acetal-protected 2-methylpropanoate which can be with several enzymes but the enantiomer ratio[26] that we
deprotected in a second step affording 2-oxoethyl 2- obtained was rather low (E¼2 3). Neither variation of
methylpropanoate (6).[17] Together with di-silylated[18] the amount of water, of pH or buffer strength nor the
glycolic acid (7) and trimethylsilyloxytrifluoromethane- variation of the organic co-solvent showed any benefit.
sulfonate (TMSOTf) as a catalyst the racemic dioxola- The reason for this inferior selectivity is the lack of sta-
none (R,S)-2 can be formed in good yields which can bility of the racemate in the presence of water. Further
21]
be easily purified by distillation (Scheme 3).[19
investigations showed that, depending on the pH, espe-
This pathway affords an easy and cheap access to the cially the side-chain ester moiety of (R,S)-2 hydrolyzes
desired racemate on a multi-ton scale without having readily in the absence of enzymes. The addition of en-
to resort to elaborate purification steps. The main im- zymes enhances this ester hydrolysis.
purity 2-oxoethyl 2-methylpropanoate (6) does not dis-
turb further process steps because it is also formed dur-
ing the resolution process (vide supra).
Alcoholysis
The experience from hydrolysis experiments indicated
that, in order to suppress the unwanted non-enzymatic
Scheme 3. Synthesis of (R,S)-2.
Scheme 4. Enzymatic hydrolysis of (R,S)-2.
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Alfred Popp et al.
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reaction, the reactant (also serving as a solvent) should Novozym 525 L (NZ525L) which is a rather dilute solu-
be a weaker nucleophile. Therefore, we screened lower tion of the enzyme in water and Novozym 435 (NZ435)
alcohols as nucleophiles [R¼Me, Et, i-Pr, n-Pr, (CH2)7 which contains the enzyme in an immobilized form. Al-
CH3]. In this screening, alcoholysis showed a similar though both formulations show an identical E value of
spectrum of products as hydrolysis (Scheme 5).
12 (Figure 1) we chose the immobilized formulation be-
Formation of the metastable hemiacetal 8 was verified cause there is only a minimum amount of water in the re-
with NMR methods. During the reaction (T<508C) al- action mixture (water coming from the enzyme formula-
most no aldehyde 6 could be detected. Instead of this, tion) and the reaction mixture can easily and quickly be
three significant NMR signals [d¼4.72 (dd, J1 ¼ separated from the catalyst after the reaction.
4.9 Hz, J2 ¼4.7 Hz, 1H, COO-CH2-CH), 4.09 (dd, J1 ¼
The fast separation of the catalyst, as mentioned
11.4, J2 ¼4.7 Hz, 1H, COO-CH2), 4.05 (dd, J1 ¼11.4, above, is of vital importance because, with an enantio-
4.9 Hz, 1H, COO-CH2)] imply the formation of 8. After meric ratio of 12, an appreciable amount of the wanted
warming the reaction mixture (e.g., removing the sol- enantiomer is also converted. This means that, after
vent) these signals were missing. Instead the NMR sig- the wanted enantiomeric excess is reached, any subse-
nals of aldehyde 6 were observed. So a quantitative quent reaction reduces the yield.
monitoring of the reaction turnover is possible.[27] The
Enzymes normally show their best activity and selec-
absolute stereochemistry of the product was established tivity at an optimal temperature. Low temperatures
by comparing the sign of the optical rotation with a sam- slow down the rate of the enzymatic reaction whereas
ple that was further processed to give the known com- the enantiomeric ratio is only slightly increased (lower-
pound 1.
ing the temperature by 10 K results in an increase:
Contrary to the behavior of (R,S)-2 in water, the race- Elow temp. ꢁEhigh temp. þ1[28]). The temperature optimum
mate is chemically reasonably stable in lower alcohols was determined in the temperature range between 20
(e.g., MeOH, EtOH). Therefore, the next step was iden- and 408C. The resulting E values (Figure 2) and reaction
tification of an appropriate enzyme and alcohol. En- rates (Figure 3) are approximately (within the error lim-
zyme screening was carried out using a binary mixture its) independent of the temperature (Table 1). The ini-
of alcohol and co-solvent [ROH/TBME¼1:1 (v/v)]. tial enzyme activity also seemed to be constant within
Under these conditions we identified two enzymes the selected temperature range. These findings indicate
which catalyzed the desired reaction with a reasonable a stable process in that the enzyme tolerates small fluc-
selectivity: pig pancreas lipase (PPL) and Candida ant- tuations in temperature which often occur in industrial
arctica lipase B (CALB). Further studies revealed that processes.
the enzyme of choice was CALB together with metha-
nol as nucleophile.
All scale-up experiments (vide supra) were carried out
at the initial substrate concentration of 15% (w/v) (ap-
There are two formulations of CALB available from prox. 0.65 mol¥Lꢀ1). With regard to a high space-time
Novozymes A/S(Bagsvaerd, Denmark) on a large scale,
yield, however, the substrate concentration had to be in-
creased (and optimized). A screening of several concen-
trations revealed no significant change in the enantio-
meric ratio up to a concentration of 40% (w/v) (Fig-
Scheme 5. Enzymatic alcoholysis of (R,S)-2.
Figure 1. Selectivity of the resolution of (R,S)-2.
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Enzymatic Kinetic Resolution of 1,3-Dioxolan-4-one and 1,3-Oxathiolan-5-one
FULL PAPERS
Figure 2. Temperature influence on the enantiomeric ratio of
the resolution of (R,S)-2.
Figure 4. Dependence of the enantiomeric ratio on the sub-
strate concentration (see also Table 2: entries 4 7).
Figure 3. Temperature influence on the reaction rate of the
resolution of (R,S)-2.
Figure 5. Dependence of the enantiomeric ratio on the sub-
strate concentration (see also Table 2: entries 8 16).
ure 4). In addition, when using NZ525L, the reaction Actually, the easiest way to purify the product by chro-
rate is constant (Table 2: entries 4 7). matographic separation of the degradation products.
We evaluated the tolerance of the enzyme towards This worked very well on a laboratory scale but could
varying solvent compositions and observed very little not be adapted to a large-scale production. The method
influence on the process (Table 2). Small fluctuations of choice for the purification of (R)-2 proved to be as fol-
of the composition, which often appear in industrial lows. After the removal of the solvents, the low-boiling
processes after the solvent recycling, have no signifi- degradation products 5b, 7b and the main fraction of 6
cant impact either on the selectivity or on the relative by distillation, the separation of the hydroxymethyl-
activity of the enzyme (entries 8, 9, 11 and 12). In con- dioxolanone 4 proved to be critical. During the resolu-
trast, the relative activity of the enzyme declines when tion compound 4 was formed at about 20% (w/w) cor-
using an excess of methanol (entries 10 and 14). This ef- responding to the amount of product.[30] In addition
fect can be compensated for by using a higher enzyme to this, 4 could not be separated by distillation because
concentration (e.g., doubling of the concentration: en- its boiling point differs only slightly from that of the
tries 13/14 vs. 15/16). Nevertheless, the enzyme does product (R)-2. However, the water solubility of the pri-
not completely loose its activity, even in pure metha- mary alcohol 4 is significantly higher than that of the di-
nol.
oxolanone. The byproduct could thus be completely re-
After we had established an optimized and robust moved by multiple or continuous extractions with wa-
process a suitable work-up procedure was developed. ter. We favor the continuous extraction method, be-
Adv. Synth. Catal. 2004, 346, 682 690
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Alfred Popp et al.
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Table 1. Temperature influence on the reaction rate.[a]
Entry
Reaction temperature [8C]
Conversion [%] after 20 h
Initial enzyme activity[b] [mmol¥minꢀ1 ¥gꢀ1
]
1
2
3
20
30
40
64
61
59
440
400
410
[a]
(R,S)-2 (0.65 mol¥Lꢀ1), naphthalene (0.7 mol equiv. as internal standard) in TBME/MeOH 1: 1 (v/v), ꢀN1Z525L (6.1 g¥Lꢀ1).
[b]
The activity was calculated using following equation[29]: initial activity¼Dnsubstrate ¥Dtꢀ1 ¥menzyme formulation
.
Table 2. Dependence of the reaction rate on the substrate concentration and the solvent composition.
Entry
Substrate
Composition of
TBME/MeOH (v/v)
Enzyme
Initial enzyme
activity[c]
concentration[a]
[%] (w/v)
concentration[b]
[g¥Lꢀ1
]
[mmol¥minꢀ1 ¥gꢀ1]
4
5
6
7
8
15
20
30
40
15
15
15
40
40
40
40
40
40
1: 1
1: 1
1: 1
1: 1
2: 1
3: 2
1: 2
2: 1
3: 2
2: 9
1: 9
2: 9
1: 9
6.1
8.2
12.3
16.4
6.1
440
430
420
440
400
400
280
420
440
430
360
520
490
9
6.1
6.1
10
11
12
13
14
15
16
16.4
16.4
9.5
10.0
19.0
20.0
[a]
Naphthalene (0.3 mol-equiv. as internal standard) at 208C.
[b]
[c]
NZ525L.
See footnote[b] in Table 1.
cause it is fast [having in mind the poor stability of similar substrates. The following examples are to evalu-
(R,S)-2 in aqueous media] and can be scaled up easily. ate the scope of the resolution process and to perform a
The final purification of the product proceeded by vac- preliminary structure-activity relationship study.
uum distillation. The forerun contained mainly the re-
All racemates were synthesized from di-silylated gly-
sidual aldehyde 6 which could be reused (together with colic acid and the appropriate aldehyde according to the
the amount separated in advance) for the preparation procedure described above in good to excellent yields.
of (R,S)-2 (Scheme 3).
For known compounds (see references in Table 3) the
Using a loop reactor and the immobilized formulation analytical data were identical to literature data. Then
of CALB (NZ435), the scale-up proved to be fairly easy we studied the suitability of the obtained racemates
and economic. The optimized reaction parameters for an enzymatic resolution. A preliminary screening
[(R,S)-2 (0.65 mol¥Lꢀ1) in TBME/MeOH 1:1 (v/v), showed that the enzyme of choice was CALB [either im-
NZ435 (8.5 g¥Lꢀ1), 25ꢂ58C, average retention time mobilized (NZ435) or as crude solution (NZ525L)].
in the reactor loop: 1.7 min] were transferred to a pi- With this enzyme moderate to good enantiomeric ratios
lot-plant scale [10 kg (R,S)-2/batch]. Neither the E value could be reached at reasonable enzyme activities (Ta-
nor the activity of the enzyme changed. The resolution ble 3). Not surprisingly, we observed that the E value
of 2 on a 1000-kg scale (seven batches overall) also dis- and the activity depended strongly on the structure of
played the same performance.
the substrate. From these substrates we derived the fol-
lowing structural requirements for the enzymatic resolu-
tion of dioxolanone derivatives:
Enzymatic Solvolysis of 1,3-Dioxolan-4-one and 1,3-
Oxathiolan-5-one Derivatives
The 2-position of the dioxolanone ring bears at least
one hydrogen atom. The experiments show that a sec-
ond substituent at the stereogenic center prevents the
conversion (compounds 13 and 14). This might be due
In order to demonstrate the broad applicability we ap-
plied the before-mentioned resolution to chemically
686
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Enzymatic Kinetic Resolution of 1,3-Dioxolan-4-one and 1,3-Oxathiolan-5-one
FULL PAPERS
to a strong steric interaction between the enzyme and
the substrate that blocks the access to the active site.
But what is more important, both compounds are cy-
clic esters of tertiary alcohols. It is known that lipases
solvolyse such esters only in rare cases.
The substrate must not be disubstituted at the 5-posi-
tion of the dioxolanone ring (compound 16).
Sterically demanding rigid substituents in the 2-posi-
tion of the dioxolanone ring increase the selectivity
of the resolution (compound 10 vs. 9, 11 and 15). But
rate. This is due to an unfavorable equilibrium posi-
tion. The reaction even stops after the conversion
has reached approximately 45% and an ee of the re-
maining enantiomer of 80%. After removing the deg-
radation products and purifying the enantiomerically
enriched substrate the subsequent resolution proceeds
smoothly and we obtained an enantiopure product ox-
athiolan 17.
The described method for the resolution of dioxola-
the a-carbon of the side-chain must not be quaternary nones can also be expanded to acyclic compounds. Until
(compound 12) likely because of unfavorable steric in- now this procedure is still limited to stable hemi-acylals,
teractions.
but further work will be done to investigate the limits of
If one oxygen atom is replaced by a sulfur atom (com- the resolution process and to collect more data for a re-
pound 17) the reaction still proceeds with a high E val- liable SAR study. With our method the enantiopure
ue (compound 17 vs. 10). During this resolution, how- hemi-acylal (R)-18 was prepared in good yields
ever, we observed a significant decrease of the reaction (Scheme 6).[31]
Table 3. Enzymatic resolution of dioxolanone- and oxathiolan-derivatives compared to compound 2.[a]
Compound
E
Initial enzyme
[a]2D0
ee [%]
activity[d] [mmol¥minꢀ1 ¥gꢀ1
]
2
12
970
680
þ19.8 (d¼1.18, neat)
>98[b]
9[29]
11
þ0.7 (d¼1.19, neat)
96[b]
10[20]
11[30]
12[31]
13
65
1380
370
30
þ3.9 (d¼1.11, neat)
90[b]
98[b]
14
þ1.9 (d¼1.13, neat)
n.d.
n.d.
n.d.
13
n.d.
0
n.d.
14[19]
15
0
n.d.
850
þ5.5 (d¼1.11, neat)
>99[c]
16
17
n.d.
65
10
n.d.
100
þ6.1 (d¼1.19, neat)
96[b]
[a]
Substrate concentration [15% (w/v)], naphthalene (0.4 mol-equiv. as internal standard) in TBME/MeOH 1: 1 (v/v), NZ435
(2.5 g¥Lꢀ1).
[b]
[c]
[d]
Determined by GC method.
Determined by HPLC method.
See footnote[b] in Table 1.
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Alfred Popp et al.
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Procedure for the Preparation of the Dioxolanone
Derivatives 2 16
Compounds known from literature were also prepared in ac-
cordance with this method.
A stirred solution of di-silylated glycolic acid (7; 0.40 mol)
and Me3SiOTf (12.0 mmol) in anhydrous dichloromethane
(250 mL) was cooled to ꢀ788C. To this solution the appropri-
ate aldehyde (0.44 mol) was added dropwise. After warming to
room temperature the solution was stirred for further 20 h at
this temperature. The reaction mixture was washed with satu-
rated aqueous sodium bicarbonate and dried over sodium sul-
fate. The solvent was removed under vacuum and the residue
was purified by distillation to give the desired products.
Isobutyric acid 4-oxo-[1,3]dioxolan-2-ylmethyl ester (2) us-
ing (6): colorless liquid, bp 55 588C/0.02 mbar; 1H NMR:
d¼5.80 (dd, J1 ¼2.8 Hz, J2 ¼3.2 Hz, 1H, CHCO2), 4.37 (dd,
J1 ¼2.8 Hz, J2 ¼12.4 Hz, 1H, COO-CH2), 4.26 (dd, J1 ¼
3.2 Hz, J2 ¼12.4 Hz, 1H, COO-CH2), 4.38 (dd, J1 ¼15.0 Hz,
J2 ¼0.8 Hz, 1H, OCH2CO), 4.29 (dd, J1 ¼15.0 Hz, J2 ¼0.6 Hz,
1H, OCH2CO), 2.60 [sept, 1H, CH(CH3)2], 1.15 [d, 6H,
CH(CH3)2]; tr,GC (min)¼19.4 (R), 19.7 (S).
Scheme 6. Preparation of the racemic hemi-acylal (R,S)-18
and subsequent enzymatic alcoholysis.
The resolution proceeded with an E value of 15, com-
parable to that of the non-rigid dioxolanones and an ini-
tial enzyme activity of 120 mmol¥minꢀ1 ¥gꢀ1 and which is
significantly lower than that of its corresponding cyclic
substrate 2 (Table 3).
2-Ethyl-2-methyl-[1,3]dioxolan-4-one (13) using butan-2-
1
one: colorless liquid, bp 140 1428C; H NMR: d¼4.37 (d,
J¼15.3 Hz, 1H), 4.32 (d, J¼15.3 Hz, 1H), 1.85 (q, J¼7.5 Hz,
2H), 1.50 (s, 3H), 1.00 (t, J¼7.5 Hz, 3H); tr,HPLC (min)¼6.6
(R), 7.1 (S).
Conclusions
2-Heptyl-[1,3]dioxolan-4-one (15) using octanal: colorless
1
liquid, bp 90 928C/7 mbar; H NMR: d¼5.60 (t, J¼4.9 Hz,
Developing an enzymatic resolution process of dioxola-
1H), 4.35 (d, J¼15.0 Hz, 1H), 4.20 (d, J¼15.0 Hz, 1H), 1.85
nones and related compounds we have established an (m, 2H), 1.40 (m, 2H), 1.25 0.85 (m, 11H); tr,GC (min)¼21.7
(R), 22.0 (S).
easy and economic method for the preparation of impor-
tant enantiopure synthetic precursors of pharmaceuti-
cals. The development of an industrial resolution of 2
shows in an impressive manner the power of enzymatic
resolutions for the production of pharmaceutical key in-
termediates (e.g., Amdoxovir). The pilot process gave
the pure product (R)-2 (1100 kg with ee>98%) with
an overall yield of 22% [enzyme load: NZ435/(R,S)-
2¼3% (w/w)].
2-Cyclohexyl-5,5-dimethyl-[1,3]dioxolan-4-one (16) using
cyclohexylcarbaldehyde: colorless liquid, bp 104 1068C/0.7
1
mbar; H NMR: d¼5.25 (d, J¼4.6 Hz, 1H), 1.90 (m, 4H),
1.80 (m, 2H), 1.50 (s, 3H), 1.40 (s, 3H), 1.30 1.10 (m, 5H).
tr,GC (min)¼21.7 (R), 22.1 (S).
2-Cyclohexyl-[1,3]oxathiolan-5-one (17): A solution of mer-
captoacetic acid (0.42 mol, 38.6 g) in anhydrous dichlorome-
thane (250 mL) was cooled to 08C. Cyclohexylcarbaldehyde
(0.42 mol, 49.2 mL) was added dropwise, the resulting solution
was brought to room temperature and stirred for 20 h at this
temperature. The reaction mixture was washed with saturated
We also synthesized a variety of (a)cyclic racemates
and employed them in our enzymatic resolution proto-
col. Our findings are the basis for future structure-activ- aqueous sodium bicarbonate and dried over sodium sulfate.
The solvent was removed under vacuum and the residue was
purified by distillation to give the pure compound as a colorless
liquid; bp 120 1248C/5 mbar; 1H NMR: d¼5.30 (d, J¼6.5 Hz,
1H), 3.65 (d, J¼16.6 Hz, 1H), 3.60 (d, J¼16.6 Hz, 1H), 2.00 (m,
1H), 1.80 (m, 4H), 1.45 1.15 (m, 6H); tr,GC (min)¼23.7 (R),
24.2 (S).
ity relationship (SAR) studies to further elaborate on
the presented substrate requirement rules.
Experimental Section
2-(Acetyloxy)-2-methoxyethyl 2-methylpropanoate (18): p-
Toluenesulfonic acid monohydrate (PTSA; 3.90 mmol,
0.75 g) was dissolved in a mixture of 2,2-dimethoxyethyl 2-
methylpropanoate (0.28 mol, 50.0 g) and acetic anhydride
(0.34 mol, 34.8 g). The solution was heated to 1208C for
12.5 h. After cooling to room temperature the reaction mixture
was washed with H2O (2ꢃ50 mL) and dried over sodium sul-
fate. After filtration the residue was purified by distillation to
give the pure compound as a colorless liquid; yield: 37.6 g
(0.18 mol, 63%); bp 52 558C/0.3 mbar; 1H NMR: d¼5.91
(dd, J1 ¼5.4 Hz, J2 ¼4.9 Hz, 1H), 4.24 (dd, J1 ¼11.6, J2 ¼
4.9 Hz, 1H), 4.07 (dd, J1 ¼11.6, J2 ¼5.4 Hz, 1H), 3.48 (s, 3H),
Spectroscopic Methods
1H NMR spectra were recorded in deuterochloroform with
TMSas internal reference on a Bruker Avance 500 spectrom-
eter operating at 400 MHz. Enantiomeric excesses were either
determined by gas chromatography (HP5890II gas chromato-
graph equipped with an FID detector; column: CP Chirasil
CB; injector temperature: 2208C; detector temperature:
2508C) or HPLC [HP 1100 HPLC equipped with an UV detec-
tor (200, 204 und 210 nm); column: Chiralpak AD-RH; isocrat-
ic operation].
688
¹ 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Adv. Synth. Catal. 2004, 346, 682 690

Enzymatic Kinetic Resolution of 1,3-Dioxolan-4-one and 1,3-Oxathiolan-5-one
FULL PAPERS
2.58 (sept, J¼7.0, 1H), 2.12 (s, 3H), 1.17 (d, J¼7.0 Hz, 6H); tr,GC
(min)¼17.7 (R), 18.6 (S).
[13] L. K. Naeger, N. A. Margot, M. D. Miller, Antimicrob.
Agents Chemother. 2003, 46, 2179 2184.
[14] A. Popp, J. Stohrer, H. Petersen, A. Gilch, J. Rockinger-
Mechlem, (Consortium fuer elektrochemische Industrie
GmbH, Germany), EP 1229127 A1; Chem. Abstr. 2002,
137, 139499.
Typical Procedure for the Preparation of (R)-(þ)-
Isobutyric Acid 4-Oxo-[1,3]dioxolan-2-ylmethyl Ester
(2)
[15] H. Spahn-Langguth, Pharm. Unserer Zeit 1996, 25, 198
219.
All enzymatic resolutions were performed according to this
method.
[16] W. H. J. Boesten, P. Riebel, G. Niederhumer, (DSM Fine
Chemicals Austria Nfg G. m. b. H.þCo. K.-G., Austria),
WO 2001092199 A1, 2001; Chem. Abstr. 2001, 136, 5726.
[17] R. Rothermel, M. Hanack, Liebigs Ann. Chem. 1991,
1013 1020.
[18] A. Wissner, Tetrahedron Lett. 1978, 2749 2752.
[19] S. Abazi, L. Parra Rapado, K. Schenk, P. Renaud, Eur. J.
Org. Chem. 1999, 477 483.
[20] W. H. Pearson, M.-C. Cheng, J. Org. Chem. 1987, 52,
1353 1355.
[21] R. Ramage, A. M. MacLeod, G. W. Rose, Tetrahedron
1991, 47, 5625 5639.
(R,S)-2 (50.0 g, 0.27 mol) was dissolved in a mixture of
TBME (185 mL) and MeOH (185 mL) in a 1-L 4-necked flask.
Novozym 435 (NZ435, 2.6 g) was added and the resulting mix-
ture was stirred vigorously at 308C. After the desired ee was
reached (GC control) the enzyme was filtered off and the sol-
vent was removed under reduced pressure. The residue was
then taken up in TBME (100 mL) and washed twice with water
(100 mL each). The organic phase was separated, dried over
sodium sulfate and then freed from solvent under vacuum.
The crude product was purified by distillation to give the prod-
uct (R)-2 as a colorless liquid; overall yield: 10.3 g (0.05 mol,
20%).
[22] K. Faber, Biotransformations in Organic Chemistry,
Springer Verlag, Berlin, Germany, 1997.
[23] S. M. Roberts, G. Casy, M.-B. Nielsen, Biocatalysis for
Fine Chemicals Synthesis, John Wiley & Son Ltd, New
York, Chichester, Brisbane, Toronto, Singapore, 1999.
[24] U. T. Bornscheuer, R. J. Kazlauskas, Hydrolases in Or-
ganic Synthesis: Regio- or Stereoselective Biotransforma-
tions, John Wiley & Son Ltd, New York, Chichester,
Brisbane, Toronto, Singapore, 1999.
[25] The following enzymes were used: lipases: Alcaligines
sp., Aspergillus niger, Aspergillus oryzae, Candida ant-
arctica A, Candida antarctica B, Candida cylindracea,
Candida lipolytica, Candida rugosa, Mucor javanicus,
Mucor miehei, Penicilliumroqueforti , Pseudomonas ce-
pacia, Pseudomonas fluorescens, Pseudomonas sp., Rhi-
zomucor miehei, Rhizopus arrhizus, Rhizopus niveus,
Thermomyces lanuginose, pig pancreas, wheat germ es-
terases: acetylcholine esterase from Elektrophorus elec-
tricus, Bacillus species, Bacillus stearothermophilus, Ba-
cillus thermoglucosidasius, Candida lipolytica, Mucor
miehei, Saccharomyces cerevisiae, Thermoanaerobium
brockii, horse liver, pig liver.
References and Notes
[1] L. J. Wilson, W. B. Choi, T. Spurling, D. C. Liotta, R. F.
Schinazi, D. Cannon, G. R. Painter, M. St. Clair, P. A.
Furman, Bioorg. Med. Chem. Lett. 1993, 3, 169 174.
[2] W. B. Choi, L. J. Wilson, S. Yeola, D. C. Lotta, R. F. Schi-
nazi, J. Am. Chem. Soc. 1991, 113, 9377 9379.
[3] D. C. Lotta, R. F. Schinazi, W. B. Choi, (Emory Universi-
ty, USA), WO 9214743 A2, 1992; Chem. Abstr. 1993, 118,
22551.
[4] D. C. Liotta, W. B. Choi, (Emory University, USA), WO
9111186 A1, 1991; Chem. Abstr. 1991, 115, 208463.
[5] L. K. Hoong, L. E. Strange, D. C. Liotta, G. W. Koszalka,
C. L. Burns, R. F. Schinazi, J. Org. Chem. 1992, 57, 5563
5565.
[6] Y. Yao, Y. F. Wang, (Altus Biologics Inc., USA), WO
2000022157 A1, 2000; Chem. Abstr. 2000, 132, 278245.
[7] H. O. Kim, R. F. Schinazi, S. Nampalli, K. Shanmugana-
than, D. L. Cannon, A. J. Alves, L. S. Jeong, J. W. Beach,
C. K. Chu, J. Med. Chem. 1993, 36, 30 37.
[26] C.-S. Chen, Y. Fujimoto, G. Girdaukas, C. J. Sih, J. Am.
Chem. Soc. 1982, 104, 7294 7299.
[27] For the calculation of the enantiomeric ratio E of a ki-
netic resolution it is essential to know either the enantio-
meric excess (ee) of the substrate and the product or the
extent of conversion c together with one enantiomeric
excess (substrate or product). In our case, the resolution
of (R,S)-2 affords only one chiral ™product∫, the remain-
ing enantiomer. The actual reaction product of the reso-
lution is the aldehyde 6, which is not chiral. Therefore for
the calculation of the E value an appropriate method for
the determination of the conversion rate was necessary.
On an industrial scale the measurement of c with
NMR spectroscopic methods is sufficient and, what is
more important, easy and fast. On a laboratory scale
we used another method. The conversion c was deter-
mined using an internal standard. This method provided
[8] A. H. Corbett, J. C. Rublein, Current Opinion in Investi-
gational Drugs 2001, 2, 348 353.
[9] P. A. Furman, D. Cleary, L. C. Trost, J. W. Bigley, G. R.
Painter, Drugs Future 2000, 25, 454 461.
[10] P. A. Furman, J. Jeffrey, L. L. Kiefer, J. Y. Feng, K. S. An-
derson, K. Borroto-Esoda, E. Hill, W. C. Copeland, C. K.
Chu, J.-P. Sommadossi, I. Liberman, R. F. Schinazi, G. R.
Painter, Antimicrobial Agents and Chemotherapy 2001,
45, 158 165.
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angle Pharmaceuticals, Inc., USA), WO 2000025797 A1,
2000; Chem. Abstr. 2000, 132, 318062.
[12] Z. Gu, M. A. Wainberg, N. Nguyen-Ba, L. L×Heureux, J.-
M. De Muys, T. L. Bowlin, R. F. Rando, Antimicrob.
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Adv. Synth. Catal. 2004, 346, 682 690
asc.wiley-vch.de
¹ 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
689

Alfred Popp et al.
FULL PAPERS
reliable and precise data. But due to the fact, that even a
small variation of the input values (ee or c) causes a sig-
nificant change in the numerical value E, all stated val-
ues were verified by plotting the ee c diagrams for sev-
eral extents of conversion so that a best-fit graph could
be generated.
J1 ¼2.5 Hz, J2 ¼2.7 Hz, 1H, CHCO2), 3.88 (dd, J1 ¼
2.5 Hz, J2 ¼12.9 Hz, 1H, COO-CH2), 3.83 (dd, J1 ¼
2.7 Hz, J2 ¼12.9 Hz, 1H, COO-CH2), 4.43 (dd, J1 ¼
14.9 Hz, J2 ¼0.9 Hz, 1H, OCH2CO), 4.29 (dd, J1 ¼
14.9 Hz, J2 ¼0.6 Hz, 1H, OCH2CO). A direct detection
of 4 with GC or HPLC methods is not possible, but after
reaction with N-methyl-N-(trimethylsilyl)trifluoroaceta-
mide (MSTFA) the silylated compound can be quantita-
tively determined by GC.
[28] E. Holmberg, K. Hult, Biotechnol. Lett. 1991, 13, 323
326.
[29] A. Liese, K. Seelbach, C. Wandrey, Industrial Biotrans-
formations, Wiley-VCH Verlag GmbH, Weinheim,
2000, p. 61.
[31] The absolute configuration was derived from the known
chirality of (R)-2 under the assumption that the stereo-
specificity of the lipase does not change under otherwise
identical reaction conditions.
[30] The hydroxymethyldioxolanone 4 was identified with
NMR spectroscopic methods: 1H NMR: d¼5.70 (dd,
690
¹ 2004 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
asc.wiley-vch.de
Adv. Synth. Catal. 2004, 346, 682 690