Synthesis of alkyl (R)-lactates and alkyl (S,S)-O-lactyllactates by alcoholysis of rac-lactide using Novozym 435

Article abstract of DOI:10.1016/j.tetlet.2006.07.034

Enzymatic alcoholysis of rac-lactide for kinetic resolution was carried out in organic solvents. Effects of organic solvent, reaction temperature, and alcohol as a nucleophile were also investigated in Novozym 435-catalyzed alcoholysis of rac-lactide. Both alkyl (R)-lactate and alkyl (S,S)-O-lactyllactate were simultaneously obtained in high yields (>45%) and high enantiopurities (>97% ee) through Novozym 435-catalyzed ring-opening of rac-lactide and subsequent enantioselective alcoholysis of the resultant alkyl O-lactyllactate.

Full text of DOI:10.1016/j.tetlet.2006.07.034

Tetrahedron Letters 47 (2006) 6517–6520
Synthesis of alkyl (R)-lactates and alkyl (S,S)-O-lactyllactates
by alcoholysis of rac-lactide using Novozym 435
Nan Young Jeon,a,b Sung-Jin Ko,a Keehoon Won,a Han-Young Kang,b Bum Tae Kim,a
Yeon Soo Leea and Hyuk Leea,*
aBioorganic Science Division, Korea Research Institute of Chemical Technology, Daejeon 305-600, Republic of Korea
bDepartment of Chemistry and Institute of Basic Science, Chungbuk National University, Chungbuk 361-763, Republic of Korea
Received 15 June 2006; revised 5 July 2006; accepted 10 July 2006
Available online 31 July 2006
Abstract—Enzymatic alcoholysis of rac-lactide for kinetic resolution was carried out in organic solvents. Effects of organic solvent,
reaction temperature, and alcohol as a nucleophile were also investigated in Novozym 435-catalyzed alcoholysis of rac-lactide. Both
alkyl (R)-lactate and alkyl (S,S)-O-lactyllactate were simultaneously obtained in high yields (>45%) and high enantiopurities (>97%
ee) through Novozym 435-catalyzed ring-opening of rac-lactide and subsequent enantioselective alcoholysis of the resultant alkyl O-
lactyllactate.
Ó 2006 Elsevier Ltd. All rights reserved.
Lactic acid (2-hydroxypropanoic acid) is one of the
simplest chiral compounds, and enantiopure (R)- and
(S)-lactic acid derivatives are important synthons for
chiral drugs.1 (S)-Lactic acid is commercially produced
through microbial fermentation, while (R)-lactic acid is
relatively difficult to obtain. In addition, ethyl (S,S)-O-
lactyllactate, which is a linear dimer of (S)-ethyl lactate,
was reported to be used as a starting material for the
alkyl lactate oligomers, which have anticancer activity.2
lactyllactate from rac-lactide, which is a cyclic dimer
produced from the dehydration of lactic acid. Lipase-
catalyzed ring-opening polymerizations of lactide into
poly(lactic acid)s have been reported.6 However, to the
best of our knowledge, no previous report has been
issued on enzymatic production of chiral compounds
from rac-lactide.
In preliminary experiments, commercially available lip-
ases7 were screened for alcoholysis of rac-lactide (rac-
1) with n-butanol (4d) in hexane/THF (90:10).8 Among
the lipases tested, a few lipases including Novozym
435 functioned in the formation of butyl O-lactyllactate
(2d), the corresponding ring-opened product. However,
their enantioselectivities were low so that SS-1 was alco-
holyzed slightly more than RR-1. Nevertheless, SS-2d
was successfully obtained in high ee when Novozym
435 was used. It turned out that this is because Nov-
ozym 435 catalyzed enantioselective alcoholysis of the
ring-opened RR-2d with an excess of 4d to generate a
butyl (R)-lactate (R-3d). It is noteworthy that Novozym
435 has quite different enantioselectivity toward cyclic 1
and its ring-opened 2. This finding prompted us to
examine a novel route for enantioselective synthesis of
SS-2 and R-3 from rac-1 by Novozym 435 (Scheme 1).
It is known that CALB demonstrates (R)-stereoselectiv-
ity toward secondary alcohols.3 Accordingly, R-3 was
produced by the alcoholysis of ring-opened 2 using Nov-
ozym 435 as shown in this experiment. The reason for
Lipases have been extensively used to obtain chiral alco-
hols and carboxylic acids, due to their excellent chiral
recognition.3 Among the various lipases, Novozym
435, which is an immobilized form of lipase B from Can-
dida antarctica (CALB), possesses the high enantioselec-
tivity for a broad range of substrates.4 Previously, we
reported the enantioselective acylations of racemic alkyl
lactates with vinyl alkanoates using Novozym 435.5 We
have succeeded in obtaining both butyl (R)-O-butanoyl-
lactate and butyl (S)-lactate in excellent yields (48%) and
enantioselectivities (>99.5% ee) on a large scale.
In the present work, Novozym 435 was applied to
obtain enantiopure alkyl (R)-lactate and alkyl (S,S)-O-
Keywords: Novozym 435; Kinetic resolution; Alkyl lactate; rac-Lac-
tide; Alkyl O-lactyllactate.
*
Corresponding author. Tel.: +82 42 860 7019; fax: +82 42 861
0307; e-mail: leeh@krict.re.kr
0040-4039/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2006.07.034

6518
N. Y. Jeon et al. / Tetrahedron Letters 47 (2006) 6517–6520
(entries 9–12). The alcoholyzed products, SS-2d and
R-3d, were obtained in high yields with high ee within
5 h at 60 °C.
O
O
OH
O
R
O
O
O
OH
RO
RO
RO
O
O
O
O
RR-1
RR-2
-3
Lipase-catalyzed alcoholyses of rac-1 with various alco-
hols were also conducted at 60 °C in hexane/THF to
afford SS-2 and R-3 with excellent yields (>45%) and
enantioselectivities (>97% ee) as shown in Table 2.11
Compared to Table 1, the times required to reach 50%
conversion were prolonged because the ratio of Nov-
ozym 435 to rac-1 was reduced for large-scaled reactions
from 2 mg/0.3 mmol to 40 mg/20 mmol. n-Butanol (4d)
showed the highest reactivity among primary alcohols
used (entries 1, 2, and 4). The reaction of 4d was com-
pleted within 20 h, giving a SS-2d and a R-3d in 48%
and 48% of isolated yields, respectively. Isopropyl alco-
hol (4c) exhibited reactivity comparable to 4d (entry 3).
Some secondary alcohols such as isopropyl alcohol were
reported to be efficient substrates for CALB.4a But
cyclohexanol (4e) had low reactivity to achieve a 50%
conversion in 260 h due to the bulkiness of 4e (entry
5). When tert-butanol (4f) or phenol (4g) was used,
rac-1 remained even after 260 h without production of
SS-2 or R-3.
Novozym 435
ROH (4)
+
+
OH
R: a: Me
b: Et
O
O
O
c:
d:
i
-Pr
O
O
n-Bu
e: Cyclohexyl
SS-1
SS-2
Scheme 1. Enzymatic alcoholysis of rac-lactide by Novozym 435.
(S)-preference of Novozym 435 toward cyclic 1 was not
clear. However, when CALB was employed for the ring-
opening reaction of racemic cyclic esters such as d- and
e-lactone derivatives, it has been reported to show (S)-
preference of alcohol moiety although its enantioselec-
tivity was low.9 Novozym 435 is expected to show the
same enantioselectivity toward cyclic 1, which has a
structure similar to that of lactones.
The enzymatic alcoholyses of rac-1 with 4d by Novozym
435 were conducted in various conditions (Table 1).10
Although in hexane/THF solvent both R-3d and SS-2d
were predominantly produced by alcoholysis, in THF it-
self only SS-2d was formed as a major product with
poor enantioselectivities (entries 1 and 2). When acetoni-
trile or toluene was used as a solvent, SS-2d was mainly
obtained (entries 3 and 4). In ethers such as i-Pr2O,
Et2O, and t-BuOMe except for THF, the two successive
alcoholyses of rac-1 produced R-3d in good yields (en-
tries 5–7). Interestingly, CH2Cl2 afforded RR-1 and
SS-2d with high ee without the formation of R-3d, which
could be applied to kinetic resolution of rac-1 to pro-
duce RR-1 and SS-2d (entry 8). Novozym 435 is known
to be very heat-tolerant and can be used even at 70–
80 °C.4a Novozym 435-catalyzed alcoholyses were also
examined at various temperatures in hexane/THF. The
higher the reaction temperature was, the faster the reac-
tion was without deterioration of enantioselectivities
When Novozym 435 was recycled for the alcoholysis of
rac-1 with 4d, its activity was decreased slowly while its
enantioselectivity was maintained (Table 3). The time
required to reach a 50% conversion increased by an
hour per cycle. As a result, in the fourth cycle the 50%
conversion was attained in 9 h.
Typical procedure for the syntheses of the alkyl (S,S)-O-
lactyllactates (SS-2a–e) and alkyl (R)-lactates (R-3a–e)
from the rac-lactide (rac-1) is as follows: To a solution
of rac-lactide12 (rac-1, 20 mmol) and alcohol (4,
60 mmol) in hexane/THF (10 mL, 90:10) was added
Novozym 435 (40 mg). The solution was shaken at
200 rpm at 60 °C. The progress of the reaction was mon-
itored by GC analyses on the chiral column (Cyclosil-
BÒ, Agilent). After completion of alcoholysis, Novozym
435 was removed by filtration and the solvent and the
Table 1. Optimization of alcoholysis of rac-lactide (1) with n-butanol (4d) by Novozym 435a
Entry
Solvent
Temp (°C)
Time (h)
GC ratio (%)b
RR-1/SS-1
RR-2/SS-2
R-3/S-3
1
2
3
4
5
6
7
8
9
Hexane/THFc
THF
Toluene
Acetonitrile
i-Pr2O
t-BuOMe
Et2O
CH2Cl2
Hexane/THFc
Hexane/THFc
Hexane/THFc
Hexane/THFc
30
30
30
30
30
30
30
30
30
40
50
60
8
8
8
8
8
8
8
8
14d
9d
7d
5d
3/0
9/>49
10/37
12/34
6/35
8/>49
6/48
17/>49
9/47
0/>49
0/>49
0/>49
0/>49
37/0
4/0
1/0
35/12
37/15
35/14
5/0
15/1
0/0
41/2
0/0
0/0
8/0
36/0
28/0
32/0
0/0
>49/0
>49/0
>49/0
>49/0
10
11
12
0/0
0/0
a Reaction conditions: rac-1 (0.3 mmol), 4d (0.9 mmol), and Novozym 435 (2 mg) in solvent (3 mL) were shaken for 8 h.
b Determined by GC (Cyclosil-BÒ).
c Hexane:THF = 90:10.
d Monitored by GC till 50% conversions.

N. Y. Jeon et al. / Tetrahedron Letters 47 (2006) 6517–6520
Table 2. Alcoholysis of rac-lactide (1) with various alcohols by Novozym 435a
6519
Entry
Alcohol
Timec (h)
SS-2b
R-3b
GC Yield (%)
GC Yield (%)
% ee
% ee
1
2
3
4
5
6
7
MeOH (4a)
EtOH (4b)
i-PrOH (4c)
n-BuOH (4d)
CyOH (4e)
t-BuOH (4f)
PhOH (4g)
160
66
24
49
48
49
48 (48)d
45
98
97
97
>99
99
49
49
47
49 (48)d
46
>99
>99
>99
>99
>99
20
260
>260
>260
No rxn
No rxn
a Reaction conditions: rac-1 (20 mmol), 4 (60 mmol), and Novozym 435 (40 mg) in hexane/THF (90:10, 10 mL) were shaken at 60 °C.
b Determined by GC (Cyclosil-BÒ).
c Monitored by GC till 50% conversions.
d Isolated yields.
Table 3. Recycling of Novozym 435 for the alcoholysis of rac-lactide
2. (a) Naganushi, Y.; Sato, K. Japan Patent 9-227388, 1997;
(b) Watanabe, M.; Murakami, M. Eur. Patent 1378502,
2004.
(1)a
Recycle Timec (h)
SS-2db
R-3db
3. Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in
Organic Synthesis; Wiley-VCH: Weinheim, 2006, pp 61–
183.
GC Yield (%) % ee GC Yield (%) % ee
1
2
3
4
5
6
7
9
49
49
49
49
>99 49
>99 49
>99 49
>99 49
>99
>99
>99
>99
4. (a) Anderson, E. M.; Larsson, K. M.; Kirk, O. Biocatal.
Biotransform. 1998, 16, 181; (b) Kirk, O.; Christensen, M.
W. Org. Process Res. Dev. 2002, 6, 446; (c) Xu, D.; Li, Z.;
Ma, S. Tetrahedron Lett. 2003, 44, 6343; (d) Jacobsen, E.
E.; van Hellemond, E.; Moen, A. R.; Prado, L. C. V.;
Anthonsen, T. Tetrahedron Lett. 2003, 44, 8453.
5. Lee, Y. S.; Hong, J. H.; Jeon, N. Y.; Won, K.; Kim, B. T.
Org. Process Res. Dev. 2004, 8, 948.
a Reaction conditions: rac-1 (0.3 mmol), 4d (0.9 mmol), and Novozym
435 (2 mg) in n-Hexane/THF (90:10, 10 mL) were shaken.
b Monitored by GC till 50% conversions.
c Determined by GC (Cyclosil-BÒ).
6. Matsumura, S.; Mabuchi, K.; Toshima, K. Macromol.
Rapid Commun. 1997, 18, 477.
remaining alcohol were evaporated under reduced pres-
sure. The corresponding products, alkyl (R)-lactates and
alkyl (S,S)-O-lactyllactates, were obtained by vacuum
distillation. The absolute configurations of the SS-2a–e
were identified by comparison with authentic com-
pounds prepared by enzymatic alcoholyses of commer-
cially available SS-1.13
7. The lipases screened are as follows: Candida antarctica
lipase B (Novozym 435, Novozymes), Candida rugosa
lipase (Sigma), porcine pancreatic lipase (Sigma), Rhizo-
mucor miehei lipase (Lipozyme IM, Novozymes), Candida
rugosa lipase (AY, Amano), Pseudomonas fluorescence
(AK, Amano), Burkholderia cepacia (PS, Amano), Asper-
gillus niger (A, Amano), Rhizopus oryzae (F-AP 15,
Amano), Penicillium camembertii (G, Amano), Mucor
javanicus (M, Amano).
8. THF (10%) was added to hexane to increase the solubility
of rac-1.
9. (a) Enzelberger, M. M.; Bornscheuer, U. T.; Gatfield, I.;
Schmid, R. D. J. Biotechnol. 1997, 56, 129; (b) Kondaveti,
L.; Al-Azemi, T. F.; Bisht, K. S. Tetrahedron: Asymmetry
2002, 13, 129.
In conclusion, we found that C. antarctica lipase B
(Novozym 435) was not very stereospecific to lactide,
but highly enantioselective to alkyl O-lactyllactate in
hexane/THF. A new synthetic route for alkyl (R)-lactate
and alkyl (S,S)-O-lactyllactates from rac-lactide was
developed. Alcoholysis of rac-lactide catalyzed by Nov-
ozym 435 produced both alkyl (R)-lactates and alkyl
(S,S)-O-lactyllactates at the same time in high yields
(>45%) and high enantiomeric purities (>97% ee).
10. The concentration effect of rac-1 was not significant (data
not shown).
11. A trace of trimer of alkyl lactate was observed after
prolonged reaction time.
12. rac-Lactide was purchased from Aldrich, USA.
25
Acknowledgment
13. SS-2a: bp 76–78 °C/2 mmHg; ½aꢀD ꢁ35.2 (c 0.015,
CHCl3); 1H NMR (CDCl3, 300 MHz) d 1.50 (d, 3H,
J = 7.2 Hz), 1.53 (d, 3H, J = 6.9 Hz) 2.79 (s, 1H), 3.76 (s,
3H), 4.36 (q, 1H, J = 6.9 Hz), 5.20 (q, 1H, J = 6.9 Hz);
13C NMR (CDCl3, 125 MHz) d 16.9, 20.5, 52.5, 66.8, 69.3,
170.7, 175.1.
This work was financially supported by grants-in aid
from the Korea Research Council for Industrial Science
and Technology (SK-0503).
25
SS-2b: bp 78–80 °C/2 mmHg; ½aꢀD ꢁ32.4 (c 0.015,
1
CHCl3); H NMR (CDCl3, 300 MHz) d 1.28 (t, 3H, J =
References and notes
7.2 Hz), 1.43–1.54 (m, 6H), 2.76 (br, 1H), 4.21 (q, 2H,
J = 7.2 Hz), 4.36–4.39 (m, 1H), 5.18 (q, 1H, J = 6.9 Hz);
13C NMR (CDCl3, 125 MHz) d 14.1, 16.9, 20.5, 61.6, 66.8,
69.5, 170.2, 175.2; MS (m/z) 191 (MH+).
1. (a) Kitazaki, T.; Tasaka, A.; Hosono, H.; Matsushita, Y.;
Itoh, K. Chem. Pharm. Bull. 1999, 47, 360; (b) Roulland,
E.; Monneret, C.; Florent, J.-C. Tetrahedron Lett. 2003,
44, 4125; (c) Kandula, S. R. V.; Kumar, P. Tetrahedron
Lett. 2003, 44, 6149.
25
SS-2c: bp 76–78 °C/5 mmHg; ½aꢀD ꢁ31.8 (c 0.014,
CHCl3); 1H NMR (CDCl3, 300 MHz) d 1.24 (s, 3H,
J = 6.6 Hz), 1.27 (d, 3H, J = 6.6 Hz), 1.46–1.69 (m, 6H),

6520
N. Y. Jeon et al. / Tetrahedron Letters 47 (2006) 6517–6520
2.76 (d, 1H, J = 5.7 Hz), 4.32–4.39 (m, 1H), 5.01–5.07 (m,
19.0, 20.5, 30.5, 65.4, 66.7, 69.5, 170.2, 175.2; MS (m/z)
219 (MH+).
SS-2e: bp 126–130 °C/2 mmHg; ½aꢀD ꢁ29.1 (c 0.022,
CHCl3); 1H NMR (CDCl3, 300 MHz)
1.25–1.85
(m, 16H), 2.72 (br, 1H), 4.35 (q, 1H, J = 6.8 Hz), 4.77–
4.84 (m, 1H), 5.14 (q, 1H, J = 7.1 Hz); 13C NMR (CDCl3,
125 MHz) d16.9, 20.6, 23.6, 25.3, 31.4, 66.7, 69.7, 74.1,
169.6, 175.2; MS (m/z) 245 (MH+).
1H), 5.09–5.16 (m, 1H); 13C NMR (CDCl3, 125 MHz) d
25
16.8, 20.6, 21.7, 66.8, 69.3, 69.6, 169.7, 175.2.
25
SS-2d: bp 131–138 °C/2 mmHg; ½aꢀD ꢁ32.7 (c 0.014,
d
CHCl3); 1H NMR (CDCl3, 300 MHz) d 0.94 (t, 3H,
J = 7.4 Hz), 1.34–1.65 (m, 10H), 2.76 (d, 1H, J = 6.0 Hz),
4.12–4.18 (m, 2H), 4.33–4.38 (m, 1H), 5.18 (q, 1H,
J = 7.1 Hz); 13C NMR (CDCl3, 125 MHz) d 13.6, 16.9,