Enzyme-mediated enantioselective hydrolysis of soluble polymer-supported dendritic carbonates

Article abstract of DOI:10.1016/j.tetlet.2008.09.030

The easy separation of optically active compounds from enzymatic kinetic resolution products by simple precipitation using poly(ethylene glycol) (PEG)-supported dendritic carbonates is disclosed. The water-soluble polymer-supported substrates were prepared by immobilization of (±)-1-phenylethanol onto a monomethoxy PEG (MPEG; av MW 5000) bearing a dendritic spacer through a carbonate linker. The enantioselective hydrolysis of the dendritic substrates of the 1st and 2nd generations using lipase from porcine pancreas (PPL; Type II, Sigma) smoothly proceeded, and a multimolecule of the corresponding (R)-alcohols was released from one molecule of the racemic substrates. The E values of the reactions at 0 °C in a mixed solvent (hexane/buffer = 9/1) were up to >200.

Full text of DOI:10.1016/j.tetlet.2008.09.030

Tetrahedron Letters 49 (2008) 6642–6645
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Enzyme-mediated enantioselective hydrolysis of soluble polymer-supported
dendritic carbonates
*
Masayuki Okudomi, Masaki Nogawa, Naoka Chihara, Makoto Kaneko, Kazutsugu Matsumoto
Department of Chemistry, Meisei University, Hodokubo 2-1-1, Hino, Tokyo 191-8506, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The easy separation of optically active compounds from enzymatic kinetic resolution products by simple
precipitation using poly(ethylene glycol) (PEG)-supported dendritic carbonates is disclosed. The water-
soluble polymer-supported substrates were prepared by immobilization of ( )-1-phenylethanol onto a
monomethoxy PEG (MPEG; av MW 5000) bearing a dendritic spacer through a carbonate linker. The
enantioselective hydrolysis of the dendritic substrates of the 1st and 2nd generations using lipase from
porcine pancreas (PPL; Type II, Sigma) smoothly proceeded, and a multimolecule of the corresponding
(R)-alcohols was released from one molecule of the racemic substrates. The E values of the reactions at
0 °C in a mixed solvent (hexane/buffer = 9/1) were up to >200.
Received 4 August 2008
Revised 3 September 2008
Accepted 8 September 2008
Available online 11 September 2008
Keywords:
Carbonates
Enantioselective hydrolysis
Hydrolase
Ó 2008 Elsevier Ltd. All rights reserved.
Poly(ethylene glycol)-supported substrates
Dendritic spacer
The kinetic resolution of racemic alcohols and esters using
hydrolytic enzymes is one of the practical methods for the prepa-
ration of optically active alcohols, and a significant number of
examples have been published.1 During the reaction process, the
enantiomers, the remaining substrate and the resulting product,
should be separated, and the tedious and wasteful purification step
by column chromatography is still a big problem for an easy oper-
ation. We noticed that poly(ethylene glycol) (PEG) was an inexpen-
sive and convenient soluble polymer,2–5 and that a PEG-supported
strategy could be suitable for an enzymatic transformation and
potentially useful for the easy isolation of the products. We have
already succeeded in the kinetic resolution of low- and middle-
molecular weight monomethoxy PEG (MPEG, av MW 550, 750,
and 5000)-supported substrates with a carbonate linker using a
hydrolytic enzyme (porcine pancreas lipase (PPL), lipase Type II
from Sigma),6,7 and the easy separation of the products has been
achieved. The reaction of MPEG5000-supported substrates, which
were soluble solids and easier to handle, was preferable in terms
of both their reactivity and their enantioselectivity. However, the
number of alcohols immobilized per gram of the substrate (the
loading capacity) is very low (ca. 0.2 mmol/g), and this drawback
is a bottleneck to an effective synthesis. In order to overcome this
problem, we decided to incorporate the basic principle of dendri-
mer chemistry8 into our strategy.9 In this Letter, we disclose the
first example of the hydrolase-mediated kinetic resolution of
PEG-supported dendritic substrates with a carbonate linker. The
substrates have a higher loading capacity (ca. 0.4–0.8 mmol/g),
and a multimolecule of the corresponding optically active alcohols
can be released from one molecule of the substrate.
We first examined the PPL-catalyzed hydrolysis of the MPEG (av
MW 5000)-supported carbonates ( )-1a (R = Ph) and 1b
(R = (CH2)2OBn) bearing two functional moieties (Scheme 1).10–12
The dendritic substrates of the 1st generation were easily synthe-
sized by coupling of the racemic imidazolides ( )-7a and 7b de-
rived from 1-phenylethanol (2a) and 4-benzyloxy-2-butanol (2b),
respectively, with the diol 6, which was prepared by a modification
procedure of a reference (Scheme 1).9 Both substrates, as white sol-
ids, are soluble in water, but not in hexane.
The reaction was carried out under the same reaction condi-
tions as those already reported.7 In a typical experiment, 125 mg
of the substrate (sub. concd 5 mM) and 10 mg of PPL were added
to a mixed solvent (hexane, 4.5 mL; 0.1 M phosphate buffer (pH
6.5), 0.5 mL) in a test tube, and stirred at 30 °C (Table 1). Fortu-
nately, the enantioselective hydrolysis of 1a smoothly proceeded
even in this case, and the corresponding optically active (R)-2a
was obtained (entries 1–4).13 After the reaction, the separation of
products was already finished, because only the resulting alcohol
(R)-2a was extracted in the hexane layer. The aqueous layer was
diluted with CH2Cl2, and the dehydration was performed with
anhydrous Na2SO4. After evaporation, the remaining 1a was easily
precipitated by pouring the residue into Et2O. As a matter of
course, the chemical hydrolysis of 1a gave (S)-2a. To the best of
our knowledge, this is a first example of the enzyme-mediated
* Corresponding author. Tel./fax: +81 42 591 7360.
E-mail address: mkazu@chem.meisei-u.ac.jp (K. Matsumoto).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.09.030

M. Okudomi et al. / Tetrahedron Letters 49 (2008) 6642–6645
6643
CO2Me
HO
CO2Me
CO2Me
MsCl, Et3N
4
CO2Me
a:
b:
R = Ph
MPEG5000-OH
OMs
3 (99%)
O
CH2Cl2
rt
Cs2CO3, DMF
70°C
R = (CH2)2OBn
5 (98%)
O
O
R
O
R
OH
O
O
O
O
Me
N
N
O
Me
( )-7a,b
DMAP, DMF
100 °C
DIBAL
O
O
R
CH2Cl2
0 °C
6 (91%)
( )-1a (98%)
( )-1b (96%)
OH
Me
Scheme 1.
Table 1
Enantioselective hydrolysis of MPEG5000-supported dendritic carbonates ( )-1a
O
R
O
O
R
O
O
O
O
Me
Me
O
O
O
Me
PPL
OH
+
O
O
O
R
R
hexane/buffer = 9/1
24 h
Me
R
O
Me
( )-1
(S)-1
(R)-2
a:
b:
R = Ph
R = (CH2)2OBn
Entry
Substrate
Temperature (°C)
ee of carbonate 1b (%)
ee of alcohol 2c (%)
Conv.
E value
1
2
3
1a
1a
1a
1a
1a
1b
30
20
10
0
0
0
82
81
72
89
92
97
0.48
0.47
0.43
0.29
0.43
0.12
44
60
142
>200
>200
75
4
40
99
5d
6d
73e,f
13f,h
98f,g
97f,i
a
The reaction was performed using 125 mg of the substrate in a test tube.
Determined by GLC analysis after the chemical hydrolysis of 1.
Determined by GLC analysis.
b
c
d
e
f
The reaction was performed on a gram scale of the substrate in a recovery flask.
Isolated in 52% yield as (S)-2a; ½a D29
ꢁ29.5 (c 1.03, MeOH).
ꢀ
The isolated yields of 2 were calculated on the basis of the amount of 1, and the ideal combined yields of both enantiomers of 2 were 200%. We assumed that the MW of
MPEG-OH was 5000.
Isolated in 46% yield; ½a D29
ꢀ
+40.1 (c 0.38, MeOH).
g
Isolated in 154% yield as (S)-2b; ½a D29
ꢀ
+2.1 (c 1.05, MeOH).
h
i
Isolated in 22% yield; ½a D29
ꢁ14.9 (c 1.08, MeOH).
ꢀ
enantioselective hydrolysis of a dendritic substrate. As expected,
lowering the temperature apparently improved the enantioselec-
tivity, while the conversion gradually decreased. For the reaction
at 0 °C, the E value14 was up to >200 and (R)-2a with a 99% ee
was obtained.15 On the other hand, we also tried to examine the
enzymatic hydrolysis of the dendritic substrate 8, which was not
supported by MPEG, under the same reaction conditions (Scheme
2). The reaction, however, did not proceed at all. This indicates that
the solubility of MPEG plays an important role in the enzymatic
hydrolysis.
This reaction was also useful in a preparative-scale operation
(>1 g of ( )-1a) using a recovery flask, and a comparable enantiose-
lectivity was obtained with a higher conversion (entry 5).16,17 The
substrate ( )-1b was also hydrolyzed with a high enantioselectiv-
ity (E value = 75) to afford the optically active (R)-2b with 97%
ee, although the conversion was apparently lower than that of 1a
(entry 6).18,19
Next, we tried to examine the enzymatic reactions of several
substrates bearing a higher loading capacity as shown in Scheme
3,20 the results of which are summarized in Table 2. The compound
( )-9a is an MPEG5000-supported dendritic carbonate of 2nd gener-
ation, and has four functional moieties (entry 1). The enzyme also
O
O
Ph
Ph
O
O
O
Me
Me
MeO
O
( )-8
Scheme 2.

6644
M. Okudomi et al. / Tetrahedron Letters 49 (2008) 6642–6645
Ph
Ph
O
O
O
Me
O
Me
Ph
O
O
Ph
O
O
Me
Me
O
O
O
O
O
O
O
O
O
O
O
O
O
Ph
Ph
Me
O
O
O
O
O
Me
O
O
Ph
Me
Ph
Me
O
O
( )-9a
( )-9b
O
O
Ph
Ph
O
O
Ph
Me
Me
O
O
O
O
O
O
O
Me
O
O
O
O
Ph
O
Me
( )-9c
Scheme 3.
catalyzed the enantioselective hydrolysis of 9a for 48 h to give (R)-
2a (E value = 36), but the reactivity was quite low (conv. = 0.03).
We assumed that the steric hindrance of 9a might inhibit the
reaction, and the elongation of the spacer could cancel the
repulsion between the reactive parts. As expected, introducing a
longer spacer (( )-9b) apparently improved the reactivity, and
the conversion was up to 0.17 (entry 2). Interestingly, the E value
also increased to 120. On the other hand, the enantioselective
hydrolysis of the substrate ( )-9c, which was constructed on PEG
(av MW 4600) as the matrix and had twice the functional groups
of MPEG, proceeded even in this case (entry 3), and the E value
was up to >200.
In conclusion, we succeeded in producing the first example of
the enzyme-mediated kinetic resolution of soluble polymer-sup-
ported dendritic carbonates to afford optically active alcohols (2a
and 2b). In our method, a multimolecule of optically active alco-
hols could be released from one molecule of the racemic sub-
strates, and the separation and isolation of the reaction products
were achieved by a simple precipitation technique. We anticipate
that the concept of the coupling of dendrimer chemistry with
PEG chemistry can provide a useful and eco-friendly protocol in
not only organic chemistry, but also medicinal chemistry for the
development of PEG-supported prodrugs, which gradually release
native drugs by enzymatic hydrolysis.
Acknowledgment
We thank Material Science Research Center (Meisei university)
for NMR analysis.
References and notes
1. (a) Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in Organic Synthesis; Wiley-
VCH: Weinheim, 1999; (b) Bommarius, A. S.; Riebel, B. R. Biocatalysis; Wiley-
VCH: Weinheim, 2004; (c) Faber, K. Biotransformations in Organic Chemistry: A
Textbook, 5th ed.; Springer: Berlin, 2004; (d) Ghanem, A.; Aboul-Enein, H. Y.
Chirality 2004, 17, 1–15; (e) Garcia-Uradiales, E.; Alfonso, I.; Gotor, V. Chem. Rev.
2005, 105, 313–354; (f) Bornscheuer, U. T. Trends and Challenge in Enzyme
Technology; Springer: Berlin, 2005; (g) Fogassy, E.; Nógrádi, M.; Kozma, D.; Egri,
G.; Pálovics, E.; Kiss, V. Org. Biomol. Chem. 2006, 4, 3011–3030; (h) Gadler, P.;
Faber, K. Trends Biotechnol. 2007, 25, 83–88; (i) Ghanem, A. Tetrahedron 2007,
63, 1721–1754; (j) Gotor, V.; Alfonso, I.; Garcia-Urdiales, E. Asymmetric Organic
Synthesis with Enzymes; Wiley-VCH: Weinheim, 2008.
2. (a) Gravert, D. J.; Janda, K. D. Chem. Rev. 1997, 97, 489–509; (b) Wentworth, P.;
Janda, K. D. Chem. Commun. 1999, 1917–1924; (c) Dickerson, T. J.; Reed, N. N.;
Janda, K. D. Chem. Rev. 2002, 102, 3325–3344; (d) Bergbreiter, D. E. Chem. Rev.
2002, 102, 3345–3384.
3. (a) Benaglia, M.; Celentano, G.; Cozzi, F. Adv. Synth. Catal. 2001, 343, 171–173;
(b) Pozzi, G.; Cavazzini, M.; Quici, S.; Benaglia, M.; Dell’Anna, G. Org. Lett. 2004,
6, 441–443; (c) Oikawa, M.; Tanaka, T.; Kusumoto, S.; Sasaki, M. Tetrahedron
Lett. 2004, 45, 787–790; (d) Reed, N. N.; Dickerson, T. J.; Boldt, G. E.; Janda, K. D.
J. Org. Chem. 2005, 70, 1728–1731; (e) Li, J.-H.; Hu, X.-C.; Liang, Y.; Xie, Y.-X.
Tetrahedron 2006, 62, 31–38; (f) Hong, S. H.; Grubbs, R. H. Org. Lett. 2007, 9,
1955–1957.
4. (a) Veronese, F. M.; Morpurgo, M. Il Farmaco 1999, 54, 497–516; (b) Greenwald,
R. B. J. Controlled Release 2001, 74, 159–171; (c) Vincent, M. J.; Duncan, R. Trends
Biotechnol. 2006, 24, 39–47.
5. López-Pelegrín, J. A.; Wentworth, P., Jr.; Sieber, F.; Metz, W. A.; Janda, K. D. J.
Org. Chem. 2000, 65, 8527–8531.
Table 2
Enantioselective hydrolysis of MPEG5000
-
and PEG4600-supported dendritic
carbonatesa
6. (a) Shimojo, M.; Matsumoto, K.; Nogawa, M.; Nemoto, Y.; Ohta, H. Tetrahedron
Lett. 2004, 45, 6769–6773; (b) Nogawa, M.; Shimojo, M.; Matsumoto, K.;
Okudomi, M.; Nemoto, Y.; Ohta, H. Tetrahedron 2006, 62, 7300–7306.
7. Okudomi, M.; Shimojo, M.; Nogawa, M.; Hamanaka, A.; Taketa, N.; Matsumoto,
K. Tetrahedron Lett. 2007, 48, 8540–8543.
8. For recent reviews, see: (a) Gittins, P. J.; Twyman, L. J. Supramol. Chem. 2003,
15, 5–23; (b) Tomalia, D. A. Chim. Oggi 2005, 23, 41–42; (c) Tomalia, D. A.
Prog. Polym. Sci. 2005, 30, 294–324; (d) Dufes, C.; Uchegbu, I. F.; Schatzlein, A. G.
Polymers in Drug Delivery; CRC Press LLC: Boca Raton, 2006; (e)
Boas, U.; Christensen, J. B.; Heegaard, P. M. H. J. Mater. Chem. 2006, 16, 3785–
3798.
Entry
Substrate
ee of (S)-9b (%)
ee of (R)-2ac (%)
Conv.
E value
1
9a
9b
9c
3
20
20
96
98
99
0.03
0.17
0.17
50
120
>200
2
3d
a
Unless otherwise noted, the reaction was performed using 125 mg of the sub-
strate in a test tube for 48 h at 0 °C.
b
Determined by GLC analysis after the chemical hydrolysis of 9.
Determined by GLC analysis.
The reaction was performed for 24 h.
c
d

M. Okudomi et al. / Tetrahedron Letters 49 (2008) 6642–6645
6645
9. The concept of PEG Chemistry with dendrimer chemistry was already reported
in the following paper: Benaglia, M.; Annunziata, R.; Cinquini, M.; Cozzi, F.;
Ressel, S. J. Org. Chem. 1998, 63, 8628–8629.
ESI-TOF MS m/z 2692.58 Da (most abundant ion; 2690.54 Da calcd for
[MeO(CH2CH2O)108C32H37O8ꢂ2Na]2+),
[MeO(CH2CH2O)84-135C32H37O8ꢂ2Na]2+
(m/z range 2163.64–3287.75 Da; z = 2).
10. The yields of the MPEG-supported compounds were based on the weights of
the starting materials. The purities were determined by a 1H NMR analysis, and
the terminal methyl group and/or the PEG-methylenes were used as the
reference.
13. GC analysis of 2a: Column, CP-Cyclodextrin-B-236-M19 (Chrompack),
0.25 mm ꢃ 50 m; injection, 160 °C; detection, 160 °C; oven, 140 °C; carrier
gas, He; head pressure, 2.4 kg/cm2; retention time, 8.9 (R) and 9.2 (S) min.
14. Chen, C. S.; Fujimoto, Y.; Girdaukas, G.; Sih, C. J. J. Am. Chem. Soc. 1982, 104,
7294–7299.
15. The conversion was calculated by ee(1)/[ee(1) + ee(2)], and the E value was
calculated by ln[(1 ꢁ conv.)(1 ꢁ ee(1))]/ln[(1 ꢁ conv.)(1 + ee(1))].
16. Although the total isolated yield (98%) of (R)- and (S)-2a was low due to the
high volatility, the number was almost twice for of that a non-dendritic
substrate.7
11. Compound ( )-1a (purity, ca. 90%): 1H NMR (500 MHz, CDCl3) d 1.59 (d,
J = 6.5 Hz, 6H), 3.38 (s, 3H), 3.44–3.80 (m, PEG–methylenes), 3.83 (t, J = 5.0 Hz,
2H), 4.08 (t, J = 5.0 Hz, 2H), 5.02 (d, J = 12.5 Hz, 2H), 5.08 (d, J = 12.0 Hz, 2H),
5.73 (q, J = 6.5 Hz, 2H), 6.86 (s, 2H), 6.91 (s, 1H), 7.27–7.39 (m, 10H); 13C NMR
(125 MHz, CDCl3) d 22.2, 58.9, 67.3, 68.8, 69.4, 70.4 (PEG), 70.5, 70.6, 71.7, 76.5,
114.1, 119.9, 125.8, 128.0, 128.4, 136.8, 140.8, 154.2, 158.9; IR (KBr)
2886, 1742, 1645, 1466, 1344, 1281, 1252, 1113, 953, 843 cmꢁ1; ESI-TOF MS
m/z 2634.30 Da (most abundant ion; 2632.50 Da calcd for
17. The absolute configurations of the products were determined by comparing
the optical rotation of 2a with the reported one; lit.21
for the (R)-enantiomer.
½ ꢀ +45 (c 5.15, MeOH)
a 2D0
[MeO(CH2CH2O)108C26H25O6ꢂ2Na]2+),
(m/z range 2040.40–3206.49 Da; z = 2).
[MeO(CH2CH2O)81–134C26H25O6ꢂ2Na]2+
18. The absolute configurations of the products were determined by comparing
12. Compound ( )-1b (purity, ca. 94%): 1H NMR (500 MHz, CDCl3) d 1.31 (d,
J = 6.0 Hz, 6H), 1.79–1.89 (m, 2H), 1.91–2.01 (m, 2H), 3.37 (s, 3H), 3.46–3.80
(m, PEG–methylenes), 3.82 (t, J = 5.0 Hz, 2H), 4.10 (t, J = 5.0 Hz, 2H), 4.46 (s,
4H), 4.97 (tq, J1 = 1.0 Hz, J2 = 6.5 Hz, 2H), 5.04 (d, J = 12.5 Hz, 2H), 5.08 (d,
J = 12.0 Hz, 2H), 6.878 (s, 1H), 6.880 (s, 1H), 6.94 (s, 1H), 7.18–7.37 (m, 10H);
13C NMR (125 MHz, CDCl3) d 20.0, 35.8, 58.9, 66.2, 67.4, 68.7, 69.5, 70.4 (PEG),
70.6, 70.7, 71.8, 72.9, 73.0, 114.1, 120.0, 127.4, 127.5, 128.2, 137.0, 138.1, 154.4,
the optical rotation of 2b with the reported one; lit.6b
MeOH) for the (S)-enantiomer.
½ ꢀ +19.0 (c 0.95,
a 2D7
19. HPLC analysis of 2b: column, CHIRALCEL OD-H (Daicel Chemical Industries,
Ltd); eluent, hexane/2-propanol = 90/10; flow rate, 0.5 mL/min; 254 nm;
temperature, 25 °C; retention time, 13 (S) and 14 (R) min.
20. The substrates were synthesized by the sequential coupling of MPEG5000-OH or
PEG4600-OH with their respective units.
159.0; IR (KBr) 2886, 1742, 1645, 1468, 1342, 1281, 1242, 1115, 964, 843 cmꢁ1
;
21. Laumen, K.; Schneider, M. P. J. Chem. Soc., Chem. Commun. 1988, 598–600.