Enzymatic deprotection of the cephalosporin 3′-acetoxy group using Candida antarctica lipase B

Article abstract of DOI:10.1021/jo902406b

(Chemical Equation Presented) Cephalosporins remain one of themost important classes of antibiotics. A useful site for derivatization involves generation of and chemistry at the 3′-hydroxymethyl position. While 3′-acetoxymethyl-substituted cephalosporins are readily available, deacetylation to access the free 30-hydroxymethyl group is problematic when the carboxylic acid is protected as an ester. Herein we report that this important transformation has been efficiently accomplished using Candida antarctica lipase B. Although this transformation is difficult to carry out using chemical methods, the enzymatic deacetylation has been successful on gram scale, when the cephalosporin is protected as either the benzhydryl or tert-butyl esters and on the corresponding sulfoxide and sulfone of the tert-butyl ester.

Full text of DOI:10.1021/jo902406b

pubs.acs.org/joc
isomer, has yet to be accomplished in a high-yielding and
Enzymatic Deprotection of the Cephalosporin
30-Acetoxy Group Using Candida antarctica Lipase B
reproducible manner (Scheme 1).3 Herein we report a selective
enzymatic approach to the deprotection of the cephalosporin
acetoxy group, using commercially available Candida antartica
lipase B (CAL B), on acrylic resin.
Leslie D. Patterson and Marvin J. Miller*
Department of Chemistry and Biochemistry, University of
Notre Dame, Notre Dame, Indiana 46556
SCHEME 1. Cephalosporin Acetate Deprotection
mmiller1@nd.edu
Received November 23, 2009
Commercially available 7-aminocephalosporanic acid (7-
ACA) was converted, in reasonable yields, to 3 using a modified
literature procedure to introduce the tert-butyl ester4 and stan-
dard Schotten-Baumann conditions to install the phenylacetyl
group as a simple representative side chain (Scheme 2). Despite
considerable effort, the 30-acetoxy substituent was unable to be
removed in acceptable yields and in the absence of double bond
isomerization using the following methods (see the Supporting
Information for details): saponification,3 KCN, HCl,5 TMSI/
trifluoroacetate/pH 7.0,6 or bis(tributyltin) oxide.7 On the
basis of these findings, enzymatic deacetylation methods were
explored.
Enzymatic deprotection of the cephalosporin acetoxy
group has been demonstrated in the literature.8 Similar to
chemical deacetylations,9 the enzymatic deacetylation reac-
tions have only been reported when the cephalosporin con-
tains a free acid.8 When making modifications to the allylic
alcohol it is usually more convenient and often necessary to
have the acid protected as an ester. This is especially true for
Cephalosporins remain one of the most important classes of
antibiotics. A useful site for derivatization involves genera-
tion of and chemistry at the 30-hydroxymethyl position.
While 30-acetoxymethyl-substituted cephalosporins are
readily available, deacetylation to access the free 30-hydroxy-
methyl group is problematic when the carboxylic acid is
protected as an ester. Herein we report that this important
transformation has been efficiently accomplished using
Candida antarctica lipase B. Although this transforma-
tion is difficult to carry out using chemical methods, the
enzymatic deacetylation has been successful on gram
scale, when the cephalosporin is protected as either the
benzhydryl or tert-butyl esters and on the corresponding
sulfoxide and sulfone of the tert-butyl ester.
(3) Wang, Y.; Yuan, H.; Wright, S. C.; Wang, H.; Larrick, J. W. BMC
Chem. Biol. [Online] 2001, 1, article 4.
(4) (a) Mangia, A.; Scandroglio, A.; Del Buttero, P. Org. Prep. Proced.
Int. 1986, 18, 13–15. (b) Stedman, R. J. J. Med. Chem. 1966, 9, 444.
(5) Galeazzi, R.; Martelli, G.; Mabbili, G.; Orena, M.; Rinaldi, S. Org.
Lett. 2004, 6, 2571–2574.
(6) Mobashery, S.; Johnston, M. Tetrahedron Lett. 1986, 27, 3333–3336.
(7) Salomon, C. J.; Mata, E. G.; Mascaretti, O. A. J. Org. Chem. 1994, 59,
7259–7266.
(8) While there is extensive early patent literature related to similar
transformations, the following references provide representative documen-
tation of the efforts related to this type of cephalosporin chemistry.
(a) Jeffery, J. D’A.; Abraham, E. P.; Newton, G. G. F. Biochem. J. 1961,
81, 591–596. (b) Negi, S.; Yamanaka, M.; Sugiyama, I.; Komatsu, Y.; Sasho,
M.; Tsuruoka, A.; Kamada, A.; Tsukada, I.; Hiruma, R.; Katsu, K.;
Machida, Y. J. Antibiot. 1994, 47, 1507–1525. (c) Carrea, G.; Corcelli, A.;
Palmisano, B.; Riva, S. Biotechnol. Bioeng. 1996, 52, 648–652. (d) Sakai, Y.;
Abe, T.; Ohbayashi, Y.; Isaka, K.; Yamamoto, K.; Tani, Y.; Kato, N. App.
Environ. Microbiol. 1996, 62, 2669–2672. (e) Keltjens, R.; Vadivel, S. K.; de
Vroom, E.; Klunder, A. J. H.; Zwanenburg, B. Eur. J. Org. Chem. 2001, 13,
2529–2534. (f) Kidwai, M.; Dave, B.; Bhushan, K. R.; Misra, P.; Saxena, R.
K.; Gupta, R.; Gulanti, R.; Singh, M. Biocat. Biotransform. 2002, 20, 377–
379. (g) Grant, J. W.; Smyth, T. P. J. Org. Chem. 2004, 69, 7965–7970.
(9) (a) Okabe, M.; Sun, R. C. Synthesis 1992, 11, 1160–1164. (b) Kukolja,
Cephalosporin antibiotics have been in use for more than 40
years and are still being employed to fight bacterial infections
despite the rising incidence of resistance.1 Decades of intense
research have given rise to an arsenal of synthetic methods that
have allowed many analogues to be prepared, including four
generations of cephalosporins that have reached clinical use.2
Although this research has led to many breakthroughs in
cephalosporin-specific chemical methodology, accessing pre-
cursors for further elaboration is still not straightforward.
Numerous derivatives have been made through modification
at the 30-hydroxymethyl group of cephalosporins. However,
the deprotection of the common 30-acetoxy precursor in the
presence of the protected cephalosporanic acid, and without
lactonization and/or double bond isomerization to give the Δ2
(1) (a) Fischbach, M. A.; Walsh, C. Science 2009, 325, 1089–1093.
(b) Walsh, C. Antibiotics: Actions, Origins, Resistance; ASM Press: Washington,
DC, 2003. (c) Bryskier, A. J. Antibiot. 2000, 53, 1028–1037. (d) Singh, G. S.
Mini Rev. Med. Chem. 2004, 4, 93–109.
(2) Chemistry and Biology of β-Lactam Antibiotics; Morin, R. B., Gorman,
M., Eds.; Academic Press: New York, 1982; Vol. 1.
ꢀ
S. J. Med. Chem. 1968, 11, 1067–1069. (c) Ganzalez, M.; Rodrı
´
guez, Z.;
guez, J. C.; Velez, H.; Valdez, B.; Lopez, M. A.; Fini, A.
Farmaco 2003, 58, 409.
ꢀ
Tolon, B.; Rodrı
´
ꢀ
ꢀ
DOI: 10.1021/jo902406b
r
Published on Web 01/25/2010
J. Org. Chem. 2010, 75, 1289–1292 1289
2010 American Chemical Society

Patterson and Miller
SCHEME 3. Synthesis of Cephalosporins 5-7
JOCNote
SCHEME 2. Synthesis of Cephalosporin 3
TABLE 1. Optimization of Cephalosporin 3 Deacetylation Using CAL Ba
SCHEME 4. Synthesis of Cephalosporins 8 and 9
entry
CAL Bb
additivec
n-butanol
n-butanol
timed
conve
1f
2
3
4
5
6
7
50
50
50
50
10
10
5
4
4
4
4
4
7
4
7
4
0
48
85
93
74
92
21
39
97h
TABLE 2. Scope of Enzymatic Deacetylation of Cephalosporins 5-9a
sec-butanol
sec-butanol, MS
sec-butanol, MS
sec-butanol, MS
sec-butanol, MS
sec-butanol, MS
sec-butanol, MS
8
5
50
9g
aAll reactions were performed on 20 mg scale and at 50 °C, unless
otherwise indicated, in 9:1 hexanes/THF, and carried out in an incu-
bated shaker. bMass %. cMS = 4 A molecular sieves. dDays. ePercent
conversions to product determined using 1H NMR spectroscopy and
reported in %. fReaction performed at 32 °C; gReaction performed on 1
g. hIsolated yield after recrystallization.
compd
n
R
yieldb (conv)c
10
11
12
13
14
0
0
0
1
2
H
N/A
52 (100)
N/A
70
benzhydryl
p-methoxybenzyl
tert-butyl
the synthesis of dual action cephalosporins, where the allylic
alcohol is modified.8g,10
tert-butyl
58
aAll reactions were performed in an incubated shaker with 50 mass %
Initially, a panel of nine lipases was screened for the enzy-
matic deprotection of the allylic alcohol of 3.11 This resulted in
no product formation under various conditions. Conversely,
extensive studies with CAL B provided an effective solution.
C. antarctica lipase B was chosen on the basis of its broad
utility and general experience in our group.12 After exploring
solvents13 and temperatures and adding n-butanol,14 cephalos-
porin 4 was obtained (Table 1). Interestingly, when water was
used as the nucleophile, low yields or no reaction was observed.
Alternatively, n-butanol was used to obtain the product in
moderate yields, and the use of sec-butanol was found to
increase the conversion from 3 to 4 significantly.15 Further
optimization showed that the addition of molecular sieves
increased the efficiency and conversion of the reaction. In
addition, different lots of CAL B gave different reaction rates
CAL B. bIsolated % yield. Percent conversions determined using 1H
NMR spectroscopy.
c
based on the “loss on drying” reported by the manufacturer.16
These observations indicate that the reaction is sensitive to
moisture. The optimized reaction can be performed on gram
scale, and a yield of 97%, after recrystallization, can be
obtained.
Several additional substrates were prepared to test the
scope of the enzymatic deprotection. The substrates were
prepared from 7-ACA using known and modified literature
procedures. Acylation of 7-ACA using phenylacetyl chloride8e
followed by protection of the acid using diphenyldiazo-
methane17 or p-methoxybenzyl bromide, prepared in situ,18
gave cephalosporins 6 and 7, respectively (Scheme 3). Cepha-
losporin 3 could be selectively oxidized to either sulfoxide 8 or
sulfone 9 using varying reaction times and amounts of m-CPBA
(Scheme 4).4
€
(10) Zhao, G.; Miller, M. J.; Franzblau, S.; Wan, B.; Mollmann, U. Biorg.
Med. Chem. Lett. 2006, 16, 5534–5537.
(11) Lipase kit from Aldrich containing: lipase from Aspergillus, Lipase from
Candida antarctica, lipase from Candida cylindracea, lipase from Mucor miehei,
lipase from Pseudomonas cepacia, lipase from Pseudomonas fluorescens, lipase
from Rhizopus arrhizus, lipase from Rhizopus niveus, lipase from hog pancreas.
(12) Mulvihill, M. J.; Gage, J. L.; Miller, M. J. J. Org. Chem. 1998, 63,
3357–3363.
These additional substrates were submitted to the pre-
viously optimized CAL B reaction conditions (Table 2).
(16) The “loss on drying” reported for two different lots of CAL B was
0.31% and 1%. The lot with 0.31% loss on drying gave faster reaction times.
See the Supporting Information for a detailed study.
(13) Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Biotechnol. Bioeng. 1987,
30, 81–87.
(14) Anilkumar, A. T.; Goto, K.; Takahashi, T.; Ishizake, K.; Kaga, H.
Tetrahedron: Asymmetry 1999, 10, 2501–2503.
(15) Miyazawa, T.; Hamada, M.; Morimoto, R.; Murashima, T.; Yamada,
T. Tetrahedron Lett. 2007, 48, 8334–8337.
(17) (a) Takaya, T.; Takasugi, H.; Murakawa, T.; Nakano, H. J. Antibiot.
1981, 34, 1300–1310. (b) Kumar, S.; Murray, R. W. J. Am. Chem. Soc. 1984,
106, 1040–1045.
(18) Mobashery, S.; Johnston, M. J. Org. Chem. 1986, 51, 4723–4726.
1290 J. Org. Chem. Vol. 75, No. 4, 2010

Patterson and Miller
JOCNote
When the acid was not protected, such as in cephalos-
porin 5, no reaction was seen; this result was not disap-
pointing as removal of the acetate from cephalosporin 10
and other free acid containing cephalosporins has been
reported.8,9 The most commonly used cephalosporin acid
protecting group is the benzhydryl ester. One difficulty in
working with cephalosporin 11 is that the alcohol can
readily cyclize to form the corresponding lactone;19 this
transformation can even occur when the acid is left un-
protected.9b Surprisingly, cephalosporin 11 was obtained
without cyclization to the lactone, and 100% conversion
was observed. The low yield reported in Table 2 reflects the
difficult isolation; recrystallization was inefficient, and
silica gel column chromatography caused lactone forma-
tion. Fortunately, the crude material was isolated very
cleanly and was suitable for use. Cephalosporin 12, con-
taining the PMB ester, gave complex mixtures and was not
explored further. Both sulfoxide, 13, and sulfone, 14, also
were substrates for CAL B; however, only moderate yields
were obtained.
molecular sieves. The reaction was sonicated for a few minutes
to help solubilization. CAL B (52.3 mg) and sec-butanol (0.5 mL,
6.746 mmol) were added. The reaction was shaken at 50 °C for
4 days in an incubated shaker. The reaction mixture was dissolved
in methanol, and the sieves and CAL B were removed using
vacuum filtration. The crude NMR indicated that the reac-
tion had gone to completion. The material was suspended
in a minimal amount of EtOAc, cooled, and filtered to give
a white solid (48.4 mg, 52%): mp 175-176 °C (lit.19 mp 178-
180 °C); IR (KBr) 3501, 1761, 1713, 1666 cm-1; 1H NMR (500
MHz, DMSO-d6) δ 3.50-3.61 (od, J = 14 Hz, 2H), 3.62 (s, 2H),
4.17-4.27 (m, 2H), 5.12 (d, J = 4.5 Hz, 1H), 5.19 (t, J = 5.5 Hz,
1H), 5.73 (dd, J = 5, 8 Hz, 1H), 6.91 (s, 1H), 7.21-7.40 (m, 11H),
7.43 (d, J = 7.5 Hz, 2H), 7.52 (d, J = 7.5 Hz, 2H), 9.16 (d,
J =8 Hz, 1H);13CNMR(125MHz, DMSO-d6) δ25.6, 41.6, 57.7,
58.9, 59.8, 78.4, 122.0, 126.5, 126.6, 126.8, 127.8, 127.9, 128.3,
128.4, 128.6, 129.0, 134.4, 135.8, 140.0, 140.1, 160.9, 165.3, 171.0;
HRMS (ESI-TOF) calcd for C29H26N2NaO5S 537.1460, found
537.1476.
(6R,7R)-tert-Butyl 3-(Hydroxymethyl)-8-oxo-7-(2-phenylacet-
amido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate 5-Oxide (13).
Cephalosporin 8 (261.4 mg, 0.565 mmol) was partially dissolved
in anhydrous toluene (40 mL). CAL B (137.9 mg), sec-butanol
(1 mL, 10.901 mmol), and 4 A molecular sieves (520.3 mg)
were added. The reaction was shaken at 50 °C in an incubated
shaker. After shaking over 4 nights, the reaction mixture
was dissolved in methanol and filtered to remove the lipase
and the sieves, and the solvent was removed in vacuo. The
material was purified using column chromatography. The
material was preloaded on silca and eluted using 2:1 CH2Cl2/
EtOAc until starting compound 8 was isolated (12.1 mg) and
then EtOAc until product 13 was isolated as a white solid (286.8
mg, 70%): mp 192-195 °C dec (lit.8e mp 198-199 °C); Rf 0.15
(2:1 EtOAc/CH2Cl2); IR (thin film) 3506, 1778, 1695, 1661, 1029
cm-1; 1H NMR (500 MHz, DMSO-d6) δ 1.49 (s, 9H), 3.54 (d,
J = 18.5 Hz, 1H), 3.55 (d, J = 14 Hz, 1H), 3.69 (d, J = 14 Hz,
1H), 3.86 (d, J = 19 Hz, 1H), 4.08 (dd, J = 5.5, 14 Hz, 1H), 4.43
(dd, J = 5.5, 14 Hz, 1H), 4.84 (d, J = 4.5 Hz, 1H), 5.13-5.17 (m,
1H), 5.77 (dd, J = 4.5, 8.5 Hz, 1H), 7.21-7.26 (m, 1H),
7.28-7.33 (m, 4H), 8.38 (d, J = 8.5 Hz, 1H); 13C NMR (125
MHz, DMSO-d6) δ 27.5, 41.5, 45.3, 58.0, 60.2, 66.2, 82.5, 123.1,
125.6, 126.6, 128.3, 129.1, 135.8, 160.0, 164.1, 171.1; HRMS
(ESI-TOF) calcd for C20H24N2NaO6S 443.1247, found
443.1243.
In conclusion, we have developed an enzymatic trans-
formation that allows the 30-acetoxy group of cephalos-
porins, containing an ester protected carboxylic acid, to
be removed in moderate to excellent yields. This metho-
dology is especially useful in preparing cephalosporin
analogues that require further modification at the allylic
alcohol.
Experimental Section
(6R,7R)-tert-Butyl 3-(hydroxymethyl)-8-oxo-7-(2-phenylacet-
amido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate (4). Com-
pound 3 (1.007 g, 2.241 mmol) was added to a 1 L Erlenmeyer
flask. Tetrahydrofuran (12 mL) was added to dissolve the
substrate. Then hexanes (108 mL) was added, initially forming
a gel which upon swirling formed a suspension. CAL B (502.0 mg),
4 A molecular sieves (1.0146 g), and sec-butanol (4 mL, 43.604
mmol) were added, and the flask was stoppered with a septum.
The reaction was shaken in an incubated shaker at 50 °C for 4-
6 days (solid that had dried on the sides of the flask was scraped
back into the reaction each day). When the reaction was com-
plete by TLC, the solid was dissolved in methylene chloride, the
lipase and sieves were filtered off using vacuum filtration, and the
solvent was removed in vacuo. The crude material was recrystal-
lized from chloroform/cyclohexane in two crops to yield cepha-
losporin 4. The material was dried under vacuum with P2O5
to yield the product as a white solid (877.1 mg, 97%): mp
170-171.5 °C (lit.8e mp 174-176 °C); Rf 0.29 (5:1 DCM/EtOAc);
(6R,7R)-tert-Butyl
3-(Hydroxymethyl)-8-oxo-7-(2-phenyl-
acetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate 5,
5-Dioxide (14). Cephalosporin 9 (99.9 mg, 0.209 mmol) was
added to a 25 mL Erlenmeyer flask and dissolved in THF
(1.2 mL, from AcroSeal bottle), and hexanes (10.8 mL, HPLC
grade) were added. Molecular sieves (4 A, 199.9 mg), CAL B
(50.0 mg), and sec-butanol (0.4 mL) were added. The reaction
was stoppered and shaken in an incubated shaker at 50 °C over
5 days. When complete by TLC, the reaction was dissolved in
methylene chloride, the CAL B and molecular sieves were
removed using vacuum filtration, and the solvent was removed
in vacuo. The material was purified using column chromato-
graphy, loading in methylene chloride and eluting with 5:1
CH2Cl2/EtOAc. The product was isolated as a white solid
(42.9 mg, 58%): mp 170.5-171.5 °C; Rf 0.16 (5:1 DCM/EtOAc);
1
IR (KBr) 3382, 1757, 1712, 1661 cm-1; H NMR (300 MHz,
CDCl3) δ 1.51 (s, 9H), 2.76 (dd, J = 6.5, 17 Hz, 1H), 3.51 (s, 1H),
3.57-3.70 (od, 2H), 3.84 (dd, J = 10.5, 12.6 Hz, 1H), 4.44 (dd, J =
4.2, 12.6 Hz, 1H), 4.89 (d, J = 5.1 Hz, 1H), 5.84 (dd, J = 4.8, 9.3
Hz, 1H), 6.15 (d, J = 9 Hz, 1H), 7.23-7.28 (m, 2H), 7.29-7.39 (m,
3H); 13C NMR (125 MHz, CDCl3) δ 27.8, 28.0, 43.7, 57.2, 59.3,
62.4, 84.4, 127.1, 128.1, 129.5, 129.7, 130.1, 133.8, 161.8, 164.8,
171.4; HRMS (ESI-TOF) calcd for C20H24N2NaO5S 427.1298,
found 427.1274.
(6R,7R)-Benzhydryl 3-(Hydroxymethyl)-8-oxo-7-(2-phenyl-
acetamido)-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate (11).
Cephalosporin 6 (101.1 mg, 0.181 mmol) was dissolved in THF
(1.5 mL), and hexanes (13.5 mL, HPLC grade) was added
followed by 4 A molecular sieves (228.2 mg). The mixture be-
came white and cloudy, and some material adhered to the
1
IR (KBr) 3512, 1782, 1718, 1660, 1332, 1158 cm-1; H NMR
(500 MHz, DMSO-d6) δ 1.47 (s, 9H), 3.56-3.64 (od, 2H), 4.01
(d, J = 18.5 Hz, 1H), 4.17-4.27 (m, 2H), 4.26 (d, J = 18.5 Hz,
1H), 5.26 (t, J = 5 Hz, 1H), 5.35 (d, J = 4.5 Hz, 1H), 5.92 (dd,
J = 4.5, 8.5 Hz, 1H), 7.2-7.31 (m, 5H), 8.86 (d, J = 8.5 Hz, 1H);
13C NMR (125 MHz, DMSO-d6) δ 27.4, 41.2, 50.8, 57.9, 59.0,
66.8, 83.0, 122.2, 126.5, 128.2, 129.2, 130.5, 135.6, 159.9, 164.1,
170.9; HRMS (ESI-TOF) calcd for C20H25N2O7S 437.1377,
found 437.1377.
(19) Keltjens, R.; Vadivel, S. K.; de Gelder, R.; Klunder, A. J. H.;
Zwanenburg, B. Eur. J. Org. Chem. 2003, 9, 1749–1758.
J. Org. Chem. Vol. 75, No. 4, 2010 1291

Patterson and Miller
JOCNote
Acknowledgment. We acknowledge the NIH (GM
025845 and AI 030988) for support of this research. We
also acknowledge Dr. M. Joyce, Dr. W. Boggess, and N.
Sevova for assistance obtaining mass spectrometry data
and both the Center for Environmental Science and
Technology and the ND Mass Spectrometry and Proteo-
mics Facility (CHE-0741793) at the University of Notre
Dame. L.D.P. acknowledges a Grace Fellowsip (2008-
2009, UND).
Supporting Information Available: Experimental methods
for compounds 1-3 and 5-9 and all 1H and 13C NMR spectra.
This material is available free of charge via the Internet at http://
pubs.acs.org.
1292 J. Org. Chem. Vol. 75, No. 4, 2010