Enzymatic Hydrolysis of Esters Containing a Tetrazole Ring

Article abstract of DOI:10.1002/chir.22360

The lipase-catalyzed enantioselective hydrolysis of acetates containing tetrazole moiety was studied. Among all tested lipases, Novozyme SP 435 allowed to obtain optically active 4-(5-aryl-2H-tetrazol-2yl)butan-2-ol and 1-(5-aryl-2H-tetrazol-2yl)-propan-2

Full text of DOI:10.1002/chir.22360

CHIRALITY 26:811–816 (2014)
Enzymatic Hydrolysis of Esters Containing a Tetrazole Ring
*
EDYTA ŁUKOWSKA-CHOJNACKA AND JOLANTA MIERZEJEWSKA
Faculty of Chemistry, Institute of Biotechnology, Warsaw University of Technology, Warsaw, Poland
ABSTRACT
The lipase-catalyzed enantioselective hydrolysis of acetates containing tetrazole
moiety was studied. Among all tested lipases, Novozyme SP 435 allowed to obtain optically active
4-(5-aryl-2H-tetrazol-2yl)butan-2-ol and 1-(5-aryl-2H-tetrazol-2yl)-propan-2-ol and their acetates
with the highest optical purities (ee = 95%-99%) and excellent enantioselectivity (E>100). Some
of the synthesized tetrazole derivatives were screened for their antifungal activity. Racemic mix-
tures of 4-[5-(4-chlorophenyl)-2H-tetrazol-2-yl)butan-2-ol as well as pure enantiomers of this com-
pound showed promising antifungal activity against F. sambucinum, F. oxysporum, C. coccodes,
and A. niger. Chirality 26:811–816, 2014. © 2014 Wiley Periodicals, Inc.
KEY WORDS: azole; enantioseparation; lipase; antifungal activity; kinetic resolution
INTRODUCTION
Herein, we report a lipase-catalyzed hydrolysis as an alter-
native method for the preparation of optically active alcohols
containing a tetrazole ring. Additionally, the evaluation of
the antifungal properties of synthesized tetrazole derivatives
is described.
Tetrazole derivatives exhibit diverse biological and pharma-
ceutical activity. This ring system is present in drugs1–4
and in many inhibitors of enzymes.5–12 Moreover, some tetra-
zole derivatives possess analgesic,13 antiinflammatory,14–16
antiallergic,17,18 antibacterial,19–24 and antifungal activity.20–31
The last one is extremely valuable, because of the growing num-
ber of systemic fungal infections caused by Aspergillus sp.,
Candida sp., Cryptococcus sp., and other species.32 This kind of
infection is especially dangerous for populations with altered
immunity. There are known tetrazole derivatives with activity
higher or comparable with common antifungal agents like
intraconazole, ketoconazole, fluconazole, and griseofulvin.20–31
An interesting group of these compounds constitute 5-(phenyl-
selanylmethyl)-1H-tetrazole and 1-(2,4-dihydroxy-thiobenzoyl)
tetrazole with high activity against various Candida spe-
cies.25,31 Other useful antifungal agents are 5-thio-substituded
tetrazole derivatives exhibiting high activity against some
Aspergillus and Penicillium species and 3-aryl-1-(5-phenyl-1H-
tetrazol-1-yl)prop-2-en-1-ones which appeared to be the most
active against Candida albicans and Aspergillus niger.20,30 Many
newly designed tetrazole derivatives possess common azole
substructures seen in other antifungals. Among the noteworthy
structures are (2R,3S)-2-(2,4-difluorophenyl)-3-(5-{2-[4-aryl-
piperazin-1-yl]-ethyl}-tetrazol-2-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol
and (2R,3S)-2-(2,4-difluorophenyl)-3-(5-{2-[4-aryl-piperazin-1-yl]-
ethyl}-tetrazol-1-yl)-1-[1,2,4]-triazol-1-yl-butan-2-ol, inhibiting growth
of Candida, Aspergillus, and Cryptococcus species.26 Moreover,
antifungal tetrazoles containing a benzotriazole and benzimid-
azole moiety are known.22–24 A broad spectrum of biological
activities of compounds containing a tetrazole ring in the
molecule prompted us to synthesize and evaluate activities
of 5-(aryltetrazolyl-2)-alkanols. Recently,33 we described a
chemoenzymatic procedure for the preparation of enantiomerically
enriched 4-(5-aryl-2H-tetrazol-2-yl)butan-2-ol and 1-(5-aryl-2H-
tetrazol-2-yl)propan-2-ol and their acetates. The enzymatic
step involves enantioselective acetylation of appropriate alco-
hols catalyzed by commercially available lipases. Screening
the biological activities of the synthesized racemic mixtures
of alcohols containing a tetrazole ring, we found interesting
properties in some of them. To develop more precise investi-
gation, single enantiomers of sufficiently high enantiomeric
purity are required, and we need better than the described33
procedure for enantiomers separation.
MATERIALS AND METHODS
Chemicals and Reagents
The 5-substituted tetrazoles were prepared in high yields (64–91%) from
commercially available nitriles, NaN3 and NH4Cl in dimethylformamide
(DMF) according to the described34 method. Amano AK (native lipase
from Pseudomonas fluorescens), Amano PS (native lipase from Burkholderia
cepacia, earlier Pseudomonas cepacia) were purchased from Sigma-Aldrich
(Germany). Novozyme SP 435 (lipase from C. antarctica-B immobilized on
a macroporous acrylic resin) was purchased from Novozymes (Denmark).
All commercially available reagents were obtained from Sigma-Aldrich
(Germany), Merck (Germany), and POCH (Poland), and were used with-
out further purification.
Apparatus and Materials
1H (400 MHz) and 13C (100 MHz) nuclear magnetic resonance (NMR)
spectra were recorded on Varian Mercury 400 MHz spectrometer (Palo
Alto, CA) in CDCl3 solution; chemical shifts (δ) are reported in ppm; cou-
pling constants (J) are given in hertz. Ee’s of the alcohols 2a-d, 4b-d. and
esters 3c-d, were determined on a Shimadzu high-performance liquid
chromatography (HPLC) apparatus (Japan) consisting of an LC-20AD
chromatograph, CTO-10AS oven, SPD-20A UV detector, and Chiralcel
OD-H column (in hexane:ethanol 95:5; 0.8 mL/min for alcohols 2a-c, hex-
ane:iso-propanol 95:5; 0.8 mL/min for alcohols 2d, 4c-d and esters 3c-d,
hexane:iso-propanol 97:3 for alcohol 4b) using the corresponding race-
mic compounds as references. Optical rotations were measured with an
ATAGO AP-300 automatic polarimeter (Japan). The reactions were moni-
tored by thin-layer chromatography (TLC) aluminum plates with silica gel
Kieselgel 60F254 (0.2 mm thickness film, Merck, Germany) using UV light
as visualizing agent and by gas chromatography (GC) with Hewlett-Packard
Model 5890 II chromatograph with helium as the carrier gas. Column
chromatography was performed using Kieselgel 60 (0.040-0.063 mm,
Merck, Germany).
*Correspondence to: Edyta Łukowska-Chojnacka, Faculty of Chemistry, Insti-
tute of Biotechnology, Warsaw University of Technology, Noakowskiego
St. 3, 00-664 Warsaw, Poland. E-mail: elukowska@ch.pw.edu.pl
Received for publication 31 March 2014; Accepted 10 June 2014
DOI: 10.1002/chir.22360
Published online 28 July 2014 in Wiley Online Library
(wileyonlinelibrary.com).
© 2014 Wiley Periodicals, Inc.

812
ŁUKOWSKA-CHOJNACKA AND MIERZEJEWSKA
JHbCH=3.6 Hz), 4.58 and 4.61 (dd, 1H, CHaHbN, JHaCH=7.6 Hz,
JHaHb=13,6 Hz), 4.42-4.49 (m, 1H, CH), 2.79 (s, 1H, OH), 1.35 (d, 3H,
CH3, JCH3CH=6.4 Hz); 13C NMR (100 MHz, CDCl3, δ): 164.23, 136.50,
129.18, 128.06, 125.47, 66.21, 59.87, 20.29.
General Procedure for the Enzyme-Catalyzed Hydrolysis of
Esters ( )-1a-d and ( )-3a-d
Acetate ( )–1a–d or ( )-3a-d (1.5 mmol) was dissolved in 20 mL of
TBME (tert-butyl methyl ether). The solution was mixed with 0.1 M phos-
phate buffer (50 mL, pH = 7.2) and 40 mg of enzyme was added. The mix-
ture was stirred at room temperature (25°C) and the conversion
monitored by TLC using toluene–ethyl acetate (5:1 v/v) as the eluent.
After an appropriate time the reaction was stopped by filtering off the
enzyme and the products extracted with CH2Cl2 (5 x 30 mL). The organic
layers were combined, washed with water (3 x 50 mL), and dried over
anhydrous MgSO4. The crude mixture was purified by chromatography
on a silica-gel column with toluene–ethyl acetate (5:1 v/v). NMR spectra
of the enantiomerically enriched alcohols (–)-(R)-2a-d, (–)-(R)-4a-d and
acetates (+)-(S)-1a-d, (–)-(S)-3a-d were identical to those given in the
literature.33
(–)-(R)-1-[5-(2-chorophenyl)-2H-tetrazol-2-yl)propan-2-ol ((–)-(R)-4d).
Oil, yield 43%. [α]D25.6 =-52.42 (c 1.49 in CH3OH, ee=98%). 1H NMR
(400 MHz, CDCl3, δ): 7,96-7.98 (m, 1H, C6H4), 7.52-7.54 (m, 1H, C6H4),
7.39-7.42 (m, 2H, C6H4), 4.74 and 4.78 (dd, 1H, CHaHbN, JHbCH=3.2 Hz),
4.63 and 4.66 (dd, 1H, CHaHbN, JHaCH=8 Hz, JHaHb=14 Hz), 4.45-4.48
(m, 1H, CH), 2.67 (s, 1H, OH), 1.35 (d, 3H, CH3, JCH3CH=6.4 Hz);
13C NMR (100 MHz, CDCl3, δ): 163.30, 133.02, 131.28, 131.23,
130.86, 126.96, 126.09, 66.21, 59.80, 20.19.
(+)-(S)-4-(5-phenyl-2H-tetrazol-2-yl)butan-2-yl acetate ((+)-(S)-1a).
Oil, yield 40%. [α]D22 = +11.94 (c 1.55 in CH3OH, ee=99%). 1H NMR
(400 MHz, CDCl3, δ): 8.11-8.14 (m, 2H, C6H5), 7.45-7.50 (m, 3H, C6H5),
4.96-5.00 (m, 1H, CH), 4.70-4.73 (m, 2H, CH2N), 2.29-2.35 (m, 2H, CH2),
2.01 (s, 3H, CH3CO), 1.29 (d, 3H, CH3, JCH3CH=6.4 Hz); 13C NMR
(100 MHz, CDCl3, δ): 170.36, 165.11, 130.26, 128.84, 127.30, 126.75,
67.94, 49.66, 35.13, 21.11, 19.85.
(–)-(R)-4-(5-phenyl-2H-tetrazol-2-yl)butan-2-ol ((–)-(R)-2a). Oil, yield
45%. [α]D24 = -31.01 (c 1.13 in CH3OH, ee=97%). 1H NMR (400 MHz,
CDCl3, δ): 8.11-8.13 (m, 2H, C6H5), 7.46-7.48 (m, 3H, C6H5), 4.78-4.85
(m, 2H, CH2N), 3.80-3.90 (m, 1H, CH), 2.20-2.25 (m, 2H, CHaHb, OH),
2.20-2.25 (m, 2H, CHaHb, OH), 2.08-2.12 (m, 1H, CHaHb), 1.26 (d, 3H,
CH3, JCH3CH=6 Hz); 13C NMR (100 MHz, CDCl3, δ): 165.02, 130.28,
128,81, 127.26, 126.74, 64.66, 50.14, 38.02, 23.61.
(+)-(S)-4-[5-(4-methylphenyl)-2H-tetrazol-2-yl]butan-2-yl acetate ((+)-
(S)-1b). Oil, yield 42%. [α]D23 = +18.35 (c 1.39 in CH3OH, ee=97%). 1H
NMR (400 MHz, CDCl3, δ): 8.00-8.02 (m, 2H, C6H4), 7.26-7.29 (m, 2H,
C6H4), 4.94-5.02 (m, 1H, CH), 4.69-4.72 (m, 2H, CH2N), 2.40 (s, 3H,
CH3), 2.23-2.37 (m, 2H, CH2), 2.01 (s, 3H, CH3CO), 1.29 (d, 3H, CH3,
JCH3CH=6.0 Hz); 13C NMR (100 MHz, CDCl3, δ): 170.37, 165,10, 140.43,
129.54, 126.67, 124.49, 67.99, 49.62, 35.13, 21.45, 21.12, 19.85.
(–)-(R)-4-[5-(4-methylphenyl)-2H-tetrazol-2-yl]butan-2-ol ((–)-(R)-2b).
Colorless crystals, mp 48-49°C, yield 43%. [α]D24 = -23.81 (c 0.97 in CH3OH,
1
ee=99%). H NMR (400 MHz, CDCl3, δ): 7.99-8.01 (m, 2H, C6H4), 7.26-7.29
(m, 2H, C6H4), 4.77-4.84 (m, 2H, CH2N), 3.84-3.86 (m, 1H, CH), 2.40 (s,
3H, CH3), 2.17-2.23 (m, 2H, CHaHb, OH), 2.07-2.13 (m, 1H, CHaHb ), 1.26
(d, 3H, CH3, JCH3CH=6 Hz); 13C NMR (100 MHz, CDCl3, δ): 165.11,
140.44, 129.53, 126.67, 124.46, 64.67, 50.07, 38.02, 23.60, 21.43.
(+)-(S)-4-[5-(4-chlorophenyl)-2H-tetrazol-2-yl]butan-2-yl acetate ((+)-
(S)-1c). Colorless crystals, mp 46-48°C, yield 48%. [α]D23 = +13.32 (c 1.54
in CH3OH, ee=99%). 1H NMR (400MHz, CDCl3, δ): 8.05-8.07 (m, 2H,
C6H4), 7.44-7.46 (m, 2H, C6H4), 4.93-5.01 (m, 1H, CH), 4.69-4.73 (m, 2H,
CH2N), 2.29-2.34 (m, 2H, CH2), 2.01 (s, 3H, CH3CO), 1.29 (d, 3H, CH3,
JCH3CH=6 Hz); 13C NMR (100 MHz, CDCl3, δ): 170.34, 164.25, 136.30,
129.16, 128.05, 125.80, 67.87, 49.74, 35.11, 21.11, 19.86.
(–)-(R)-4-[5-(4-chlorophenyl)-2H-tetrazol-2-yl]butan-2-ol ((–)-(R)-2c).
Colorless crystals, mp 70-72°C, yield 45%. [α]D24 =-26.54 (c 1.22 in CH3OH,
1
ee=98%). H NMR (400 MHz, CDCl3, δ): 8.04-8.08 (m, 2H, C6H4), 7.43-7.46
(m, 2H, C6H4), 4.74-4.88 (m, 2H, CH2N), 3.84-3.89 (m, 1H, CH), 2.19-2.23
(m, 1H, CHaHb), 2.06-2.12 (m, 2H, CHaHb, OH), 1.27 (d, 3H, CH3, JCH3CH=6
Hz); 13C NMR (100 MHz, CDCl3, δ): 164.18, 136.31, 129.16, 128.05, 125.80,
64.71, 50.22, 37.98, 23.68.
(+)-(S)-4-[5-(2-chlorophenyl)-2H-tetrazol-2-yl]butan-2-yl acetate ((+)-
(S)-1d). Colorless crystals, mp 42-44°C, yield 39%.[α]D23 = +23.26 (c 1.72
in CH3OH, ee=99%). 1H NMR (400 MHz, CDCl3, δ): 7.91-7.94 (m, 1H,
C6H4), 7.51-7.53 (m, 1H, C6H4), 7.35-7.42 (m, 2H, C6H4), 4.96-5.01 (m,
1H, CH), 4.74-4.78 (m, 2H, CH2N), 2.31-2.38 (m, 2H, CH2), 2.01 (s, 3H,
CH3CO), 1.29 (d, 3H, CH3, JCH3CH=6.4 Hz); 13C NMR (100 MHz, CDCl3,
δ): 170.34, 163.30, 132.97, 131.28, 131.04, 130.77, 126.88, 126.40, 67.92,
49.81, 35.10, 21.12, 19.83.
(–)-(R)-4-[5-(2-chlorophenyl)-2H-tetrazol-2-yl]butan-2-ol ((–)-(R)-2d).
Oil, yield 44%. [α]D24 =-23.88 (c 1.38 in CH3OH, ee=99%). 1H NMR
(400 MHz, CDCl3, δ): 7.92-7.95 (m, 1H, C6H4), 7.52-7.54 (m, 1H, C6H4),
7.37-7.41 (m, 2H, C6H4), 4.84-4.90 (m, 2H, CH2N), 3.86-3.90 (m, 1H, CH),
2.20-2.28 (m, 1H, CHaHb), 2.06-2.15 (m, 1H, CHaHb), 2.02 (s, 1H, OH),
1.26 (d, 3H, CH3, JCH3CH=6.4 Hz); 13C NMR (100 MHz, CDCl3, δ):
163.23, 132.99, 131.29, 131.09, 130.80, 126.92, 126.40, 64.73, 50.29, 38.01,
23.62.
(–)-(S)-1-(5-phenyl-2H-tetrazol-2-yl)propan-2-yl acetate ((–)-(S)-3a).
Oil, yield 46%. [α]D23.4 = -12.81 (c 1.64 in CH3OH, ee=nd). 1H NMR
(400 MHz, CDCl3, δ): 8.13-8.15 (m, 2H, C6H5), 7.46-7.50 (m, 3H, C6H5),
5.42-5.47 (m, 1H, CH), 4.77-4.79 (m, 2H, CH2N), 2.01 (s, 3H, CH3CO),
1.36 (d, 3H, CH3, JCH3CH=6.4 Hz); 13C NMR (100 MHz, CDCl3, δ):
170.03, 165.21, 130.35, 128.86, 127.19, 126.79, 67.84, 56.27, 20.89, 17.43.
(–)-(R)-1-(5-phenyl-2H-tetrazol-2-yl)propan-2-ol ((–)-(R)-4a). Color-
less crystals, mp 53-55°C, yield 45%. [α]D24 = -58.48 (c 1.18 in CH3OH,
1
ee=nd). H NMR (400 MHz, CDCl3, δ): 8.06-8.09 (m, 2H, C6H5), 7.45-7.47
(m, 3H, C6H5), 4.66 and 4.69 (dd, 1H, CHaHbN, JHbCH=3.6Hz), 4.57 and
4.61 (dd, 1H, CHaHbN, JHaCH=7.6 Hz, JHaHb=13.6 Hz), 4.43-4.47 (m, 1H,
CH), 3.02 (s, 1H, OH), 1.34 (d, 3H, CH3, JCH3CH = 6.4 Hz); 13C NMR
(100 MHz, CDCl3,δ): 165.06, 130.41, 128.85, 126.93, 126.77, 66.18, 59.78, 20.22.
(–)-(S)-1-[5-(4-methylphenyl)-2H-tetrazol-2-yl)propan-2-yl acetate ((–)-
(S)-3b). Oil, yield 43%. [α]D25.5 = -9.81 (c 1.68 in CH3OH, ee=95.5%). 1H
NMR (400 MHz, CDCl3, δ): 8.00-8.03 (m, 2H, C6H4), 7.26-7.29 (m, 2H,
C6H4), 5.41-5.46 (m, 1H, CH), 4.77 and 4.80 (dd, 1H, CHaHbN, JHbCH=1.6
Hz), 4.73 and 4.76 (dd, 1H, CHaHbN, JHaCH=2.4 Hz, JHaHb=14 Hz), 2.40
(s, 3H, CH3), 2.00 (s, 3H, CH3CO), 1.35 (d, 3H, CH3, JCH3CH=6.8 Hz);
13C NMR (100 MHz, CDCl3, δ): 170.03, 164.20, 140.52, 129.54, 126.71,
124.39, 67.86, 56.21, 21.45, 20.89, 17.43.
(–)-(R)-1-[5-(4-methylphenyl)-2H-tetrazol-2-yl)propan-2-ol ((–)-(R)-4b).
Colorless crystals, mp 85-87°C, yield 43%. [α]D25.5 = -52.59 (c 1.41 in
CH3OH, ee=95.5%). 1H NMR (400 MHz, CDCl3, δ): 7.96-7.98 (m, 2H,
C6H4), 7.25-7.27 (m, 2H, C6H4), 4.64 and 4.68 (dd, 1H, CHaHbN,
JHbCH=3.6 Hz), 4.56 and 4.59 (dd, 1H, CHaHbN, JHaCH=7.6 Hz,
JHaHb=13,6 Hz), 4.42-4.46 (m, 1H, CH), 3.01 (s, 1H, OH), 2.40 (s, 3H,
CH3C6H4), 1.33 (d, 3H, CH3, JCH3CH=6.4 Hz); 13C NMR (100 MHz,
CDCl3, δ): 165.16, 140.61, 129.55, 126.70, 124.14, 66.19, 59.73, 21.45, 20.20.
(–)-(S)-1-[5-(4-chlorophenyl)-2H-tetrazol-2-yl)propan-2-yl acetate ((–)-
(S)-3c). Colorless crystals, mp 66-68°C, yield 47%. [α]D25.7 =-7.07 (c 1.63 in
1
CH3OH, ee=98%). H NMR (400 MHz, CDCl3, δ): 8.05-8.08 (m, 2H, C6H4),
(–)-(R)-1-[5-(4-chlorophenyl)-2H-tetrazol-2-yl)propan-2-ol ((–)-(R)-4c).
Colorless crystals, mp 100-102°C, yield 39%. [α]D25.5 = -53.5 (c 1.37 in
CH3OH, ee=98%). 1H NMR (400 MHz, CDCl3, δ): 8.01-8.04 (m, 2H,
C6H4), 7.42-7.45 (m, 2H, C6H4), 4.66 and 4.69 (dd, 1H, CHaHbN,
7.43-7.47 (m, 2H, C6H4), 5.40-5.46 (m, 1H, CH), 4.72-4.81 (m, 2H, CH2N),
2.00 (s, 3H, CH3CO), 1.35 (d, 3H, CH3, JCH3CH=6.4 Hz); 13C NMR
(100 MHz, CDCl3, δ): 170.00, 164.36, 136.38, 129.16, 128.09, 125.70, 67.79,
56.37, 20.88, 17.45.
Chirality DOI 10.1002/chir

HYDROLYSIS OF TETRAZOLE DERIVATIVES
813
(–)-(S)-1-[5-(2-chorophenyl)-2H-tetrazol-2-yl)propan-2-yl acetate ((–)-
(S)-3d). Oil, yield 47%. [α]D25.5 = -11.72 (c 1.66 in CH3OH, ee=99%).
1H NMR (400 MHz, CDCl3, δ): 7.92-7.96 (m, 1H, C6H4), 7.50-7.55 (m,
1H, C6H4), 7.35-7.42 (m, 3H, C6H5), 5.41-5.49 (m, 1H, CH), 4.84 and
4.87 (dd, 1H, CHaHbN, JHbCH=4.4 Hz), 4.78 and 4.81 (dd, 1H,
CHaHbN, JHaCH=6.8 Hz, JHaHb=14 Hz), 2.01 (s, 3H, CH3CO), 1.37
(d, 3H, CH3, JCH3CH=6.8 Hz); 13C NMR (100 MHz, CDCl3, δ): 170.01,
163.39, 133.04, 131.29, 131.10, 130.78, 126.88, 126.32, 67.86, 56.40, 20.88,
17.43.
toluene:ethyl acetate (5:1 v/v) as the eluent and by GC. After
an appropriate time reactions were stopped by filtering off
the enzyme. Enantiomerically enriched products were sepa-
rated by silica gel column chromatography. Enantiomeric
excesses (ee) of alcohols (–)-2a-d and (–)-4b-d, and esters
(–)-3c-d were determined by HPLC analysis using a Chiralcel
OD-H column. This column was inappropriate to the detection
of ee’s of the remaining esters (+)-1a-d and (–)-3b; therefore
they were hydrolyzed to the corresponding alcohols (+)-2a-d
and (–)-4b. The reaction was performed in MeOH with 1M
NaOH solution at room temperature. It seems reasonable to
assume that the ee of acetates (+)-1a-d and (–)-3c-d were the
same or even higher than alcohols prepared from them. This
column was also unsuitable for detection of the optical purity
of alcohol (–)-4a and ester (–)-3a; therefore the results of the
enzymatic hydrolysis of ester ( )-3a are unknown.
Taking into consideration the results of the enzymatic
hydrolysis of esters ( )-1a-d, the most effective catalyst
was Novozyme SP 435, which catalyze reactions in good
stereoselectivity and in a relatively short time, giving excel-
lent conversion rates. Indeed, the resolution was complete
and optically pure products were obtained after 20–24 h. Fur-
thermore, the data presented in Table 1 indicate that chloro
and methyl substituents improve the enantioselectivity of
the reaction without significant change of their times. For
comparison, when these reactions were performed in the
presence of other lipases (Amano PS and Amano AK), the
decrease in conversion, selectivity, and extending the reac-
tion times were observed. Even, after 2 w, the resolutions
hardly reached conversions close to 13.5–43%. The reactions
with Amano PS as catalyst were the most sluggish and almost
not selective.
A similar observation was done for kinetic resolution of
esters ( )-3a-d. The results of these studies are summarized
in Table 2. As can be seen, immobilized lipase (Novozyme SP
435) was a more effective catalyst than the two other native
enzymes (Amano AK and Amano PS). The reactions catalyzed
by Novozyme SP 435 required shorter times (21–25 h) to reach
50% conversion, and achieved the highest enantioselectivity
values (E > 100).
Comparison of the results in Tables 1 and 2 clearly shows
that the distance between the acetoxy group and the tetrazole
ring does not significantly affect the selectivity of enzymes.
Additionally, in all cases (R)-stereopreference was observed.
The absolute configurations of the products, that is, alcohols
(–)-2a-d, (–)-4a-d and esters (+)-1a-d, (–)-3a-d, were deter-
mined by comparing their specific rotation values with those
described earlier.33 According to them, the alcohols and un-
changed esters have (R) and (S) configuration, respectively.
In comparison to our previous article33 we found that
reversing direction of the reaction, and the use of hydrolysis
reaction instead of acetylation, significantly affects the
Antifungal Activity Assay
The antifungal activity of compounds ( )-1a-d, ( )-2a-d, (–)-(R)-2a-d,
(+)-(S)-2a-d, ( )-3a–d, (–)-(S)-3d, ( )-4a-d, (–)-(R)-4a-d, and (+)-(S)-
4a-d against mold strains Fusarium oxysporum MF 5, Fusarium
sambucinum MF 1, Aspergillus niger ATCC 16404, Colletotrichum coccodes
MC 1 was determined by measuring the inhibition of radial growth on an
agar medium in a Petri dish.35,36 The compound was dissolved in di-
methyl sulfoxide (DMSO) and added to a potato dextrose agar (PDA,
Merck) medium to give a final concentration of 128 μg ml-1 (w/v) of
tested compound and less than 0.2 % (v/v) of DMSO. The 6-mm diameter
mycelial disk was cut out of the actively growing fungal mycelium on
PDA and placed at the center of each Petri dish. The final DMSO concen-
tration was identical in both control (without tested compound) and
treated cultures. The radial fungal growth was measured after several
days of incubation at 25 °C. The inhibitory activity of each compound
was calculated as a percentage of the positive control growth using the
following equation: Inhibition = ((control – treatment)/control) × 100%.37
Each experiment was repeated twice and the data presented here are
the average of two experiments. As a control to the antifungal activity
assay the plates with amphotericin B (BioShop) at a final concentration
of 21 μg ml-1 (w/v) were used.
RESULTS AND DISCUSSION
The racemic mixtures of 4-(5-aryl-2H-tetrazol-2-yl)butan-2-yl
acetate ( )-1a-d and 1-(5-aryl-2H-tetrazol-2-yl)propan-2-yl
acetate ( )-3a-d were prepared from the appropriate5-
substituted tetrazoles according to the procedure reported
earlier.33 The synthesis involved three steps: preparation of
appropriate ketones, reduction of carbonyl compounds to cor-
responding alcohols, and acetylation of hydroxy group with
acetic anhydride in anhydrous pyridine solution.
Kinetic resolution of the racemic mixtures of acetates ( )-
1a-d and ( )-3a-d was performed by lipase-catalyzed hydro-
lysis. The reactions were carried out at room temperature in
a two-phase tert-butyl methyl ether (TBME)-phosphate buffer
of pH=7.2 system (Scheme 1).
The control experiment revealed that the reaction did not
proceed in the absence of the enzyme. Three commercially
available lipases: Novozyme SP 435 from Candida antarctica,
Amano AK from Pseudomonas fluorescens, and Amano PS from
Burkholderia cepacia (earlier Pseudomonas cepacia) were
used as catalysts and the influence of lipase type on the
enantioselectivity of the reaction was investigated. The prog-
ress of the reactions was monitored on TLC plates using
Scheme 1. Lipase-catalyzed hydrolysis of acetate ( )-1a-d and ( )-3a-d.
Chirality DOI 10.1002/chir

814
ŁUKOWSKA-CHOJNACKA AND MIERZEJEWSKA
TABLE 1. Results of lipase-catalyzed hydrolysis of esters ( )-1a-d
Entry
1
Ar
C6H5-
4-CH3-C6H4-
4-Cl-C6H4-
2-Cl-C6H4-
C6H5-
4-CH3-C6H4-
4-Cl-C6H4-
2-Cl-C6H4-
C6H5-
4-CH3-C6H4-
4-Cl-C6H4-
2-Cl-C6H4-
Enzyme
Time (h)
ca (%)
eealk.b (%)
eeest.b (%)
Ea
Novozyme SP 435
24
20
23
50
49
50
50
43
41
40
37
27
13
17
32
97
99
98
99
99
97
93
95
85
77
67
71
99
97
99
99
76
69
62
57
32
12
14
33
>100
>100
>100
>100
>100
>100
56
23
2
3
Amano AK
Amano PS
336
336
336
336
312
312
312
312
69
17
9
6
8
aConversion (c) and E values were calculated from the enantiomeric excess of substrate (+)-1a-d (eeest.) and product (-)-2a-d (eealk.) using the formula: c=eesub
(eesub+eeprod), E=Ln[(1-eesub)(eeprod/eesub+eeprod)]/Ln[(1+eesub)(eesub+eeprod)].
/
bDetermined by HPLC analysis using a Chiracel OD-H column.
TABLE 2. Results of lipase-catalyzed hydrolysis of esters ( )-3a-d
Entry
1
Ar
C6H5-
4-CH3-C6H4-
4-Cl-C6H4-
2-Cl-C6H4-
C6H5-
4-CH3-C6H4-
4-Cl-C6H4-
2-Cl-C6H4-
C6H5-
4-CH3-C6H4-
4-Cl-C6H4-
2-Cl-C6H4-
Enzyme
Time (h)
ca(%)
eealk.b(%)
eeest.b(%)
Ea
Novozyme SP 435
21
25
23
nd
50
50
50
nd
51
33
49
nd
43
26
61
nd
95
98
98
nd
92
97
88
nd
87
93
34
nd
95
98
99
nd
95
48
83
nd
67
32
54
nd
>100
>100
>100
nd
89
>100
41
nd
29
38
23
2
3
Amano AK
Amano PS
336
336
95
336
432
432
95
336
3
aConversion (c) and E values were calculated from the enantiomeric excess of substrate (-)-3b-d (eeest.) and product (-)-4b-d (eealk.) using the formula: c=eesub
/
(eesub+eeprod), E=Ln[(1-eesub)(eeprod/eesub+eeprod)]/Ln[(1+eesub)(eesub+eeprod)].
bDetermined by HPLC analysis using a Chiracel OD-H column.
nd, no data.
selectivity of enzymes. In hydrolysis the most stereoselective
catalyst is Novozyme SP 435, while in the acetylation lipase
Amano AK. Furthermore, taking into account the time of
reactions, the lipase-mediated hydrolysis approach appeared
to be better than the transesterification reaction. The enzy-
matic hydrolysis runs faster, and the pure enantiomers of
alcohols 2a-d and 4a-d can be obtained after 20–25 h of the
reaction.
Synthesized tetrazole derivatives were tested for antimicro-
bial activity. An agar-diffusion method was used for determina-
tion of the antibacterial activity against Gram-negative bacteria
Escherichia coli, Salmonella typhimurium, Pseudomonas
aeruginosa as well as Gram-positive bacteria Bacillus subtilis
and Staphylococcus aureus. Unfortunately, all the tested
compounds did not reveal appreciable antibacterial
activity, since the MIC (minimal inhibitory concentration)
values were 512 μg. These compounds were also evaluated
for antifungal activity against the yeast Candida albicans
using a broth microdilution susceptibility procedure38 and
against mold strains Fusarium sambucinum, Fusarium
oxysporum, Colletotrichum coccodes, Aspergillus niger as
described in the Materials and Methods. In the case of
C. albicans, only compounds ( )-2c, (–)-(R)-2c, and
Chirality DOI 10.1002/chir
(+)-(S)-2c showed very weak antifungal activity (MIC value of
128 μgml-1). Some of the synthesized compounds showed
promising antifungal activity against the tested mold strains.
The median inhibitory concentration (IC50) was considered
as the tetrazole derivative concentration that resulted in 50%
inhibition of the fungal growth when compared to the control
group. The results are given in Table 3. As can be seen, only
compounds ( )-1c, ( )-2c, (R)-(–)-2c, (S)-(+)-2c, and ( )-3c
showed promising antifungal activity against all tested mold
strains when applied to the culture medium at concentration
128 μg ml-1 to reach IC50. We also observed that even a
-1
64 μg ml concentration of compound ( )-2c was effective
for 50% growth inhibition of A. niger ATCC16404. The com-
pounds ( )-1a-b, 2a, (–)-(R)-2b, (+)-(S)-2d, ( )-3c, (+)-(S)-3d,
( )-4a-d, 4c, and (+)-(S)-4d effectively inhibited the growth
of at least one of the tested mold strains. Comparison of all
results clearly shows that esters and alcohols substituted
by chlorine at para position of the aromatic ring possess
the highest antifungal activity. Moreover, tetrazole deriva-
tives with a longer alkyl chain (1a-d and 2-a-d) show
better antifungal activity. It is also worth mentioning that
in most cases the antifungal activity of both enantiomers
is similar.

HYDROLYSIS OF TETRAZOLE DERIVATIVES
815
TABLE 3. Antifungal activity (% of growth inhibition) of the tetrazole derivatives at concentration of 128 μg ml -1
Entry
Compound
F. sambucinum MF 1
F. oxysporum MF 5
C. coccodes MC 1
A. niger ATCC 16404
1
2
3
4
5
( )-1a
28.5
51.1
54.3
33.3
5
3.7
7.8
44.2
42.9
41.8
60.5
60.2
61.6
16.7
7.6
28.6
9.3
38.3
49.6
8.6
26.9
6.8
nd
nd
14
16.7
16.7
47.1
42.9
41
3.1
7.2
51.9
51.1
60.9
43,6
20.2
24.5
25.7
41.3
35
26.1
55.2
54.7
55.8
18.4
20.9
23.1
38.7
45.1
58.1
45.1
48.6
7.9
43.3
47.6
57.8
43.1
53.1
53.9
55.6
36.9
43.8
44.4
72
66.7
67.5
46.6
47.5
52
41.1
45.1
60.8
43.6
55.6
53.7
nd
37.3
59.9
67.3
33.3
28.4
32.4
33.2
47
( )-1b
( )-1c
( )-1d
( )-2a
(-)-(R)-2a
(+)-(S)-2a
( )-2b
6
7
8
(-)-(R)-2b
(+)-(S)-2b
( )-2c
(-)-(R)-2c
(+)-(S)-2c
( )-2d
54.3
36.2
83
85
80.6
27.5
30.5
31.6
18.3
39.7
61.5
28.6
43.2
4.7
(-)-(R)-2d
(+)-(S)-2d
( )-3a
9
10
11
12
( )-3b
( )-3c
( )-3d
(+)-(S)-3d
( )-4a
13
14
15
16
(-)-(R)-4a
(+)-(S)-4a
( )-4b
(-)-(R)-4b
(+)-(S)-4b
( )-4c
(-)-(R)-4c
(+)-(S)-4c
( )-4d
(-)-(R)-4d
(+)-(S)-4d
control*
nd
nd
nd
nd
nd
61.8
40
35.1
28.4
22.4
49.7
48.2
46.7
12.8
14.3
11.4
64.7
39.6
11.1
25
72.2
66.8
65.5
17.9
11.3
17.3
88.2
38
59.7
63.6
56.8
55.2
39.9
53.9
88.9
8.3
83.1
17
nd, no data.
*Amphotericin B-standard antifungal agent at a concentration of 21 μg ml-1.
3. Alonen A, Finel M, Kostiainen R. The human UPD-glucuronosyltransferese
UGT1A3 is highly selective towards N2 in the tetrazole ring of losartan,
candesartan, and zolasartan. Biochem Pharmacol 2008;76:763–772.
4. Alonen A, Jansson J, Kallonen S, Kiriazis A, Aitio O, Finel M, Kostiainen
R. Enzyme-assisted synthesis and structure characterization of glucuronic
acid conjugates of losartan, candesartan, and zolarsartan. Bioorg Chem
2008;36:148–155.
5. Brandstetter TW, Davis B, Hyett D, Smith C, Hackett L, Winchester BG,
Fleet GWJ. Tetrazoles of manno- and rhamno-pyranoses: inhibition of
glycosidases by tetrazoles and other mannose mimics. Tetrahedron Lett
1995;36:7511–7514.
6. Davis BG, Brandstetter TW, Hackett L, Winchester BG, Nash RJ, Watson
AA, Griffiths RC, Smith C, Fleet GWJ. Tetrazoles of manno- and rhamno-
pyranoses: contrasting inhibition of mannosidases by [4.3.0] but of
rhamnosidase by [3.3.0] bicyclic tetrazoles. Tetrahedron 1999;55:4489–4500.
7. Vinh TK, Ahmadi M, Delgado POP, Perez SF, Walters HM, Smith HJ,
Nicholls PJ, Simons C. 1-[(Benzofuran-2-yl)phenylmethyl]-triazoles and –
tetrazoles- potent competitive inhibitors of aromatase. Bioorg Med Chem
Lett 1999;9:2105–2108.
8. Liljebris C, Larsen SD, Ogg D, Palazuk BJ, Bleasdale JE. Investigation of
potential bioisosteric replacements for the carboxyl groups of
peptidomimetic inhibitors of protein tyrosine phosphatase 1B: identifica-
tion of a tetrazole-containing inhibitor with cellular activity. J Med Chem
2002;45:1785–1798.
CONCLUSION
In summary, the enzymatic hydrolysis of racemic esters
containing a tetrazole ring is a very efficient and convenient
method for obtaining enantiomerically enriched secondary
alcohols and their acetates. The best results from the point
of view of enantioselectivity and enantiopurity of the formed
products were achieved by hydrolysis in the presence of
Novozyme SP 435. Some tetrazole derivatives possess inter-
esting antifungal activity against F. sambucinum, F. oxysporum,
C. coccodes, and A. niger. An especially active compound
proved to be 4-[5-(4-chlorophenyl)-2H-tetrazol-2yl]butan-2-ol.
ACKNOWLEDGMENTS
This work was supported in part by the European Union in
the framework of the European Social Fund through the
Warsaw University of Technology Development Programme,
realized by the Center for Advanced Studies and in part by
Warsaw University of Technology.
LITERATURE CITED
1. Herr RJ. 5-Substituted-1H-tetrazoles as carboxylic acid isosteres: medicinal
chemistry and synthetic methods. Bioorg Med Chem 2002;10:3379–3393.
2. Aureggi V, Sedelmeier G. 1,3-Dipolar cycloaddition: click chemistry for
the synthesis of 5-substituted tetrazoles from organoaluminum azides
and nitriles. Angew Chem Int Ed 2007;46:8440–8444.
9. Sun DX, Liu L, Heinz B, Kolykhalov A, Lamar J, Jahnson RB, Wang QM,
Yip Y, Chen S-H. P4 cap modified tetrapeptidyl α-ketoamides as potent
HCV NS3 protease inhibitors. Bioorg Med Chem Lett 2004;14:4333–4338.
10. Al-Hourani BJ, Sharma SK, Mane JY, Tuszynski J, Baracos V, Kniess T, Suresh
M, Pietzsch J, Wuest F. Synthesis and evaluation of 1,5-diaryl-substituted
Chirality DOI 10.1002/chir

816
ŁUKOWSKA-CHOJNACKA AND MIERZEJEWSKA
tetrazoles as novel selective cyclooxygenase-2 (COX-2) nhibitors. Bioorg Med
Chem Lett 2011;21:823–1826.
25. Matysiak J, Niewiadomy A, Krajewska-Kułak E, Mcik-Niewiadomy G.
Synthesis of some 1-(2,4-dihydroxythiobenzoyl)imidazoles, -imidazolines
and tetrazoles and their potent activity against Candida species. IL
Farmaco 2003;58:455–461.
26. Upadhayaya RS, Sinha N, Jain S, Kishore N, Chandra R, Arora SK. Opti-
cally active antifungal azoles and antifungal activity of (2R,2S)-2-(2,4-
difluorophenyl)-3-(5-{2-[4-aryl-piperazin-1-yl]-ethyl}-tetrazol-2-yl/1-yl)-1-[1,2,4]-
triazol-1-yl-butan-2-ol. Bioorg Med Chem 2004;12:2225–2238.
11. Quan ML, Ellis CD, He MY, Liauw AY, Woerner FJ, Alexander RS, Knabb
RM, Lam PYS, Luettgen JM, Wong PC, Wright MR, Wexler RR.
Nonbenzamidine tetrazole derivatives as factor Xa inhibitors. Bioorg
Med Chem Lett 2003;13:369–373.
12. Yuan H, Silverman RB. New substrates and inhibitors of γ-aminobutyric acid
aminotransferase containing bioisosteres of the carboxylic acid group: design,
synthesis, and biological activity. Bioorg Med Chem 2006;14:1331–1338.
13. Rajasekaran A, Thampi PP. Synthesis and analgesic evaluation of some
5-[â-(10-phenothiazinyl)ethyl]-1-(acyl)-1,2,3,4-tetrazoles. Eur J Med Chem
2004;39:273–279.
14. Juby PF, Hudyma TW, Brown M. Preparation and antiinflammatory proper-
ties of some 5-(2-anilinophenyl)tetrazoles. J Med Chem 1968;11:111–117.
15. Juby PF, Hudyma TW. Preparation and antiinflammatory properties of
some 1-substituted 3-(5-tetrazolulmethyl)indoles and homologs. J Med
Chem 1969;12:396–401.
16. Maxwell JR, Wasdahl DA, Wolfson AC, Stenberg VI. Synthesis of 5-aryl-
2H-tetrazoles, 5-aryl-2H-tetrazole-2-acetic acids, and [(4-phenyl-5-aryl-4H-
1,2,4-triazol-3-yl)thio]acetic acids as possible superoxide scavengers and
anti-inflammatory agents. J Med Chem 1984;27:1565–1570.
27. Upadhayaya RS, Jain S, Sinha N, Kishore N, Chandra R, Arora SK. Synthe-
sis of novel substituted tetrazoles having antifungal activity. Eur J Med
Chem 2004;39:579–592.
28. Khanage SG, Mohite PB, Pandhare RB, Raju SA. Investigation of pyrazole
and tetrazole derivatives containing 3,5 disubstituted-4H-1,2,4-triazole as a
potential antitubercular and antifungal agent. Biointerface Res Appl Chem
2012;2:277–283.
29. Malik MA, Al-Thabaiti SA, Malik MA. Synthesis, structure optimization
and antifungal screening of novel tetrazole ring bearing acyl-hydrazones.
Int J Mol Sci 2012;13:10880–10898.
30. Mohite PB, Bhaskar VH. Synthesis and antifungal activity of 3-aryl-1-
(5-phenyl-1H-tetrazol-1yl)prop-2-en-1-one. Orbital Elec J Chem Campo
Grande 2010;2:311–315.
31. Libero FM, Xavier MCD, Victoria FN, Nascente PS, Savegnago L, Perin G,
Alves D. Synthesis of novel selenium and tellurium-containing tetrazoles: a
class of chalcogen compounds with antifungal activity. Tetrahedron Lett
2012;53:3091–3094.
32. Kathiravan MK, Salake AB, Chothe AS, Dudhe PB, Watode RP, Mukta
MS, Gadhwe S. The biology and chemistry of antifungal agents: a review.
Bioorg Med Chem 2012;20:5678–5698.
17. Ellis GP, Shaw D. Benzopyrones. 7. Synthesis and antiallergic activity of
some 2-(5-tetrazolyl)chromones. J Med Chem 1972;15:865–867.
18. Nohara A, Kuriki H, Saijo T, Sugihara H, Kanno M, Sanno Y. Studies on
antianaphylactic agent. 5. Synthesis of 3-(1H-tetrazol-5-yl)chromones, a
new series of antiallergic substances. J Med Chem 1977;20:141–145.
19. Essery JM. Preparation and antibacterial activity of α-(5-tetrazolyl)
benzylpenicillin. J Med Chem 1996;12:703–705.
33. Łukowska-Chojnacka E, Berna U, Plenkiewicz J. Lipase-catalyzed
enantioseparation of alcohols containing a tetrazole ring. Tetrahedron:
Asymmetry 2012;23:136–143.
20. Dhayanithhi V, Syed SS, Kumaran K, Sankar KRJ, Ragavan RV, Goud PSK,
Kumari NS, Pati HN. Synthesis of selected 5-thio-substituted tetrazole
derivatives and evaluation of their antibacterial and antifungal activities.
J Serb Chem Soc 2011;76:165–175.
21. Varadaraji D, Suban SS, Ramasamy VR, Kubendiran K, Raguraman JSKG,
Nalilu SK, Pati HN. Synthesis and evaluation of a series of 1-substituted
tetrazole derivatives as antimicrobial agents. Org Commun 2010;3:45–56.
22. Jeyanthi P, Sheela K, Pazhanisamy P. Synthesis and biological activities of
2-(α-p-substituted phenyl-α-benzimidazolo)methyl-1,2,3,4-tetrazole. Int J
Pharm Chem Sci 2013;2:950–955.
23. Uttarwar RB, Nawale RB, Shamkuwar PB. Synthesis and pharmacological
screening of derivatives of benzimidazole linked with quinoline and tetra-
zole. J Chem Pharm Res 2013;5:41–46.
34. Finnegan WG, Henry RA, Lofquist R. An improved synthesis of 5-substituted
tetrazoles. J Am Chem Soc 1958;80:3908–3911.
35. Liu W, Shi HM, Jin H, Zhao HY, Zhou GP, Wen F, Yu ZY, Hou TP. Design,
synthesis and antifungal activity of a series of novel analogs based an
diphenyl ketones. Chem Biol Drug Des 2009;73:661–667.
36. Borowiecki P, Milner-Krawczyk M, Brzeziska D, Wielechowska M,
Plenkiewicz J. Synthesis and antibacterial activity of imidazolium and
triazolium chiral ionic liquids. Eur J Org Chem 2013;2013:712–720.
37. Nguefack J, Leth V, Zollo PHA, Mathur SB. Evaluation of five essential
oils from aromatic plants of Cameroon for controlling food spoilage and
mycotoxin producing fungi. Int J Food Microbiol 2004;94:329–334.
38. National Committee for Clinical Laboratory Standards (NCCLS). Approved
Standard (M27-A2), NCCLS, Wayne, PA, 2002.
24. Rajasekaran A, Murugesan S, Rajagopal KA. Antibacterial, antifungal and
anticonvulsant evaluation of novel newly synthesized 1-[2-(1H-tetrazol-5-yl)
ethyl]-1H-benzo[d][1,2,3]triazoles. Arch Pharm Res 2006;29:535–540.
Chirality DOI 10.1002/chir