Enzymatic removal of carboxyl protecting groups. 1. Cleavage of the tert-butyl moiety

Article abstract of DOI:10.1021/jo050114z

(Chemical Equation Presented) A recent discovery that a certain amino acid motif (GGG-(A)X-motif) in lipases and esterases determines their activity toward tertiary alcohols prompted us to investigate the use of these biocatalysts in the mild and selective removal of tert-butyl protecting groups in amino acid derivatives and related compounds. An esterase from Bacillus subtilis (BsubpNBE) and lipase A from Candida antarctica (CAL-A) were identified as the most active enzymes, which hydrolyzed a range of tert-butyl esters of protected amino acids (e.g., Boc-Tyr-O_tBu, Z-GABA-O_tBu, Fmoc-GABA-O _tBu) in good to high yields and left Boc, Z, and Fmoc-protecting groups intact.

Full text of DOI:10.1021/jo050114z

Enzymatic Removal of Carboxyl Protecting
Groups. 1. Cleavage of the tert-Butyl
Moiety
synthesis of polyfunctional molecules is that a given
functional group has to be protected or deprotected
selectively in the presence of functionalities of similar
reactivity as well as functionalities sensitive to acids,
bases, oxidation, or reduction. The application of biocata-
lysts in the protecting group chemistry may offer excel-
lent alternatives to chemical methods because enzymes
(i) carry out highly chemo- and regioselective transforma-
tions, (ii) usually operate at neutral pH values, and (iii)
combine a high selectivity for the reactions they catalyze
and the structure they recognize with a broad substrate
tolerance.2
Marlen Schmidt,†,‡ Efrosini Barbayianni,
‡,§
Irene Fotakopoulou,‡ Matthias H o¨ hne,
,|
†
|
Violetta Constantinou-Kokotou,
Uwe T. Bornscheuer,*,† and George Kokotos*,§
Department of Technical Chemistry and Biotechnology,
Institute of Chemistry and Biochemistry, Greifswald
University, Soldmannstr. 16, D-17487 Greifswald, Germany,
Laboratory of Organic Chemistry, Department of Chemistry,
University of Athens, Panepistimiopolis, GR-15771,
Athens, Greece, and Chemical Laboratories, Agricultural
University of Athens, Iera Odos 75, Athens 11855, Greece
Among the biocatalysts, hydrolases have attracted
special interest for their chemo-, regio-, and enantiose-
lectivities and have found interesting applications in
organic synthesis, in particular for the production of
gkokotos@cc.uoa.gr; uwe.bornscheuer@uni-greifswald.de
Received January 20, 2005
3
enantiopure organic molecules. Indeed, some hydrolases
were already found to be suitable for the selective
2
removal of protecting groups. Lipases were shown to
4
hydrolyze heptyl esters of peptides, and an enzyme from
Sphingomonas paucimobilis SC16113 was able to enan-
tioselectively cleave N-benzyloxycarbonyl (Cbz) groups
5
from protected amino acids and related compounds. For
tert-butyl esters, an alkaline serine protease (thermitase)
6
could be used to cleave differently N-protected peptides,
and a lipase from Burkholderia sp. YY62 was able to
hydrolyze tert-butyl octanoate.7
Recently, we discovered that a certain amino acid motif
GGG(A)X-motif, in single-letter amino acid code where
(
G denotes glycine and X denotes any amino acid; in a
few enzymes, one glycine is replaced by an alanine, A)
located in the oxyanion binding pocket of lipases and
A recent discovery that a certain amino acid motif (GGG-
8
(
A)X-motif) in lipases and esterases determines their activity
esterases determines activity toward tertiary alcohols.
toward tertiary alcohols prompted us to investigate the use
of these biocatalysts in the mild and selective removal of
tert-butyl protecting groups in amino acid derivatives and
related compounds. An esterase from Bacillus subtilis
All enzymes bearing this motif (e.g., lipase from Candida
rugosa, pig liver esterase, acetyl choline esterases, and
a p-nitrobenzyl esterase from Bacillus subtilis (Bsubp-
NBE)) were active toward several acetates of tertiary
alcohols, while enzymes bearing the more common GX-
motif did not hydrolyze the model compounds. Conse-
quently, the search for a hydrolase active toward the
sterically demanding tertiary alcohol moiety is consider-
ably facilitated, as it can be simply based on the existence
of the GGG(A)X-motif. Thus, the trial and error approach
in testing all available lipases and esterases is not
necessary any more.
(BsubpNBE) and lipase A from Candida antarctica (CAL-
A) were identified as the most active enzymes, which
hydrolyzed a range of tert-butyl esters of protected amino
t
t
t
acids (e.g., Boc-Tyr-O Bu, Z-GABA-O Bu, Fmoc-GABA-O Bu)
in good to high yields and left Boc, Z, and Fmoc-protecting
groups intact.
The synthesis of complex, polyfunctional organic mol-
ecules, such as oligonucleotides, oligosaccharides, and in
general natural products, requires the proper introduc-
tion and removal of protecting groups. For this purpose,
a large number of protecting groups as well as chemical
methods for protection and deprotection of various func-
tionalities have been developed and are widely used in
(
2) (a) Kadereit, D.; Waldmann, H. Chem. Rev. 2001, 101, 3367. (b)
Waldmann, H.; Sebastian, D. Chem. Rev. 1994, 94, 911.
(
3) Bornscheuer, U. T.; Kazlauskas, R. J. Hydrolases in Organic
Synthesis - Regio- and Stereoselective Biotransformations; Wiley-
VCH: Weinheim, 1999.
(4) (a) Braun, P.; Waldmann, H.; Vogt, W.; Kunz, H. Synthesis 1990,
105. (b) Braun, P.; Waldmann, H.; Vogt, W.; Kunz, H. Liebigs Ann.
Chem. 1991, 165. (c) Braun, P.; Waldmann, H.; Kunz, H. Synlett 1992,
1
synthetic organic chemistry. A major problem for the
3
9.
(5) (a) Nanduri, V. B.; Goldberg, S.; Johnston, R.; Patel, R. N.
*
To whom correspondence should be addressed. (G.K.) Tel: +30
2
10 7274462. Fax: +30 210 7274761. (U.T.B.) Tel: +49 3834 864367.
Enzyme Microb. Technol. 2004, 34, 304. (b) Patel, R. N.; Nanduri, V.
B.; Brzozovski, D. B.; McNamee, C.; Banerjee, A. Adv. Synth. Catal.
2003, 345, 830.
Fax: +49 3834 86 80066.
†
Greifswald University.
‡
These authors contributed equally to this study.
(6) Schultz, M.; Hermann, P.; Kunz, H. Synlett 1992, 37.
(7) Yeo, S. H.; Nihira, T.; Yamada, Y. Biosci. Biotechnol. Biochem.
1998, 62, 2312.
§
University of Athens.
|
Agricultural University of Athens.
(
1) (a) Kocienski, P. J. Protecting Groups in Organic Synthesis, 3rd
(8) (a) Henke, E.; Pleiss, J.; Bornscheuer, U. T. Angew. Chem., Int.
Ed. 2002, 41, 3211. (b) Henke, E.; Bornscheuer, U. T.; Schmid, R. D.;
Pleiss, J. ChemBioChem 2003, 4, 485.
ed.; Wiley and Sons: New York, 1999. (b) Greene, T. W.; Wuts, P. G.
M. Protecting Groups; Georg Thieme Verlag: Stuttgart, 1994.
1
0.1021/jo050114z CCC: $30.25 © 2005 American Chemical Society
Published on Web 03/23/2005
J. Org. Chem. 2005, 70, 3737-3740
3737

SCHEME 1. Hydrolysis of tert-Butyl
γ-(tert-Butoxycarbonylamino)butyrate (1) Using
GGG(A)- and GX-Motif Enzymes
TABLE 1. Hydrolysis of tert-Butyl
γ-(tert-Butoxycarbonylamino)butyrate (1) by Various
Hydrolases
enzyme
motif
conversion
BsubpNBE
CAL-A
PLE
GGG(A)X
GGG(A)X
GGG(A)X
GGG(A)X
GX
+
+
-
-
-
-
-
-
PCE
PFE
CAL-B
BstE
GX
GX
GX
RMIM
In this paper, we have therefore selected enzymes
bearing the GGG(A)X-motif and investigated their ap-
plicability in the selective removal of the tert-butyl
protecting group in amino acid derivatives and related
compounds.
of tert-butyl esters were optimized using BsubpNBE and
CAL-A by the addition of various cosolvents. Toluene,
n-hexane, diethyl ether, and to a smaller extent metha-
nol, chloroform, and dimethyl sulfoxide facilitated the
enzymatic hydrolysis. Acetonitrile, acetone, ethyl acetate,
ethanol, n-propanol, and 2-propanol turned out to be
inappropriate cosolvents (data not shown).
A variety of hydrolases containing either the GGG(A)X-
9
10
motif (BsubpNBE, CAL-A, PLE, PCE ) or the GX motif
(
9
CAL-B, PFE, BstE, RMIM, serving as negative control)
To expand the applicability of this method, a variety
of tert-butyl esters were prepared by conventional chemi-
cal methods. The results of their hydrolyses by Bsubp-
NBE and CAL-A are summarized in Table 2. BsubpNBE
hydrolyzed substrates 1-4, 10, and 11 (Table 2) in good
to high yields. A variety of urethane-type N-protecting
groups (Boc, Z, Fmoc), usually used in protecting group
chemistry, remained intact under the conditions of the
enzymatic hydrolysis. Thus, we demonstrated that the
tert-butyl moiety of substrate 1 may be selectively
removed in the presence of the Boc group by enzymatic
hydrolysis. In addition, BsubpNBE selectively hydrolyzes
the tert-butyl ester of substrate 11, leaving the tert-butyl
ether intact. However, BsubpNBE is unable to remove
the tert-butyl ester group from the dipeptide substrate 5
were used in an initial screening for activity toward tert-
butyl γ-(tert-butoxycarbonylamino) butyrate 1 serving as
model substrate (Scheme 1). Compound 1 was chosen as
model, as both the C-tert-butyl and N-tert-butoxycarbonyl
protecting groups may be removed by chemical means
1
under acidic conditions. The tert-butoxycarbonyl group
may be selectively removed in the presence of tert-butyl
11
esters by using 1 M HCl in EtOAc, TBAF in refluxing
1
2
13
THF, 4 M HCl in anhydrous dioxane, and concen-
1
4
trated H
removal of C-tert-butyl esters in the presence of the Boc
group may be achieved by using the CeCl ‚7H O-NaI
C-tert-Butyl esters may
2 4 3
SO in t-BuOAc or MeSO H. The selective
3
2
1
5
16
2 2 2
system or ZnBr in CH Cl .
also be cleaved selectively, in the presence of C-tert-butyl
ethers, by using montmorillonite KSF clay.17
t
(Boc-Val-Gly-O Bu). Substrates 6 and 7, based on phe-
The results of the screening are summarized in Table
nylalanine and tyrosine moieties, were easily hydrolyzed
by BsubpNBE and the products of the enzymatic hy-
drolysis were isolated in satisfactory yields. In contrast,
substrates 8 and 9 were hydrolyzed in low yields,
indicating that BsubpNBE rather prefers the aromatic
side chains of phenylalanine and tyrosine than the small
aliphatic chains of alanine and valine. Under similar
conditions CAL-A seems to hydrolyze effectively only
substrates 2 and 6. However, when we used for the
CAL-A hydrolysis only buffer at elevated temperature (50
°C), in the absence of any cosolvent, better yields of
isolated products were observed (substrates 1-4).
Compared to the earlier work using thermitase-
catalyzed deprotection of the tert-butyl moiety from
1
. Two enzymes containing the GGG(A)X-motif, Bsubp-
9
NBE and lipase A from Candida antarctica (CAL-A,
Novozymes), hydrolyzed the substrate totally or partly,
respectively. However, two other enzymes containing the
GGG(A)X motif, PLE and PCE, did not show activity,
presumably because their specific activity was too low
or they do not accept amino acid derivatives in general.
Next, the reaction conditions for the enzymatic hydrolysis
(
9) BsubpNBE, recombinant p-nitrobenzyl esterase from Bacillus
subtilis produced in E. coli; CAL-A, lipase A from Candida antarctica
Novozymes); PLE, pig liver esterase (Roche); PCE, recombinant
(
esterase from Pyrobaculum calidifontis produced in E. coli; CAL-B,
lipase B from Candida antarctica (Novozymes); PFE, recombinant
esterase from Pseudomonas fluorescens produced in E. coli; BstE,
recombinant esterase from Bacillus stearothermophilus produced in
E. coli; RMIM, immobilized lipase from Rhizomucor miehei (No-
vozymes)).
6
peptides, the use of these esterases has the advantage
that esterases in general are more stable toward organic
solvents. In addition, the thermitase was only used for
the deprotection of peptides, and it is unclear whether
nonpeptidic substrates are accepted by this protease.
Although it appears that our methodology requires
relatively large amounts of enzyme, it must be noted
that these are crude preparations (in case of BsubpNBE
the protein content of the lyophilisate is approximately
(
10) Hotta, Y.; Ezaki, S.; Atomi, H.; Imanaka, T. Appl. Environm.
Microbiol. 2002, 68, 3925.
11) Gibson, F. S.; Bergmeier, S. C.; Rapoport, H. J. Org. Chem.
994, 59, 3216.
12) Routier, S.; Sauge, L.; Ayerbe, N.; Coudert, G.; Merour, J. Y.
(
1
(
Tetrahedron Lett. 2002, 43, 589.
(
(
13) Han, G.; Tamaki, M.; Hruby, V. J. J. Pept. Res. 2001, 58, 338.
14) Lin, L. S.; Lanza, T., Jr.; de Lazlo, S. E.; Truong, Q.; Kame-
necka, T.; Hagmann, W. K. Tetrahedron Lett. 2000, 41, 7013.
4
0% and the overall esterase content is estimated to
(
15) Marcantoni, E.; Massaccesi, M.; Torregiani, E. J. Org. Chem.
001, 66, 4430.
16) Wu, Y.; Limburg, D. C.; Wilkinson, D. E.; Vaal, M. J.; Hamilton,
G. S. Tetrahedron Lett. 2000, 41, 2847.
17) Yadav, J. S.; Subba Reddy, B. V.; Sanjeeva Rao, K.; Harikishan,
K. Synlett 2002, 5, 826.
2
approximately 15%). In addition, the reactivity of es-
terases toward the sterically demanding esters of tert-
iary alcohols is approximately 100-1000-fold lower com-
pared to natural triglycerides.8a Despite this aspect, we
(
(
3738 J. Org. Chem., Vol. 70, No. 9, 2005

TABLE 2. Hydrolysis of Various Esters by BsubpNBE and CAL-A
a
Yield of isolated product. b Mixture of buffer and n-hexane (37 °C). c Buffer (50 °C); n.d., not determined.
have demonstrated that two enzymes, BsubpNBE and
CAL-A, can be used in synthetic organic chemistry to
cleave tert-butyl esters from various substrates.
Synthesis of Substrates. 4-(9H-Fluoren-9-ylmethoxycar-
bonylamino)butyric Acid tert-Butyl Ester (3). To a stirred
solution of Fmoc-GABA (0.33 g, 1 mmol) and tert-butyl alcohol
(
2 2
0.3 mL, 3 mmol) in CH Cl (2 mL), 4-(dimethylamino)pyridine
(
0.01 g, 0.1 mmol), and subsequently N,N′-dicyclohexylcarbodi-
Experimental Section
imide (0.25 g, 1.2 mmol) were added at 0 °C. The reaction
mixture was stirred for 1 h at 0 °C and overnight at room
temperature. After filtration, the solvent was evaporated under
reduced pressure, and EtOAc (20 mL) was added. The organic
layer was washed consecutively with brine, 1 N HCl, brine, 5%
Melting points were determined on a Buchi apparatus and
are uncorrected. Specific rotations were measured at 25 °C on a
Perkin-Elmer polarimeter using a 10 cm cell. NMR spectra were
recorded on a 200 MHz spectrometer. All amino acid derivatives
were purchased from Fluka and Bachem. TLC plates (silica gel
NaHCO
3 2 4
, and brine, dried over Na SO , and evaporated under
reduced pressure. The residue was purified by column chroma-
6
0 F254) and silica gel 60 (70-230 or 230-400 mesh) for column
1
tography using CHCl
(CDCl ) δ 7.68 (m, 2H), 7.60 (m, 2H), 7.35 (m, 4H), 5.03 (m, 1H),
4.41 (d, 2H, J ) 6.6 Hz), 4.22 (t, 1H, J ) 6.6 Hz), 3.24 (m, 2H),
2.28 (t, 2H, J ) 7.0 Hz), 1.81 (m, 2H), 1.47 (s, 9H); C NMR
(CDCl ) δ 172.6, 156.4, 143.9, 141.2, 127.8, 127.6, 127.1, 126.9,
3
as eluent: yield 0.17 g (43%); oil; H NMR
chromatography were purchased from Merck. Visualization of
spots was effected with UV light and/or phosphomolybdic acid
and/or ninhydrin, both in EtOH stain. Esterase BsubpNBE was
produced recombinantly (see below), but is also available from
J u¨ lich Fine Chemicals (Esterase BS2, www.juelich-chemi-
cals.com), CAL-A was purchased from Novozymes (Denmark).
3
1
3
3
125.3, 125.0, 119.9, 80.4, 66.5, 47.2, 40.4, 32.7, 28.0, 25.1. Anal.
J. Org. Chem, Vol. 70, No. 9, 2005 3739

Calcd for C23
H
27NO
4
: C, 72.42; H, 7.13; N, 3.67. Found: C, 72.25;
Expression of Recombinant Esterases in Escherichia
coli. Clones containing the expression vector encoding genes for
esterases (BsubpNBE, BstE, PCE, and PFE) were used to
inoculate 5 mL of an overnight culture (LB-media supplemented
with 100 µg/mL of ampicillin). Five hundred microliters of the
overnight culture was used to inoculate 500 mL LB-Amp. The
culture was incubated at 37 °C and 200 rpm to a cell density
of OD600 0.4-0.6, and enzyme expression was induced by
addition of L-rhamnose solution (end concentration 0.2% w/v).
After 4 h of further incubation at 37 °C, cells were harvested by
centrifugation (15 min, 4 °C, 8000 g) and washed twice with 50
mL sodium phosphate buffer (50 mM, pH 7.5). Cells were
resuspended in 20 mL phosphate buffer and disrupted by
sonification with cooling on ice. Cell debris was removed by
centrifugation (15 min, 4 °C, 8000 g), the supernatant was frozen
at -80 °C and then lyophilized. Specific activities of crude
extracts were determined spectrophotometricaly using p-nitro-
phenyl acetate for activity and the Bradford reagent for protein
content.
H, 7.23; N, 3.72.
Substrates 1, 2, 4, and 6-9 were prepared according to the
above-described procedure. Their analytical data were in ac-
1
5
cordance with those reported in the literature (substrate 1,
1
8
19
15,20
15,21
substrate 2, substrate 4, substrate 6,
substrate 7,
1
5,22
15,23
substrate 8,
substrate 9
).
(
S)-(2-tert-Butoxycarbonylamino-3-methylbutyryl-
amino)acetic Acid tert-Butyl Ester (5). To a stirred solution
t
of H-Gly-O Bu‚HCl (0.42 g, 2.5 mmol) and Boc-Val-OH (0.54 g,
2
2 2 3
.5 mmol) in CH Cl (25 mL) were added Et N (0.76 mL, 5.5
mmol) and subsequently 1-(3-dimethylaminopropyl)-3-ethyl car-
bodiimide (0.53 g, 2.8 mmol) and 1-hydroxybenzotriazole (0.34
g, 2.5 mmol) at 0 °C. The reaction mixture was stirred for 1 h at
0
°C and overnight at room temperature. The solvent was
evaporated under reduced pressure, and EtOAc (20 mL) was
added. The organic layer was washed consecutively with brine,
1
N HCl, brine, 5% NaHCO
evaporated under reduced pressure. The residue was purified
by column chromatography using CHCl as eluent: yield 0.59 g
); H NMR (CDCl ) δ 6.61 (t,
H, J ) 4.8 Hz), 5.14 (d, 1H, J ) 8.8 Hz), 4.00-3.92 (m, 3H),
.15 (m, 1H), 1.43 (br s, 18H), 0.96 (d, 3H, J ) 6.6 Hz), 0.91 (d,
3 2 4
, and brine, dried over Na SO , and
3
1
(
71%); oil; [R]
D
) -7.3 (c 1, CHCl
3
3
General Method for Enzymatic Hydrolysis. To a stirred
solution of the substrate (0.15-0.20 mmol) in n-hexane (1 mL)
and CH OH (100 µL) was added a solution of the enzyme (50
3
1
2
3
7
1
3
H, J ) 6.8 Hz); C NMR (CDCl
9.8, 59.7, 41.9, 30.9, 28.2, 27.4, 19.2, 17.6. Anal. Calcd for
: C, 58.16; H, 9.15; N, 8.48. Found: C, 58.47; H, 8.95;
3
) δ 171.7, 168.7, 155.8, 82.2,
mg) in phosphate buffer (9 mL, 50 mM pH 7.4). The reaction
mixture was stirred for 24-60 h at 37 °C. After acidification
until pH 6 and extraction with EtOAc (3 × 5 mL), the organic
16 30 2 5
C H N O
N, 8.36.
layers were combined and washed with 5% NaHCO (3 × 5 mL).
3
tert-Butyl 4-tert-Butoxybenzoate (11). To a stirred solution
The aqueous layer was acidified until pH 6 and extracted with
EtOAc (3 × 10 mL). The combined organic layers were dried
of benzoic acid (1.0 g, 7.2 mmol) in dioxane (29 mL) was added
concentrated H SO (1.44 mL) dropwise under vigorous stirring
2 4
2 4
over Na SO , and the organic solvent was removed under
at 0 °C. Isobutylene was bubbled through the mixture for 1 h at
°C. The reaction mixture was stirred for 1 h at 0 °C and
overnight at room temperature. The mixture was cooled at 0
C, and aqueous sodium hydroxide (1 N, 50 mL) was added
reduced pressure to give the product.
-tert-Butoxybenzoic Acid. To a stirred solution of tert-
butyl 4-tert-butoxybenzoate 11 (45 mg, 0.18 mmol) dissolved in
0
4
°
1
5
5
3
mL of n-hexane and 100 µL CH OH was added a solution of
0 mg BsubpNBE (dissolved in 9 mL sodium phosphate buffer,
0 mM pH 7.4). The reaction mixture was stirred for 60 h at 37
slowly, followed by ethyl acetate (30 mL). The organic layer
was separated and the aqueous phase washed with ethyl acetate
(
2 × 30 mL). The combined organic layers were washed with
°C followed by workup as described above for the general
brine, dried over Na SO , and evaporated under reduced pres-
2 4
1
method: yield 15 mg (61%); white solid; mp 132-135 °C;
NMR (CDCl
Hz), 1.44 (s, 9H); 13C NMR (CDCl ) δ 168.3, 161.4, 130.7, 127.1,
H
sure. The residue was purified by column chromatography
3
) δ 8.04 (d, 2H, J ) 5.6 Hz), 7.05 (d, 2H, J ) 5.6
using petroleum ether (60-80 °C)/EtOAc as eluent: yield 0.68
1
g (38%); oil; H NMR (CDCl
3
) δ 7.91 (dd, 2H, J ) 8.8, 2.2 Hz),
3
1
3
11 14 3
122.7, 79.3, 28.3. Anal. Calcd for C H O : C, 68.02; H, 7.27.
Found: C, 67.76; H, 7.46.
The other products of the enzymatic hydrolysis were identified
by their analytical data in comparison with authentic samples.
6
.99 (dd, 2H, J ) 8.8, 2.2 Hz), 1.58 (s, 9H), 1.38 (s, 9H);
C
NMR (CDCl
3
) δ 165.5, 159.5, 130.6, 122.6, 80.6, 79.3, 28.8, 28.1.
Anal. Calcd for C15
22 3
H O : C, 71.97; H, 8.86. Found: C, 71.72;
H, 9.06.
Substrate 10 was prepared according to above-described
procedure. Its analytical data were in accordance with those
reported in the literature.
2
4
Acknowledgment. We thank the Deutsche Luft-
und Raumfahrt (DLR, Bonn, Germany) for a grant (GRC
(
18) Muller, D.; Zeltser, I.; Bitan, G.; Gilon, C. J. Org. Chem. 1997,
2, 411.
19) Parrish, J. P.; Chun Jung, Y.; Il Shin, S.; Woon Jung, K. J.
Org. Chem. 2002, 67, 7127.
20) Konda, Y.; Takahashi, Y.; Arima, S.; Sato, N.; Takeda, K.;
Dobashi, K.; Baba, M.; Harigaya, Y. Tetrahedron 2001, 57, 4311.
21) Doherty, G. A.; Kamenecka, T.; McCauley, E.; Van Riper, G.;
0
1/011) as well as the Greek Secretariat for Research
6
(
and Technology. U.T.B. thanks the Fonds der Chemis-
chen Industry (Frankfurt, Germany) for financial
support.
(
(
Mumford, R. A.; Tong, S.; Hagmann, W. K. Bioorg. Med. Chem. Lett.
JO050114Z
2
002, 12, 5, 729.
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(
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3740 J. Org. Chem., Vol. 70, No. 9, 2005