Ezymatic Removal of Protecting Groups
purified by column chromatography using petroleum ether
Although it appears that our methodology requires
relatively large amounts of enzyme, it must be noted that
these are crude preparations. In case of BS2 the protein
content of the lyophilized E. coli extract is approximately
40% and the overall esterase content is estimated to be
approximately 15%. Still, these values are higher than
those reported for most commercial biocatalyst prepara-
tions.3 In addition, the preparation does not contain any
competing hydrolase activity, which allows the use of the
cheap crude preparation.
In conclusion, using a wide variety of substrates it has
been demonstrated that methyl and benzyl ester protect-
ing groups can be cleaved by BS2 and CAL-A under
extremely mild conditions, avoiding side reactions and
leading to high isolated yields of the corresponding
carboxylic acid. In general, BS2 provides products in
higher yields than CAL-A, exhibiting broader substrate
tolerance. CAL-A may be used in cases where BS2 is not
effective, for example, in the hydrolysis of long chain
substrates. Most importantly, for several examples no
alternative chemical method exists. Thus, the presently
introduced enzymatic protocols present general applica-
bility, may serve as tools in cases where chemical
methods are incompatible, and are anticipated to find
wide use in organic synthesis.
(60-80 °C)/EtOAc (8:2) as eluent. Yield 0.15 g (91%); white
1
solid; mp 68-70 °C. H NMR (200 MHz, CDCl3): δ 7.36 (m,
5H), 7.11 (t, 1H, J ) 5.8 Hz), 5.13 (s, 2H), 3.35 (m, 2H), 2.90
(t, 2H, J ) 7.4 Hz), 2.24 (t, 2H, J ) 7.4 Hz), 1.91 (m,2H), 1.60
(m, 2H), 1.26 (m, 22H), 0.89 (t, 3H, J ) 6.2 Hz). 13C NMR (50
MHz, CDCl3): δ 199.2, 172.7, 160.3, 135.7, 128.6, 128.31,
128.25, 66.5, 38.6, 36.7, 31.9, 31.5, 29.6, 29.5, 29.4, 29.3, 29.0,
24.4, 23.1, 22.7, 14.1. Anal. Calcd for C27H43NO4: C, 72.77; H,
9.73; N, 3.14. Found: C, 72.86; H, 9.57; N, 3.23.
General Method for Enzymatic Hydrolysis. To a stirred
solution of the substrate (0.15-0.20 mmol) in n-hexane (1 mL)
and CH3OH (100 µL) was added a solution of the enzyme (50
mg) in phosphate buffer (9 mL, 50 mM pH 7.4). The reaction
mixture was stirred for 24-72 h at 37 °C. After acidification
until pH 6 and extraction with EtOAc (3 × 5 mL), the organic
layers were combined and washed with 5% NaHCO3 (3 × 5
mL). The aqueous layer was acidified until pH 6 and extracted
with EtOAc (3 × 10 mL). The combined organic layers were
dried over Na2SO4, and the organic solvent was removed under
reduced pressure to give the product.
(S)-2-(2-(tert-Butoxycarbonyl)hexanamido)acetic Acid.
Oil; [R]D ) +3.0 (c 1.6, CHCl3). 1H NMR (200 MHz, CDCl3): δ
7.85 (br s, 1H), 7.18 (m, 1H), 5.43 (d, 1H, J ) 9.2 Hz), 4.33 (m,
1H), 4.07 (m, 2H), 1.69 (m, 2H), 1.43 (s, 9H), 1.32 (m, 4H),
0.89 (t, 1H, J ) 6.6 Hz). 13C NMR (50 MHz, CDCl3): δ 173.1,
172.5, 156.1, 80.7, 54.2, 41.3, 32.5, 28.2, 27.6, 22.3, 13.9. Anal.
Calcd for C13H24N2O5: C, 54.15; H, 8.39; N, 9.72. Found: C,
54.38; H, 8.44; N, 9.59.
The data for the other products of enzymatic hydrolysis are
summarized in Supporting Information.
Experimental Section
Synthesis of Substrates. The synthesis of methyl and
benzyl esters of carboxylic acids and protected amino acids is
described in Supporting Information.
Expression of Recombinant Esterase BS2 in Escheri-
chia coli. Clones containing the expression vector encoding
the gene for esterase BS2 were used to inoculate 5 mL of an
overnight culture (LB media supplemented with 100 µg/mL
ampicillin). Then, 500 µL of the overnight culture was used to
inoculate 500 mL of 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 of sodium
phosphate buffer (50 mM, pH 7.5). Cells were resuspended in
20 mL of phosphate buffer and disrupted by sonification with
cooling on ice. Cell debris was removed by centrifugation (15
min, 4 °C, 8000 g), and the supernatant was frozen at -80 °C
and then lyophilized. Specific activities of crude extracts were
determined spectrophotometrically using p-nitrophenyl acetate
for activity and the Bradford reagent for protein content.
Benzyl 4-(2-Hydroxyhexadecanamido)butanoate. To a
stirred solution of 2-hydroxy-hexadecanoic acid (0.27 g, 1
mmol) and HCl‚H-GABA-OBn (0.23 g, 1 mmol) in CH2Cl2 (10
mL) were added Et3N (0.3 mL, 2.2 mL) and subsequently 1-(3-
dimethylaminopropyl)-3-ethyl carbodiimide (WSCI) (0.21 g, 1.1
mmol), and 1-hydroxybenzotriazole (HOBt) (0.14 g, 1 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 (10 mL) was added. The
organic layer was washed consecutively with brine, 1 N HCl,
brine, 5% NaHCO3, and brine, dried over Na2SO4, and
evaporated under reduced pressure. The residue was purified
by column chromatography using CHCl3/CH3OH (95:5) as
eluent. Yield 0.27 g (61%); white solid; mp 89-91 °C. 1H NMR
(200 MHz, CDCl3): δ 7.36 (m, 5H), 6.72 (t, 1H, J ) 5.8 Hz),
5.13 (s, 2H), 4.06 (m, 1H), 3.32 (m, 2H), 2.85 (b, 1H), 2.42 (t,
2H, J ) 7 Hz), 1.95-1.60 (m, 4H), 1.26 (m, 24H), 0.88 (t, 3H,
J ) 6.6). 13C NMR (50 MHz, CDCl3): δ 174.1, 173.1, 135.7,
128.6, 128.3, 128.2, 72.1, 66.4, 38.4, 34.9, 31.9, 31.6, 29.7, 29.62,
29.58, 29.5, 29.4, 29.3, 25.0, 24.6, 22.7, 14.1. Anal. Calcd for
C27H45NO4: C, 72.44; H, 10.13; N, 3.13. Found: C, 72.23; H,
10.35; N, 3.25.
Acknowledgment. We thank the Deutsche Luft-
und Raumfahrt (DLR, Bonn, Germany) for a grant (GRC
01/011) and the Greek Secretariat for Research and
Technology (Athens, Greece). U.T.B. thanks the Fonds
der Chemischen Industry (Frankfurt, Germany) for
financial support.
Benzyl 4-(2-Oxohexadecanamido)butanoate (5, Table
2). To a stirred solution of 2-hydroxy-amide (0.17 g, 0.4 mmol)
in CH2Cl2 (10 mL) was added Dess-Martin periodinane (0.21
g, 0.5 mmol). The reaction mixture was stirred for 1 h at room
temperature. The organic layer was washed with 5% NaHCO3
(10 mL) and then the organic solvent was dried over Na2SO4
and evaporated under reduced pressure. The residue was
Supporting Information Available: Experimental pro-
cedures and characterization data. This material is available
JO051004V
J. Org. Chem, Vol. 70, No. 22, 2005 8733