P. ClapØs et al.
duced and purified in our lab and in a fermentation plant facility by fol-
lowing previously described methodologies.[14] All other chemicals and
reagents that were used in this work were of analytical grade.
purified by semipreparative RP-HPLC on a Perkin–Elmer 25025 mm
column, filled with C18, 5 mm-type stationary phase, by using the following
procedure. First, the column was equilibrated with 0.1% (v/v) TFA in
H2O. Then, the sample was adjusted to pH 2–3with TFA and loaded
onto the column. The salts were eliminated by washing with of 0.1% (v/
v) TFA in H2O (100 mL). Then, the product was eluted with a gradient
of 0.1% (v/v) TFA in CH3CN (8 to 48% v/v in 30 min). The flow rate
was 10 mLminÀ1, and the products were detected at 215 nm. The pure
fractions were pooled and lyophilized for further characterization by
NMR spectroscopy.
NMR spectroscopy: 1H (500.13MHz) and 13C (125.76 MHz) NMR spec-
tra were recorded on an AVANCE 500 Bruker spectrometer equipped
with a high-sensitivity cryogenically cooled triple-resonance TCI probe-
head for samples that were dissolved in [D6]DMSO and CD3OD solu-
tions. Full structural and stereochemical characterization of all com-
pounds was carried out with the aid of 2D COSY, NOESY, HSQC, and
HMBC experiments and from NOE data that was obtained from selec-
tive 1D NOESY experiments that were recorded with a mixing time of
500 ms. The 13C NMR spectroscopic peaks were assigned by HSQC and
HMBC experiments. For complete signal assignments, see the Supporting
Information.
Enzyme activity: Threonine aldolase activity was measured spectrophoto-
metrically as described in a previous work.[14] One unit of TA activity was
defined as the amount of enzyme that catalyzed the formation of 1 mmol
of acetaldehyde (1 mmol of NADH oxidized) per minute at 378C.
Aldol additions catalyzed by SHMT and lTA
Enzymatic aldol additions in emulsions: Aldol additions of glycine to al-
dehydes in emulsion systems were conducted in 10 mL screw-caped test
tubes as follows:[22] The aldehyde (0.15 mmol), oil (0.3g), and surfactant
(0.2 g) were mixed with a vortex mixer for 15 s (2400 rpm). Then, a gly-
cine solution (2 mL; 0.175, 0.350, or 0.700 mmol, depending on the ex-
periment) at pH 6.5 for SHMT or pH 8 for LTA was added dropwise
while stirring at 4, 10, or 258C with a vortex mixer. This was followed by
the addition of a solution of pyridoxal-5-phosphate (190 mL, 0.3 mmol).
Finally, the reaction was started by adding the enzyme (20 U SHMT or
3 0 U lTA). The final reaction volume was 2.5 mL. The test tubes were
placed on a horizontal shaking bath (100 rpm) at constant temperature.
The reactions were followed by HPLC until the peak of the product
reached a maximum.
AHCTRE(GNU 2S,3R,4R)-2-Amino-4-(benzyloxycarbonylamino)-3-hydroxypentanoic
Enzymatic aldol additions in DMF/H2O 1:4: The aldehyde (0.15 mmol)
was dissolved in DMF (0.5 mL). Then, the glycine solution (2 mL; 0.175,
0.350, or 0.700 mmol, depending on the experiment) at pH 6.5 for SHMT
or pH 8 for lTA was added while stirring at 4, 10, or 258C with a vortex
mixer. This was followed by the addition of a solution of pyridoxal-5-
phosphate (190 mL, 0.3 mmol). The rest of the experimental procedure
was the same as above.
acid ammonium salt (6a): The title compound was obtained according to
the general procedure. Yield: 170 mg (30%), 99% pure, as determined
by HPLC; retention factor, k=6.91; [a]2D0 =À14.8 (c=0.49 in DMSO);
1H NMR (500.13MHz, [D 6]DMSO): d=7.34 (s, 1H), 7.25 (d, J=8.3Hz,
1H), 5.03 (s, 1H), 3.73 (s, 1H), 3.54 (dd, J=8.5 Hz, 1H), 3.02 (d, J=
8.5 Hz, 1H), 1.12 ppm (d, J=6.8 Hz, 1H); 13C NMR (125.76 MHz,
[D6]DMSO): d=169.4 (C1), 156.8 (C6), 136.9 (C ipso), 128.3(C arom),
127.7 (C arom), 72.1 (C3), 65.5 (C7), 53.1 (C2), 47.6 (C4), 17.4 ppm (C5)
HPLC analyses: HPLC analyses were performed on
a Lichrograph
HPLC system (Merck, Germany) fitted with XBridge C18 5 mm
(Waters). Samples (50 mg, for reactions in emulsions or 50 mL for reac-
tions in DMF/H2O 1:4) were withdrawn from the reaction medium, dis-
solved in methanol (1 mL), which contained HOAc (2.5% v/v) to stop
any enzymatic reaction, and then subsequently analyzed by HPLC. The
solvent system was as follows: solvent A: 0.1% (v/v) trifluoroacetic acid
(TFA) in H2O, solvent B: 0.095% (v/v) TFA in H2O/CH3CN 1:4. HPLC
conditions for reaction monitoring with underivatized adducts were: gra-
dient elution from 0 to 70% solvent B in 30 min was used; flow rate:
1 mLminÀ1; detection at 215 nm. Retention factors for each compound
are provided.
AHCTRE(GNU 2S,3S,4R)-2-Amino-4-(benzyloxycarbonylamino)-3-hydroxypentanoic
acid ammonium salt (6b): The title compound was obtained according to
the general procedure. Yield: 163mg (20%); 99% pure and a mixture of
diastereomers, as determined by HPLC; retention factor, k=7.19; [a]D20
=
À18.0 (c=0.47 in DMSO); 1H NMR (500.13MHz, [D 6]DMSO): d=7.42
(d, J=8.5 Hz, 1H), 7.36 (m, 5H), 5.01 (s, 2H), 3.8 (dd, J=7.5 Hz, 1H),
3.62 (m, 1H), 3.18 (d, J=4.0 Hz, 1H), 1.10 (d, J=6.6 Hz, 1H), minor sig-
nals that corresponded to the l-erythro diastereomer: 3.56 (dd, J=
8.2 Hz, 1H), 3.05 (d, J=8.1 Hz, 1H), 1.12 ppm (d, J=6.8 Hz, 1H);
13C NMR (125.76 MHz, [D6]DMSO): d=168.9 (C1), 155.9 (C6), 137.0 (C
ipso), 128.0 (C arom), 127.7 (C arom), 72.0 (C3), 65.3 (C7), 55.6 (C2),
48.7 (C4), 16.6 (C5), minor signals that corresponded to the l-erythro dia-
stereomer: 169.4 (C1), 156.8 (C6), 53.1 (C2), 47.6 (C4), 17.4 ppm (C5).
The l-erythro/l-threo aldol adducts from N-Cbz-glycinal (3) and N-Cbz-
3-aminopropanal (4) could not be separated by HPLC under the condi-
tions described above. Baseline separation of both diasteroisomers was
accomplished after derivatization of the free a-amino function with a
tert-butyloxycarbonyl group. Samples (50 mg) were withdrawn from the
reaction medium and dissolved in di-tert-butyl dicarbonate solution
(0.6m; 200 mL). Then Et3N (6m; 50 mL) was added to this solution and
the mixture was incubated for 1 h at 258C in a horizontal shaker. After
this time, the total volume was transferred to an HPLC vial with MeOH/
HOAc (250 mL, 1:0.025) and analyzed subsequently by HPLC. HPLC
conditions were as follows: gradient elution from 10 to 80% solvent B in
35 min, the eluents and other conditions were as described above. Reten-
tion factors for each derivatized compound are provided.
AHCTRE(GNU 2S,3R,4S)-2-Amino-4-(benzyloxycarbonylamino)-3-hydroxypentanoic
acid ammonium salt (7a): The title compound was obtained according to
the general procedure. Yield: 406 mg (29%), 99% pure, as determined
by HPLC. Retention factor, k=4.25, [a]2D0 =+14.3( c=0.56 in DMSO);
1H NMR (500.13MHz, [D 6]DMSO): d=7.88 (d, J=7.8 Hz, 1H), 7.33 (s,
5H), 5.01 (s, 2H), 3.75 (s, 1H), 3.68 (t, J=4.8 Hz, 1H), 3.29 (d, J=
4.6 Hz, 1H), 1.07 ppm (d, J=4.6 Hz, 3H); 13C NMR (125.76 MHz,
[D6]DMSO): d=168.6 (C1), 155.6 (C6), 137.2 (C ipso), 128.2 (C arom),
127.6 (C arom), 71.9 (C3), 65.0 (C7), 54.2 (C2), 48.7 (C4), 16.1 ppm (C5).
AHCTRE(GNU 2S,3S,4S)-2-Amino-4-(benzyloxycarbonylamino)-3-hydroxypentanoic
Scale-up: Enzymatic reactions on the 100 mg scale were conducted in
screw-capped flasks (250 mL), which were placed in a reciprocal shaker
(50 rpm) and thermostated at the required temperature (25 or 48C). The
procedure was similar to that described for the analytical reactions and
substrate concentrations, temperature, enzyme units, and incubation
times were selected based upon the acceptor aldehyde and are summar-
ized in Table 1.
acid amonium salt (7b): The title compound was obtained according to
the general procedure; yield: 266 mg (27%), 99% pure and a mixture of
diastereomers, as determined by HPLC; retention factor, k=4.39; [a]D20
=
+5.4 (c=0.48 in DMSO); 1H NMR (500.13MHz, [D 6]DMSO): d=7.90
(d, J=7.61 Hz, 5H), 7.34 (m, 1H), 7.23 (d, J=8.4 Hz, 1H), 5.01 (s, 2H),
3.81 (t, J=5.2 Hz, 1H), 3.76 (m, 1H), 3.22 (d, J=5.1 Hz, 1H), 1.08 (d,
J=6.7 Hz, 3H), minor signals that corresponded to the l-erythro diaste-
reomer: 7.90 (d, J=7.61 Hz, 1H), 3.68 (t, J=4.9 Hz, 1H), 3.27 (d, J=
5.0 Hz, 1H), 1.06 ppm (d, J=6.8 Hz, 3H); 13C NMR (125.76 MHz,
[D6]DMSO): d=168.9 (C1), 155.6 (C6), 137.1 (C quat), 128.3 (C arom),
127.6 (C arom), 71.0 (C3), 65.1 (C7), 54.8 (C2), 49.6 (C4), 16.6 (C5),
minor signals that corresponded to the l-erythro diastereomer: 168.6
(C1), 137.2 (C ipso), 71.8 (C3), 65.0 (C7), 54.2 (C2), 48.7 (C4), 16.1 ppm
(C5).
The enzymatic reactions were stopped by the addition of MeOH/HOAc
39:1 (v/v) and the crude was purified and filtrated through Celite/activat-
ed charcoal (95:5); the filtration cake was cleaned with MeOH. The fil-
trate was evaporated under vacuum to eliminate the MeOH. The residue
that was obtained was dissolved in H2O and the unreacted aldehyde and
other hydrophobic impurities were extracted with EtOAc (3150 mL).
The aqueous layer that contained the product was freeze dried and then
4654
ꢀ 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 4647 – 4656