11742 J. Am. Chem. Soc., Vol. 119, No. 49, 1997
Kimura et al.
General Procedure for the Synthesis of erythro-â-Hydroxy-L-r-
amino Acid by LTA. To a 50-mL solution of 1 M glycine, 10 mM
DTT, and 0.05 mM PLP containing 30% DMSO (pH 7.5) was added
1 mmol of an aldehyde and 100 U of LTA. The reaction mixture was
gently shaken at 37 °C for a period of time. After the mixture was
boiled at 100 °C for 10 min to terminate the reaction, 200 mL of EtOH
was added to the reaction mixture and the mixture was incubated at 4
°C for 10 h. The precipitate was collected and washed with 50 mL of
EtOH to recover glycine (95-82%). The filtrate was concentrated in
vacuo and applied to Dowex-1 (OH- form, 50 mL). After being washed
with 200 mL of H2O, the product containing glycine was eluted with
20% acetic acid. The collected fractions were concentrated in vacuo,
and the residue was purified on ODS (H2O) to give the desired product.
(2S,3R)-3-Hydroxyleucine: 17% yield, 92% de before crystalliza-
tion; 1H NMR (D2O, 500 MHz) δ 0.93 (3 H, d, J ) 6.5 Hz, CH3), 0.94
(3 H, d, J ) 6.5 Hz, CH3), 1.90 (1 H, dsep, J ) 9.1 and 6.5 Hz,
CH(CH3)2), 3.49 (1 H, dd, J ) 9.1 and 3.1 Hz, â-H), 3.87 (1 H, d, J
) 3.1 Hz, R-H).
200 mL of EtOH and the mixture left at 4 °C for 3 h. The precipitate
was collected and washed with 50 mL of EtOH to recover glycine
(87.6%). The filtrate was concentrated and applied to Dowex 1 (OH-
form; 50 mL). After washing with 200 mL of H2O, the product
containing glycine was eluted with 20% acetic acid. The fraction was
collected and evaporated in vacuo. The residue was suspended with
toluene and evaporated in vacuo. This procedure was repeated three
times to remove H2O. To the residue containing a 7:1:1 mixture of
glycine, D-threonine, and D-allo-threonine (based on NMR analysis)
(185 mg) was added chlorophosphoric acid,18 and the mixture was
heated at 60 °C for 2 h. To the solution was added 0.5 mL of H2O
and 1.5 mL of 1 N HCl, and the mixture was heated at 100 °C for 15
min. The mixture was then directly applied to Dowex 1 (OH-form;
50 mL) and washed with 1 L of H2O. Glycine and unreacted threonine
were eluted with 0.5 M formic acid, and the crude phosphorylated
products were eluted with 0.7-1.0 M formic acid. The fractions were
collected and evaporated in vacuo to give a 1:1 mixture of O-phospho-
D-threonine and O-phospho-D-allo-threonine (74.9 mg).
(2S,3R)-2-Amino-4-(benzyloxy)-3-hydroxybutyric Acid (γ-Ben-
zyloxy-L-allo-threonine): 36% yield, 88% de before crystallization;
1H NMR (CD3OD/D2O ) 65/35, 500 MHz) δ 3.70 (2 H, d, J ) 4.0
Hz, CH2OBn), 3.84 (1 H, d, J ) 4.3 Hz, R-H), 4.26 (1 H, dd, J ) 4.3
and 4.0 Hz, â-H), 4.55 (2 H, s, OCH2Ph), 7.29-7.38 (5 H, m, Ph).
(2S,3R)-2-Amino-5-(benzyloxy)-3-hydroxypentanoic Acid (γ-Ben-
zyloxymethyl-L-allo-threonine): 10% yield, 88% de before crystal-
O-Phospho-D-allo-threonine: 1H NMR (500 MHz, D2O) δ 1.43 (3
H, d, J ) 6.5 Hz, CH3), 4.12 (1 H, brs, R-H), 4.81-4.88 (1 H, m,
â-H).
1
O-Phospho-D-threonine: H NMR (500 MHz, D2O) δ 1.35 (3 H,
d, J ) 6.5 Hz, CH3), 4.28 (1 H, brs, R-H), 4.71-4.78 (1 H, m, â-H).
Wheat germ acid phosphatase (10 U) was added to a 10-mL solution
of the crude phosphorylated product (45 mg, 0.226 mmol) in 100 mM
citrate buffer (pH 5.6). The reaction mixture was shaken at 37 °C for
5 days and then applied to Dowex 50W-X8 (H+ form; 50 mL).
Unreacted O-phospho-D-threonine was recovered by washing with H2O
(11.6 mg, 43%), and D-allo-threonine was eluted with 3 N NH4OH.
The desired fraction was evaporated in vacuo and recrystallized with
H2O-MeOH to give pure D-allo-threonine (12.1 mg, 45%), which had
1
lization; H NMR (D2O, 400 MHz) δ 1.74-1.89 (2 H, m, CH2CH2-
OBn), 3.68-3.73 (2 H, m, CH2CH2OBn), 3.81 (1 H, d, J ) 3.7 Hz,
R-H), 4.26 (1 H, dt, J ) 9.5 and 3.7 Hz, â-H), 4.55 (1 H, d, J ) 11.4
Hz, CH2OBn), 4.58 (1 H, d, J ) 11.4 Hz, CH2OBn), 7.37-7.46 (5 H,
m, Ph).
Crystallization of the above three products from EtOH-H2O gave
diastereomerically pure products (based on the NMR signal of R-H) in
∼62-65% yield.
1
an H NMR spectrum identical to that of the authentic sample.
General Procedure for the Synthesis of 4,5-O-Isopropylidene-
Protected Polyoxamic Acids. To a solution of the enzyme (ap-
proximately 500 U of LTA or 11 000 U of DTA)20 in 50 mM potassium
phosphate buffer (LTA) or 50 mM Tris (DTA), pH ) 7.5 (25 mL),
was added 3.8 g (51 mM) of glycine, 10 mg (40 µM) of PLP, 50 mg
(0.3 mM) of DTT, and 500 mg of MgCl2‚6H2O (for DTA).
To this solution (DTA) was added 1.14 g (8.76 mM) of (R)-
isopropylideneglyceraldehyde.11 After sonication for about 10 s, the
mixture became homogeneous and was shaken at 37 °C for 16 h. After
precipitation of excess glycine with ethanol (125 mL), filtration, and
evaporation of the solvent, the total crude product was suspended in
water and applied to a reverse phase RP18 column. Elution with water
gave 1.5 g (84% yield) of the desired product 3 (Scheme 4) as a
diastereomeric mixture (see Tables 2 and 3).
(2S,3R,4S)-2-Amino-3-hydroxy-4,5-O-isopropylidenepentanoic Acid.
To prepare the diastereomeric product, compound 2 (0.249 g) (Scheme
4) was recrystallized from EtOH-H2O (2:1 v/v) to give 0.12 g of the
title compound in a diastereomerically pure form: mp 205-206 °C
(dec) (EtOH-H2O); [R]D ) + 18° (c 1.17 H2O); 1H NMR (D2O, 400
MHz) δ 4.41 (1H, ddd, J ) 7.8, 6.5, and 4.7 Hz, C4H), 4.18 (1H, dd,
J ) 9.0 and 6.5 Hz, C3H), 3.98 (1H, dd, J ) 9.0 and 4.7 Hz, C4HH),
3.98 (1H, dd, J ) 7.8 and 3.0 Hz, C4HH), 3.94 (1H, d, J ) 3.0 Hz,
C2H), 1.43 (3H, s, CH3), 1.36 (3H, s, CH3); 13C NMR δ (D2O, 400
MHz) 173.3, 112.7, 77.65, 73.4, 68.7, 59.1, 28.0, 26.3; FAB HRMS
calcd for C8H15NO5 206.1028 (MH+), found 206.1035.
General Procedure for the Synthesis of threo-â-Hydroxy-D-r-
amino Acid by DTA. To a 50-mL solution of 1 M glycine, 10 mM
DTT, 0.05 mM PLP, and 10 mM MgCl2 in H2O (pH 7.5) was added
1 mmol of an aldehyde and 10 U of DTA. The reaction mixture was
gently shaken at 37 °C for a period of time. After the reaction mixture
was boiled at 100 °C for 10 min to terminate the reaction, 200 mL of
EtOH was added and the mixture left at 4 °C for 10 h. The precipitate
was collected and washed with 50 mL of EtOH to recover glycine (95-
89%). The filtrate was purified on ODS (H2O) to give the desired
product.
(2R,3R)-3-Hydroxyleucine: 49% yield, 86% de before crystalliza-
tion; 1H NMR (D2O, 500 MHz) δ 0.89 (3 H, d, J ) 6.8 Hz, CH3), 0.94
(3 H, d, J ) 6.8 Hz, CH3), 1.69 (1 H, oct, J ) 6.8 Hz, CH(CH3)2),
3.65 (1 H, dd, J ) 6.8 and 3.5 Hz, â-H), 3.66 (1 H, d, J ) 3.5 Hz,
R-H).
(2R,3R)-2-Amino-4-(benzyloxy)-3-hydroxybutyric Acid (γ-Ben-
1
zyloxy-D-threonine): 80% yield, 40.0% de before crystallization; H
NMR (D2O, 500 MHz) δ 3.65-3.70 (2 H, m, CH2OBn), 3.70 (1 H, d,
J ) 3.6 Hz, R-H), 4.26 (1 H, ddd, J ) 8.9, 4.4, and 3.6 Hz, â-H), 4.65
(2 H, s, OCH2Ph), 7.34-45 (5 H, m, Ph).
(2R,3R)-2-Amino-5-(benzyloxy)-3-hydroxypentanoic Acid (δ-
1
Benzyloxymethyl-D-threonine): 35% yield, 64% de; H NMR (D2O,
400 MHz) δ 1.77-1.85 (1 H, m, CH2CH2OBn), 1.92-2.01 (1 H, m,
CH2CH2OBn), 3.62 (1 H, d, J ) 4.5 Hz, R-H), 3.68-3.73 (2 H, m,
CH2CH2OBn), 4.23 (1 H, dt, J ) 9.6 and 4.5 Hz, â-H), 4.57 (2 H, t,
J ) 11.6 Hz, CH2OBn), 7.38-7.46 (5 H, m, Ph).
Crystallization of the above three products from EtOH-H2O gave
diastereomerically pure products (in ∼60-65% yield), based on the
NMR signal of R-H.
D-allo-Threonine. To a 50 mM solution of 1 M glycine, 10 mM
DTT, 0.05 mM PLP, and 10 mM MgCl2 in H2O (pH 7.5) were added
1 mmol of acetaldehyde and 10 U of DTA. The reaction mixture was
gently shaken at 37 °C for 21 h. To the reaction mixture was added
Acknowledgment. This research was supported by the NIH
(Grant GM-44154). We thank Professor Dale Boger for
providing some of the aldehydes used in this study.
JA9720422
(20) The amount of the enzyme units is increased to obtain a reasonable
yield because isopropylideneglyceraldehyde is unstable at room temperature
(t1/2 ) 24 h). The product is, however, quite stable.