Enantioselective catabolism of racemic serine: Preparation of d-serine using whole cells of Fusobacterium nucleatum

Article abstract of DOI:10.1016/j.tetasy.2011.07.026

Incubation of racemic serine with the anaerobic bacterium Fusobacterium nucleatum yielded d-serine in high enantiomeric excess (>95%). Selective degradation of the l-amino acid was most efficient when F. nucleatum was resuspended in a buffer at high cell densities (ca. 50-100 g damp cells/L); a single incubation effectively removed almost all l-serine from racemic mixtures at initial concentrations up to 800 mM, the solubility limit. The product d-amino acid was separated from the metabolic end-products (acetate, butyrate and lactate) and buffer components by a single cation-exchange step. After recrystallization, 83% of the d-serine in the initial racemate was recovered with >99% ee (HPLC) and 98% purity (HPLC). This anaerobic microbial approach provides a viable complementary method for generating d-serine, a valuable chiral starting material for chemical synthesis.

Full text of DOI:10.1016/j.tetasy.2011.07.026

Tetrahedron: Asymmetry 22 (2011) 1473–1478
Contents lists available at SciVerse ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Enantioselective catabolism of racemic serine: preparation of D-serine
using whole cells of Fusobacterium nucleatum
⇑
Mohammad Ramezani , Robert L. White
Department of Chemistry, Dalhousie University, 6274 Coburg Road, P. Box 15000, Halifax, Nova Scotia, Canada B3H 4R2
a r t i c l e i n f o
a b s t r a c t
Article history:
Incubation of racemic serine with the anaerobic bacterium Fusobacterium nucleatum yielded D-serine in
Received 18 July 2011
Accepted 28 July 2011
Available online 1 September 2011
high enantiomeric excess (>95%). Selective degradation of the
nucleatum was resuspended in a buffer at high cell densities (ca. 50–100 g damp cells/L); a single incu-
bation effectively removed almost all -serine from racemic mixtures at initial concentrations up to
800 mM, the solubility limit. The product -amino acid was separated from the metabolic en -products
(acetate, butyrate and lactate) and buffer components by a single cation-exchange step. After recrystal-
lization, 83% of the -serine in the initial racemate was recovered with >99% ee (HPLC) and 98% purity
L-amino acid was most efficient when F.
L
D
D
D
(HPLC). This anaerobic microbial approach provides a viable complementary method for generating
serine, a valuable chiral starting material for chemical synthesis.
D-
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
lective catabolism of
unmetabolized
supplemented with less expensive and readily available racemic
amino acids. In this way,
-alanine (Candida maltosa20 and Arthro-
bacter sp. NJ-2621), -aminoadipic acid (Pseudomonas putida),22
-aspartic acid (Pseudomonas dacunhae),23 (R)-baclofen (Streptomy-
ces halstedii),24 branched chain -amino acids (Proteus vulgaris),25
-histidine (Proteus vulga-
-homoserine (Arthrobacter nicotinovorans),27
-lysine
-methionine (Proteus vulgaris),29
-proline (Candida sp. PRD-234)21
and the aromatic amino acids -phenylalanine (and ring-
L
-amino acids has been utilized to isolate
D
-amino acids from microbial cultures
As primary members of the chiral pool,1
a-amino acids are valu-
able substrates for the synthesis of chiral compounds.2–4 Of the
common amino acids, the individual enantiomers of serine are par-
ticularly versatile substrates for chemical synthesis,4,5 serving as
starting points for the preparation of important chiral synthons,
such as Garner’s aldehyde6 and various other serine derivatives.3,7
D
D
-a
D
D
D
-glutamic acid (Lactobacillus brevis),26
D
While
oxidation state and reactivity of the terminal carbons in the back-
bone of -enantiomer of serine also
-serine,8 the more expensive
D
-amino acids have been prepared by controlling the
ris),25
D
D
(Comamonas testosteroni),28
D
L
D
D D
-ornithine (Proteus vulgaris),25
has been a preferred starting point for the synthesis of chiral com-
pounds of diverse structures and properties. For example, over the
D
substituted analogs),
D-tyrosine and D-tryptophan (Rhodotorula
past few years,
D
-serine has been used for the synthesis of enzyme
graminis homogenate)30 have been obtained in high yield and enan-
tiomeric excess.
inhibitors,9 nonproteinogenic amino acids,10 neuroexcitatory
kainoid amino acids,11 lysinoalanine,12 the cyclopeptide alkaloid
paliurine,13 substituted nitrogen-containing heterocycles,14
sphingosine,15 and conformationally constrained analogs of diethyl-
enetriaminepentaacetic (DTPA) acid used as ligands in radiophar-
maceuticals and contrast agents for medical imaging.16
In other instances, genetic modifications have incorporated the
gene for
production of
hanced glutamate racemase (E. coli)21,32,33 and
(E. coli)34 activities for the preparation of
-glutamate,
anine and -serine. Most recently, the incorporation of a gene
encoding an amino acid racemase into -amino acid producing
strains of Corynebacterium glutamicum35 resulted in the extracellu-
lar accumulation of similar amounts of the - and -enantiomers of
arginine, lysine and ornithine. The modified -serine producing
strain, however, excreted more -serine (81 mM; vs 37 mM -ser-
ine), and the enantiomeric enrichment was attributed to the spe-
cific transport of -serine.
D
-amino acid transaminase into Escherichia coli for the
-phenylalanine and other
-amino acids,31 and en-
-serine deaminase
-phenylal-
D
D
L
D
D
Although fermentation is a common source of
only a few examples of the excretion and accumulation of
acids in microbial cultures are known [e.g., -alanine by
Corynebacterium fasciens18 and
-poly(glutamic acid) enriched in
-glutamate by Bacillus strains19]. As an alternative, the enantiose-
L
-amino acids,17
-amino
D
D
L
D
c
D
L
D
L
D
L
⇑
Corresponding author. Tel.: +1 902 494 6403; fax: +1 902 494 1310.
E-mail address: robert.white@dal.ca (R.L. White).
D
An alternative microbial approach is indicated by the preferred
utilization of amino acid substrates by several anaerobic
Present address: Pharmaceutical Research Center, School of Pharmacy, Mashhad
University of Medical Sciences, PO Box 91775-1365, Mashhad, Iran.
0957-4166/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2011.07.026

1474
M. Ramezani, R. L. White / Tetrahedron: Asymmetry 22 (2011) 1473–1478
Fusobacterium species.36–39 The rapid, efficient and selective utiliza-
tion of
-serine36 demonstrated the ability of Fusobacterium
nucleatum to remove one isomer when supplied with a racemic
amino acid. Herein, we demonstrate the gram-scale feasibility of
using this readily available, stable anaerobic bacterium to prepare
excess (i.e., 99%, 95%, 86%, and 95% ee, respectively) remained, con-
firming the rapid and efficient utilization of -serine and the ability
of F. nucleatum to tolerate high concentrations of
-serine.35 By
-enantiomer was observed
L
L
D
contrast, incomplete utilization of the
L
at similar concentrations of racemic methionine (1000 mM)29
and mandelic acid (>300 mM),43 and was attributed to product
inhibition and enzyme inactivation. On the other hand, incomplete
D
-serine from racemic serine, a strategy first realized when Pasteur
obtained ‘tartrique gauche’ from racemic tartaric acid using yeast.40
degradation of
maintaining a constant pH for the resuspension buffer.20
In principle, the efficiency of -serine preparation is greatest at
L-alanine by Candida maltosa was overcome by
2. Results and discussion
D
high initial concentrations of the racemate. DL-Serine at 900 mM,
however, crystallized from the autoclaved medium, defining a
practical upper concentration limit for the initial racemic amino
acid that is greater than the solubility of DL-serine in water (ca.
50 g/L or 475 mM).44 Subsequent resuspension experiments were
conducted at 800 mM DL-serine (i.e., 84 g/L). Similar initial concen-
trations of racemate have been employed in other whole-cell
2.1. Development of bacterial resuspension conditions
During growth on peptone medium, F. nucleatum preferentially
and efficiently utilized the L
-enantiomer of serine.36 The peptone
medium, however, is a complex mixture, and a large proportion of
the medium constituents remain after the growth of F. nucleatum
is complete. Rather than attempting to isolate
D-serine from
processes for the preparation of
2400 mM21 -aminoadipic acid (31 mM),22
(2500 mM),23 (R)-baclofen (14 mM),24 branched chain
acids (ca. 800 mM),25 -glutamic acid (680 mM),26,32
-histidine
(650 mM),25 -homoserine (510 mM),27 -lysine (680 mM),28
(670 mM),29 (300 mM),43
-mandelic acid
-ornithine (750 mM),25 -proline (870 mM),21 and aromatic
-amino acids (ca. 6 mM).30
D
-alanine (2250 mM20 and
-aspartic acid
-amino
this complicated matrix, F. nucleatum cells grown on peptone
medium were collected and resuspended20,26 in a buffered aqueous
solution of salts, vitamins and racemic amino acid at pH 7.4,41 where
they remained viable under anaerobic conditions for at least
three days.
By using an anaerobic organism, an exchange of atmospheric
gases with individual cells is neither needed nor desired, and the
cell density attained by growth of F. nucleatum on peptone medium
(yield of approximately 5 g damp cells/L) could be concentrated 5-
to 40-fold by varying the volume of the resuspension buffer. At
approximately 200 g damp cells/L, however, the resuspension mix-
ture became very viscous and difficult to manipulate. Typical
experiments were conducted at 50 g damp cells/L, and the rate of
)
D
-a
D
D
D
D
D
D
D
D
D
-methionine
D
D
L
-serine utilization was higher than that observed during the
growth of F. nucleatum on the peptone medium.36 In order to opti-
mize the incubation parameters, samples from resuspension mix-
tures were removed at time intervals and centrifuged; the
supernatants were analyzed by HPLC to determine the utilization
of serine and the enantiomeric excess (ee) of the residual
acid.
D-amino
In a static resuspension experiment (330 mM DL-serine; 35 g
damp cells/L), -serine in the culture fluid reached 59% and 85%
ee, after 16 and 24 h, respectively. By contrast, -serine at 99% ee
D
D
was present at both 16 and 24 h in a parallel, magnetically stirred
resuspension mixture. The agitation may have overcome the ten-
dency of F. nucleatum to aggregate,42 promoting faster and more
complete utilization of
L-serine. All subsequent resuspension
experiments, therefore, were stirred.
In a series of incubations of DL-serine (initial concentrations of
20–900 mM) with resuspended F. nucleatum cells, the serine con-
centration decreased to approximately 50% of the initial value
within 16 h (Table 1). At higher initial concentrations of DL-serine
(i.e., 330, 500, 700, and 900 mM), D-serine in high enantiomeric
Table 1
Incubations of DL-serine with resuspended cells of F. nucleatum
Initial
Cell
density
(g/L)
Initial DL-serine
mmol per g
damp cells
Time
(h)
Residual serine
% initial concn
DL-serine
(mM)
20
60
15
22
26
26
35
50
50
50
1.3
2.7
3.8
7.7
9.3
10
12
12
12
12
16
14
14
14
52
39
61
57
54
57
62
65
100
200
330
500
700
900
Figure 1. Effect of resuspended cell density on D-serine enrichment from an initial
concentration of 800 mM DL-serine. (A) F. nucleatum was resuspended at 17 (d), 30
(ꢀ), 52 (j), and 100 (N) g damp cells/L. The corresponding loading factors were 47,
27, 15, and 8 mmol DL-serine/g damp cells. The ee (%) was determined by HPLC. (B)
14
18
At the times indicated, samples were removed and analyzed by HPLC to determine
residual serine concentrations.
Variation of the initial rate of
cells.
L-serine utilization with the density of resuspended

M. Ramezani, R. L. White / Tetrahedron: Asymmetry 22 (2011) 1473–1478
1475
The results of the initial experiments (Table 1) indicated that
L
-
graph), further demonstrating the reproducibility of initial
L
-serine
serine was utilized effectively when up to 18 mmol racemate per g
damp F. nucleatum cells were provided. Whether larger loadings of
racemic amino acid were possible was investigated systematically
at 800 mM DL-serine. At cell densities from 17 to 100 g damp
utilization under resuspension conditions. In Expt. 2, the cells were
transferred to fresh 800 mM DL-serine at 24 h when approximately
80% of the initial
rate of -serine utilization was about 70% of the initial rate, and
residual -serine of 89% ee was obtained. The total -serine utilized
in each experiment was similar, about 13 mmol -serine/g damp
cells. At the highest loading factors used in previous experiments
(Fig. 1A), the total utilization of -serine was 12 and 15 mmol/g
damp cells. The similar values from the four independent experi-
ments indicate an inherent capacity for -serine utilization by F.
L-serine had been utilized. After the transfer, the
L
cells/L (Fig. 1A), the initial rate of
L
-serine utilization increased
D
L
proportionally with increasing cell density (Fig. 1B), and similar
specific rates of approximately 0.4 mM/h/g damp cells were
L
observed. As
L-serine utilization proceeded, only minor decreases
L
in rate were observed (Fig. 1A), showing little or no inhibition by
the accumulation of metabolic products (Section 2.2). Residual
L
D
-serine at >95% ee, however, was attained only at the two highest
nucleatum and define a maximum loading factor of approximately
26 mmol DL-serine/g damp cells. In the experiment conducted at
27 mmol DL-serine/g damp cells (Fig. 1A), the F. nucleatum cells
cell densities (Fig. 1A) corresponding to loading factors of 8 and
15 mmol DL-serine/g damp cells. At loading factors of 27 and
47 mmol DL-serine/g damp cells, a smaller proportion of
L-serine
functioned almost at maximum capacity, utilizing 12 mmol
L-ser-
was utilized, yielding -serine with only 87% and 45% ee, respec-
tively. Overall, the results indicate an upper limit of approximately
20 mmol DL-serine/g damp cells for the efficient utilization
D
ine/g damp cells (i.e., 93% of the initial
ine in 87% ee.
L-serine) and yielding
D
-ser-
of
L
-serine, yielding
The capacity of F. nucleatum cells to utilize larger amounts of
-serine was assessed by incubating F. nucleatum with 800 mM
DL-serine and subsequently transferring the cells to fresh 800 mM
DL-serine. In Expt. 1, residual -serine of 97% ee was present at 35 h
D
-serine at >95% ee.
2.2. Identification of metabolic end-products
L
The supernatant from a resuspension experiment (initially
800 mM DL-serine) was titrated with aqueous NaOH to convert car-
boxylic acid end-products to nonvolatile salts and lyophilized.46 In
addition to residual serine, 1H and 13C NMR analysis of the lyoph-
ilized residue revealed acetate and lactate as major components,
and a minor amount of butyrate (relative amounts approximately
10:10:1). The accumulation of these three carboxylic acids in cul-
tures of F. nucleatum is well known, but various ratios have been
reported,37,39 perhaps reflecting different culture conditions. A
few other resonances in the NMR spectra were just discernable
above the baseline, validating the use of resuspension conditions
D
(Fig. 2), reproducing the previous results obtained under identical
conditions (Fig. 1A). Upon resuspension of these 35-h cells in fresh
800 mM DL-serine, utilization of
the initial rate determined in the first suspension (Fig. 2). Further-
more, the utilization of -serine was incomplete, ultimately yielding
-serine enriched mixture (47% ee). On the other hand, the contin-
ued utilization of -serine in the second solution of racemate demon-
strated that the capacity of the F. nucleatum cells was greater than
the approximately 8 mmol -serine/g damp cells utilized during
the initial 35-h incubation. To make the most of the unused capacity
of F. nucleatum cells for -serine utilization and optimize the effi-
ciency of -serine production, a second resuspension might be more
L-serine continued at about 40% of
L
a
D
L
L
to avoid complications during the isolation of D-serine after L-ser-
ine catabolism. The detection of carboxylic acids as the major
metabolites suggested that simple cation-exchange chromatogra-
L
D
phy would be sufficient to separate
D-serine from metabolic prod-
effective at a lower loading factor of racemic amino acid. Alterna-
tively, the second resuspension might be effective in removing min-
ucts and residual media components.
or amounts of
L
-enantiomer from a partially resolved mixture.45
2.3. Pathway of L-serine catabolism
The accumulation of lactate in cultures of F. nucleatum has been
linked to serine catabolism,47 and a pathway for the conversion of
L
-serine to the two major end-products, acetate and lactate, is
presented in Scheme 1. In the F. nucleatum genome, the genes for
pyruvate synthase, -lactate dehydrogenase, phosphate acetyltrans-
ferase, and acetate kinase are clustered,48 and the gene for
-serine
ammonia-lyase (or dehydratase) has been annotated in the genome
of three F. nucleatum subspecies.48,49 The iron–sulfur enzyme
-ser-
L
L
L
ine ammonia-lyase has also been detected in a proteomic study of
Fusobacterium varium50 and purified from several species of anaero-
bic bacteria.51 -Serine is not a substrate for the isolated enzyme,51
D
accounting for the enantioselectivity observed for serine catabolism
by F. nucleatum. Given the documented instability of -serine ammo-
nia-lyase upon exposure to air,51 the use of the isolated enzyme for
-serine degradation is less practical than the current whole cell
L
L
approach using oxygen-tolerant whole cells of F. nucleatum.52
The observed accumulation of approximately equal quantities
of acetate and lactate suggests that the oxidation of pyruvate to
acetyl-CoA and carbon dioxide is balanced by the reduction of
pyruvate to lactate (Scheme 1). Maintaining a balance between
the oxidative and reductive processes provides a constant supply
of NAD+ as the electron acceptor for the oxidation of pyruvate to
acetyl-CoA, enabling subsequent ATP generation. The small
amount of butyrate formed suggests that the acetyl-CoA to buty-
rate reaction sequence46,48 is less important for the replenishment
of NAD+ when pyruvate is readily available. In both the butyrate
and lactate pathways, the coenzyme redox balance is maintained
Figure 2. Progress of D-serine enrichment when F. nucleatum (52 g damp cells/L)
was resuspended successively in 800 mM DL-serine. In Expt. 1 (j) and Expt. 2 (d),
cells were transferred to fresh solutions of DL-serine at 24 and 35 h of incubation,
respectively. The ee (%) was determined by HPLC.
In a duplicate experiment (Fig. 2, Expt. 2), the initial rate of
L-serine utilization was similar to that in Expt. 1 (previous para-

1476
M. Ramezani, R. L. White / Tetrahedron: Asymmetry 22 (2011) 1473–1478
crease in economic value is realized. Overall, anaerobic biodegra-
dation is an attractive alternative for the preparation of -serine,
a valuable member of the chiral pool.
H
NH3
O
D
O
HOH2C
4. Experimental
4.1. General
L
-serine
ammonia lyase
(dehydratase)
EC 4.3.1.17
Melting points (uncorrected) were determined in open capillary
tubes on a GallenKamp apparatus and optical rotations were mea-
sured on a Perkin-Elmer 141 polarimeter at 589 nm. The 1H and 13
C
O
O
NMR spectra (250.1 and 62.9 MHz, respectively) of samples dis-
solved in D2O were acquired on a Bruker AC 250F spectrometer.
Chemical shifts (d) are reported in ppm referenced to HOD
(4.80 ppm) for proton spectra and external dioxane (66.66 ppm)
for carbon spectra. Multiplicities assigned to carbon resonances
were determined from DEPT spectra. Electrospray ionization mass
spectrometry (ESI-MS and ESI-MS/MS) was performed on a Ther-
H3C
O
pyruvate
synthase
EC 1.2.7.1
L-lactate
dehydrogenase
EC 1.1.1.27
NAD
NADH
O
H
OH
O
mo-Finnigan LCQ Duo ion trap using flow-injection (20 lL metha-
nol–water/min). Maximum centrifugal forces are given. Spot tests
for amino acids were performed by treating dried applications of
sample solutions on filter paper with ninhydrin reagent (0.25% in
acetone) and heating at 100 °C for a few minutes.54 DL-Serine was
obtained from Sigma-Aldrich (Oakville, ON).
O
+
CO2
H3C
SCoA
H3C
phosphate
acetyltransferase
EC 2.3.1.8
4.2. HPLC determination of amino acids
ADP ATP
O
O
The utilization of amino acids in culture fluids was monitored by
HPLC after derivatization using o-phthalaldehyde and mercap-
toethanol, whereas derivatives formed using o-phthalaldehyde
2
H3C
O
H3C
OPO3
acetate kinase
EC 2.7.2.1
and N-acetyl-L-cysteine were used for the determination of enantio-
meric compositions by HPLC.36 Enantiomeric excesses (% ee) were
calculated from peak areas corrected for the different fluorescent
Scheme 1. Catabolism of L
-serine: regeneration of the coenzyme NAD+ and the
formation of acetate and lactate. Butyrate (not shown) is formed from acetyl-CoA.46
response of each enantiomer (i.e., peak area of
D-enantiomer =
1.06 ꢀ peak area of the
L
-enantiomer), determined using triplicate
while one acetyl-CoA (i.e., one ATP) is generated from two mole-
cules of pyruvate. Only a single enzyme-catalyzed step, however,
is required for the reduction of pyruvate to lactate.
injections of racemic serine at two different concentrations.
4.3. Microorganism, general growth conditions, and media
2.4. Preparation of D-serine
Fusobacterium nucleatum (ATCC 25586) was maintained and
cultured at 37 °C under anaerobic conditions as described.36 Liquid
cultures in the peptone medium36 were initiated with F. nucleatum
cells grown for 24 h on a 9-cm sheep-blood agar plate and sus-
pended in sterile peptone medium (3 mL).
The viability of enantioselective catabolism for the preparation
-serine was demonstrated under optimized conditions:
of
D
800 mM DL-serine; 5 g damp cells in 100 mL resuspension buffer;
and an incubation time of 42 h. The supernatant from the incuba-
tion mixture was applied to the cation-exchange resin in order to
The buffer for resuspension experiments41 contained (g/L):
K2HPO4, 9.0; KH2PO4, 6.0; MgSO4ꢁ7H2O, 0.2; CaCl2ꢁ2H2O, 0.02; so-
separate the residual
The treatment of amino acid containing fractions with charcoal
and recrystallization from aqueous ethanol gave -serine as an
D-amino acid from the carboxylate anions.
dium acetate, 0.3; and
L
-cysteineꢁHCl, 0.4. DL-Serine was dissolved
in freshly prepared resuspension buffer, and the pH was adjusted
to 7.3–7.4 with 3 M NaOH. The solution was autoclaved and sup-
plemented with vitamin solution 1 (10 mL/L), vitamin solution 2
(1 mL/L), and trace metal solution (1 mL/L), each autoclaved sepa-
rately. Vitamin solution 1 contained p-aminobenzoic acid, thiamin
chloride hydrochloride, riboflavin, nicotinic acid, pyridoxal hydro-
chloride, inositol, calcium pantothenate, each at 0.2 g/L, and was
adjusted to pH 7.0 with 1 M NaOH. Vitamin solution 2 contained
DL-thioctic acid, biotin, haemin, folic acid, each at 0.1 g/L. The trace
metal solution contained (g/L): FeSO4ꢁ7H2O, 4.0; MnSO4ꢁ2H2O,
0.15; and Na2MoO4ꢁ2H2O, 0.15.
D
optically active white solid (83% recovery from the racemate;
98% pure by HPLC) with an ee greater than 99% (by HPLC).
3. Conclusion
Without genetic modifications, F. nucleatum demonstrated an
inherent capacity to selectively catabolize large quantities of
ine from a racemic mixture. Depletion of the -amino acid was
most efficient at high densities of resuspended bacterial cells, con-
ditions easily achieved with the anaerobic bacterium. The acetate
and lactate that accumulated in the culture fluids as major cata-
L-ser-
L
4.4. Development of resuspension conditions
bolic products were easily separated from residual D-serine, which
was recovered in high yield and >99% ee. The optimized process
has several advantages over a reductive animation process53
employing isolated enzymes and the expensive, unstable
b-hydroxypyruvate. Racemic serine is a low cost starting point;
The peptone medium (500 mL) was inoculated with F. nuclea-
tum cell suspension from one agar plate and incubated for 24 h.
The damp cells (approximately 2.5 g) were harvested aseptically
in air by centrifugation (8200g, 15 min) and resuspended with
upon conversion of the racemate to D-serine, about a fivefold in-

M. Ramezani, R. L. White / Tetrahedron: Asymmetry 22 (2011) 1473–1478
1477
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trifuged (15,400g, 10 min). Supernatants were stored at ꢂ15 °C for
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4.5. Identification of metabolic end-products
The supernatant from
a serine resuspension experiment
(800 mM, 48 h incubation) was titrated to pH 9.5 with 5 M NaOH
and lyophilized. A portion of the lyophilzed residue (ca. 100 mg)
was dissolved in D2O for NMR analysis. Resonances in the NMR
spectra were assigned by chemical shift comparisons with stan-
dard samples. The relative amounts of the end-products were cal-
culated from the integrated areas of the 1H NMR signals for the
methyl groups of acetate, butyrate and lactate at d 1.84, 0.81 and
1.26 ppm, respectively.
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8. (a) Maurer, P. J.; Takahata, H.; Rapoport, H. J. Am. Chem. Soc. 1984, 106, 1095–
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4.5.1. Residue from serine catabolism
1H NMR d 4.04 (q, J = 6.9 Hz), 1.26 (d, J = 6.7 Hz) [lactate]; 2.08
(t, J = 7.3 Hz), 1.55–1.41 (m), 0.81 (t, J = 7.3 Hz) [butyrate]; 1.84
(s) [acetate]; 3.94–3.81 (AB of ABX, JAB = 12.2 Hz), 3.74 (X of ABX,
apparent t, splittings of 4.9 and 4.3 Hz) [serine]. 13C NMR d 185.3
(s), 71.2 (d), 22.8 (q) [lactate]; 184.2 (s), 26.0 (q) [acetate]; 175.9
(s), 63.2 (t), 59.1 (d) [serine]; 42.3, 22.0, 16.0 [butyrate].
Biomol. Chem. 2009, 7, 1355–1360;
a-glucosidase: (b) Ribes, C.; Falomir, E.;
Carda, M.; Marco, J. A. J. Org. Chem. 2008, 73, 7779–7782; endoththelin
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a-galactosidase: (d)
a
4.6. Preparation of D-serine by cell suspensions
2003, 44, 7649–7653; neuraminidase: (f) Hanessian, S.; Bayrakdarian, M.; Luo,
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F. nucleatum resuspended in a phosphate buffer (5 g damp cells/
100 mL) containing DL-serine (8.4 g, 800 mM) was incubated anaer-
obically with gentle magnetic stirring for 42 h at 37 °C and centri-
fuged (8200g, 15 min). The supernatant was applied to Amberlite
IR-120 (2.5 ꢀ 50 cm column, H+ form) at a flow rate of 5.0 mL/
min. The column was washed with water (500 mL) and eluted with
0.5 M aqueous ammonia (3 L). Fractions (200 mL) containing ami-
no acid (ninhydrin spot test) were combined and evaporated to
dryness in vacuo. The residue (approximately 3.5 g) was dissolved
in hot water (100 mL), treated with decolorizing charcoal (2 g), and
filtered through Celite. Concentration of the filtrate in vacuo and
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the addition of ethanol yielded
D
-serine (3.47 g, 83% recovery),
mp 213–215 °C, ½a D25
ꢃ
¼ ꢂ15:7 (c 1, 1 M HCl), 98% chemical purity
by HPLC, >99% ee by HPLC, Ref. 55 mp 217–219 °C, Ref. 56
½
a 2D3
ꢃ
¼ ꢂ15:0 (c 4, 1 M HCl). 1H NMR d 3.98–3.86 (overlapping AB
quartets of ABX, JAB = 12.2 Hz, 2H), 3.80 (X of ABX, apparent t, split-
tings of 4.9 and 4.3 Hz, 1H). 13C NMR d 175.1, 62.8, 59.0; ESI+MS
(MeOH–H2O, 1:1, 20 l
L/min) m/z 106 [M+H]+; CID of m/z 106:
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Acknowledgements
We thank the Natural Sciences and Engineering Research Coun-
cil of Canada (NSERC) and the Innovations in Chemistry Fund,
Department of Chemistry, Dalhousie University, for financial sup-
port; and the Ministry of Health and Medical Education, Iran, for
partial support to M.R. We are grateful to NMR-3 and MMSlab
for access to spectrometers, the NRC Institute of Marine Biosci-
ences for the use of the polarimeter, Dr. H. N. Shah for the F. nucle-
atum culture, Dr. S. F. Lee for providing advice and facilities to
culture anaerobic bacteria, and S. E. MacIntosh for assistance with
preliminary experiments.
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