Kinetic chiral resolutions of 1,2-diols and desymmetrization of glycerol catalyzed by glycerol kinase

Article abstract of DOI:10.1021/jo980122y

Enantioselective phosphorylation catalyzed by glycerol kinase (EC 2.7.1.30) facilitated the kinetic chiral resolution of 3-chloro-1,2- propanediol, 3-fluoro-1,2-propanediol, 3-butene-1,2-diol, and 1,2,4- butanetriol. Both enantiomers of each compound were isolated is free diol or triol form, in excellent enantiomeric purity (91 to > 99.5% ee) and in moderate to good yield (60-94% of theoretical). The enantioselectivities of glycerol kinase from different sources were compared using 1,2,4-butanetriol as the substrate. The effect of elevated temperatures on enzymatic activity, stability, and enantioselectivity were studied, and procedures for the isolation of diol and triol products were optimized. Glycerol kinase- catalyzed phosphorylation facilitated the three-step chemoenzymatic conversion of glycerol to (S)-1,2-O-isopropylideneglycerol in 83% yield and > 99.5% ee).

Full text of DOI:10.1021/jo980122y

J . Org. Chem. 1998, 63, 4039-4045
4039
Kin etic Ch ir a l Resolu tion s of 1,2-Diols a n d Desym m etr iza tion of
Glycer ol Ca ta lyzed by Glycer ol Kin a se
H. Keith Chenault,* Laura F. Chafin, and Sebastian Liehr
Department of Chemistry and Biochemistry, University of Delaware, Newark, Delaware 19716
Received J anuary 23, 1998
Enantioselective phosphorylation catalyzed by glycerol kinase (EC 2.7.1.30) facilitated the kinetic
chiral resolution of 3-chloro-1,2-propanediol, 3-fluoro-1,2-propanediol, 3-butene-1,2-diol, and 1,2,4-
butanetriol. Both enantiomers of each compound were isolated in free diol or triol form, in excellent
enantiomeric purity (91 to >99.5% ee) and in moderate to good yield (60-94% of theoretical). The
enantioselectivities of glycerol kinase from different sources were compared, using 1,2,4-butanetriol
as the substrate. The effect of elevated temperatures on enzymatic activity, stability, and
enantioselectivity were studied, and procedures for the isolation of diol and triol products were
optimized. Glycerol kinase-catalyzed phosphorylation facilitated the three-step chemoenzymatic
conversion of glycerol to (S)-1,2-O-isopropylideneglycerol in 83% yield and >99.5% ee).
9
10,11
In tr od u ction
synthons, such as glycidol and epihalohydrins.
Enan-
tiomerically pure 3-chloro-1,2-propanediol has been pre-
pared by chemical degradation of methyl 5-chloro-5-
deoxy-R-L-arabinofuranoside and methyl 6-chloro-6-deoxy-
1
Glycerol kinase (EC 2.7.1.30) catalyzes the phospho-
rylation of glycerol to form (R)-glycerol 1-phosphate (sn
or L-glycerol 3-phosphate). Because it is both regiospe-
cific and completely stereoselective in the reaction it
catalyzes, we felt it might be useful for the kinetic chiral
resolution of 1,2-diols and the desymmetrization of
12
R-D-glucopyranoside and by chemical transformation of
13
D-mannitol via the intermediate, 2,3-O-isopropylidene-
14
glyceraldehyde.
3-Chloro-1,2-propanediol of varying
optical purity has been produced by lipase or protease-
2
-substituted 1,3-propanediols. In pioneering work by
7,15,16
catalyzed resolution,
destructive fermentation of the
2
Crans and Whitesides, 1,2-diols were resolved using
glycerol kinase. However, the reactive enantiomer of
most compounds was isolated as its phosphate ester and
not as the free diol. The unreactive enantiomers were
recovered in low yield and moderate enantiomeric purity.
Furthermore, all of the resolutions were catalyzed by
glycerol kinase from Saccharomyces cerevisiae, an en-
zyme which is no longer commerically available.
Previous work on the substrate specificity of glycerol
kinase has shown that it accepts a number of substitu-
ents in place of the hydroxyl group of glycerol distal to
the site of phosphorylation.3 It is less tolerant, however,
of structural changes at C-2, although 2-fluoro-1,3-
9,17
racemate,
microbial transformation of 1,3-dichloro-2-
propanol, recrystallization of a diastereomeric deriva-
tive, and asymmetric dihydroxylation of 3-chloropro-
pene. Optically active 3-butene-1,2-diol has served as
a chiral synthon in the synthesis of oscillatoxin, lipoxin
B, thienamycin, and ethambutol. It or a derivative
thereof has been prepared by either enzymatic or
18
11
19
20
21
22
22
16
22,23
chemical
resolution, by Wittig methylidenation of (R)-
or (S)-2,3-O-isopropylideneglyceraldehyde, or by trans-
1
4
(7) Iriuchima, S.; Kojima, N. Agric. Biol. Chem. 1982, 46, 1153-
,4
1157.
(8) Paradisi, G.; Bombarda, C.; Polacci, C. Eur. Patent 575 776, 1993;
Chem. Abstr. 1994, 120, 164225.
3
propanediol, 2-methylpropane-1,2,3-triol, and 2-ethyl-
propane-1,2,3-triol are reported to be substrates. We
report here on the kinetic chiral resolution of 3-chloro-
(9) Suzuki, T.; Kasai, N. Bioorg. Med. Chem. Lett. 1991, 1, 343-
5
346.
(
10) Kawakami, Y. J . Org. Chem. 1982, 47, 3581-3585.
(
11) Ellis, M. K.; Golding, B. T.; Maude, A. B.; Watson, W. P. J .
1
,2-propanediol, 3-fluoro-1,2-propanediol, 3-butene-1,2-
Chem. Soc., Perkin Trans. 1 1991, 747-755.
(
12) J ones, H. F. Chem. Ind. 1978, 533.
diol, and 1,2,4-butanetriol, as well as on efforts to
desymmetrize 2-substituted 1,3-propanediols.
(
13) Porter, K. E.; J ones, A. R. Chem.-Biol. Interact. 1982, 41, 95-
1
4
04.
(
14) J urczak, J .; Pikul, S.; Bauer, T. Tetrahedron 1986, 42, 447-
Optically active 3-chloro-1,2-propanediol has been used
88.
6
as a chiral synthon in the synthesis of natural products
(
15) (a) Poppe, L.; Nov a´ k, L.; Kajt a´ r-Peredy, M.; Sz a´ ntay, C.
and drugs7 and is readily converted into other chiral
,8
Tetrahedron: Asymmetry 1993, 4, 2211-2217. (b) Pantali, V.; Melbye,
A. G.; Alvik, T.; Anthonsen, T. Tetrahedron: Asymmetry 1992, 3, 65-
7
2.
(
1) (a) Thorner, J . W.; Paulus, H. In The Enzymes, 3rd ed.; Boyer,
(16) Boaz, N. W.; Zimmerman, R. L. Tetrahedron: Asymmetry 1994,
P. D., Ed.; Academic: New York, 1973; Vol. 8, Chapter 14, pp 487-
5, 153-156.
5
1
08. (b) Faber, H. R.; Pettigrew, D. W.; Remington, S. J . J . Mol. Biol.
989, 207, 637-639.
(17) Suzuki, T.; Kasai, N.; Yamamoto, R.; Minamiura, N. J . Ferment.
Bioeng. 1993, 73, 443-448.
(
2) Crans, D. C.; Whitesides, G. M. J . Am. Chem. Soc. 1985, 107,
(18) Nakamura, T.; Yu, F.; Mizunashi, W.; Watanabe, I. Agric. Biol.
Chem. 1991, 55, 1931-1935.
7
019-7027.
3) Eisenthal, R.; Harrison, R.; Lloyd, W. J .; Taylor, N. R. Biochem.
J . 1972, 130, 199-205.
4) (a) Crans, D. C.; Whitesides, G. M. J . Am. Chem. Soc. 1985, 107,
(
(19) Becker, H.; Sharpless, K. B. Angew. Chem., Int. Ed. Engl. 1996,
35, 448-451.
(
(20) Walkup, R. D.; Cunningham, R. T. Tetrahedron Lett. 1987, 28,
4019-4022.
7
5
008-7018. (b) Knight, W. B.; Cleland, W. W. Biochemistry 1989, 28,
728-5734.
(21) Rama Rao, A. V.; Reddy, E. R.; J oshi, V. V.; Yadav, J . S.
Tetrahedron Lett. 1987, 28, 6497-6500.
(22) Rama Rao, A. V.; Subhas, D.; Gurjar, M. K.; Ravindranathan,
T. Tetrahedron 1989, 45, 7031-7040.
(
(
5) Grunnet, N.; Lundquist, F. Eur. J . Biochem. 1967, 3, 78-84.
6) (a) Ishibashi, H. Tetrahedron Lett. 1993, 34, 489-492. (b) Lamm,
B.; Simonsson, R.; Sundell, S. Tetrahedron Lett. 1989, 30, 6423-6426.
S0022-3263(98)00122-4 CCC: $15.00 © 1998 American Chemical Society
Published on Web 05/19/1998

4
040 J . Org. Chem., Vol. 63, No. 12, 1998
Ta ble 1. Activity of Un n a tu r a l Su bstr a tes w ith Glycer ol Kin a se fr om Va r iou s Sp ecies
Chenault et al.
a
relative activity, %
compound
C. mycoderma
S. canus
Cellulomonas sp.
E. coli
Arthrobacter sp.
a
Initial rates of phosphorylation of 20 mM racemic substrate determined by continuous spectrophotometric assay are reported as a
percent of the rate of phosphorylation of 10 mM glycerol (υ ) 100). Activities reported in parentheses were determined by an end-point
assay for ADP released. Relative activity is <0.01%. Relative activity is <0.003%. d Relative activity is <0.001%. e The relative activities
observed with glycerol kinase from the five species listed above were 0.013, 0.072, 0.042, and 0.012, respectively. However, attempted
preparative-scale phosphorylation of 2-chloro-1,3-propanediol using glycerol kinase from S. canus produced no diol phosphate and resulted
in only hydrolysis of ATP.
b
c
formation of (S)-O-benzylglycidol24 or diethyl (R,R)-(+)-
compound were recovered in good yield and generally in
99 to >99.5% ee.
tartrate.21 Enantiomerically pure 1,2,4-butanetriol has
2
5
20
been used in syntheses of pederin, oscillatoxin A, and
araguspongine D26 and is most commonly obtained by the
reduction of (R)- or (S)-malic acid.2
Resu lts a n d Discu ssion
1,24-27
Because glycerol kinasesespecially that from bacterial
sourcessis a metabolic enzyme, we expected it to exhibit
generally higher enantioselectivity than lipases and
esterases, which are degradative enzymes. Because of
the enantioselectivity displayed by glycerol kinase with
its natural substrate, glycerol, we believed that one
enantiomer of unnatural 1,2-diols would undergo glycerol
kinase-catalyzed phosphorylation to give products analo-
gous to (R)-glycerol 1-phosphate. We were further en-
couraged by the low Michaelis constant of glycerol kinase
Activity of Glycer ol Kin a se w ith Un n a tu r a l Su b-
str a tes. A continuous spectrophotometric assay was
used to measure the activity of glycerol kinase with a
number of short-chain diols and triols as potential
substrates. In the assay, ADP generated from ATP
during phosphorylation of substrate was rephosphory-
lated by phosphoenolpyruvate (PEP) and pyruvate ki-
nase, and the pyruvate produced was reduced in situ by
NADH and lactate dehydrogenase. Potential substrates
were tested with glycerol kinases from Candida myco-
derma, Streptomyces canus, Cellulomonas sp., Escheri-
chia coli, and Arthrobacter sp., all of which are commer-
cially available. These enzymes exhibited specific activities
for glycerol (K ) 0.01 and 0.035 mM for glycerol kinase
m
from Escherichia coli and Candida mycoderma, respec-
tively). Efficient binding of substrate makes it more
likely that the reactive enantiomer will be completely
consumed, leaving behind unreacted 1,2-diol that is
essentially enantiomerically pure.
-
1
of 9-44 units mg when assayed at 25 °C with 10 mM
glycerol as substrate.
An end-point assay for ADP released during glycerol
kinase-catalyzed phosphorylation was used to verify very
low activities observed by the continuous spectrophoto-
metric assay with some of the unnatural substrates. This
assay allowed for larger quantities of glycerol kinase to
be used without substantial interference from a back-
ground reaction.
In this work, we compare the substrate specificities and
enantioselectivities of commercially available glycerol
kinases from a variety of biological sources. We examine
the effect of temperature on enzymatic activity, stability,
and enantioselectivity and report optimized conditions
for the isolation of both enantiomers of resolved 1,2-diols
in the free alcohol form. Both enantiomers of each
Initial rates of phosphorylation of unnatural substrates
(20 mM racemate) were determined relative to the initial
rates observed with 10 mM glycerol (Table 1). All of the
unnatural substrates tested exhibited relatively low
activities. 1,2,4-Butanetriol, 3-chloro-1,2-propanediol,
and 3-fluoro-1,2-propanediol exhibited the highest activi-
ties among the unnatural substrates. 3-Butene-1,2-diol,
(
23) (a) Neagu, C.; Hase, T. Tetrahedron Lett. 1993, 34, 1629-1630.
b) Kusakabe, M.; Kato, H.; Sato, F. Chemistry Lett. 1987, 2163-2166.
24) Takano, S.; Tomita, S.; Iwabuchi, Y.; Ogasawara, K. Synthesis
988, 610-611.
25) Willson, T. M.; Kocienski, P.; J arowicki, K.; Isaac, K.; Hitchcock,
(
(
1
(
P. M.; Faller, A.; Campbell, S. F. Tetrahedron 1990, 46, 1767-1782.
(
(
26) B o¨ rjesson, L.; Welch, C. J . Tetrahedron 1992, 48, 6325-6334.
27) (a) Thiam, M.; Slassi, A.; Chastrette, F.; Amouroux, R. Synth.
3
-butyne-1,2-diol, and 4-chloro-1,3-butanediol also showed
some activity with glycerol kinase from at least some of
the species tested. Among the symmetric 2-substituted
Commun. 1992, 22, 83-95. (b) Pawlak, J .; Nakanishi, K.; Iwashita,
T.; Borowski, E. J . Org. Chem. 1987, 52, 2896-2901.

Kinetic Chiral Resolutions of 1,2-Diols
,3-propanediols tested, only 2-chloro-1,3-propanediol
J . Org. Chem., Vol. 63, No. 12, 1998 4041
1
Sch em e 1
gave any indication of being a substrate. However,
further investigation revealed that 2-chloro-1,3-pro-
panediol only stimulated ATPase activity within glycerol
kinase (see Desymmetrization of Glycerol below). 2-Meth-
yl-1,3-propanediol and 2-(hydroxymethyl)-1,3-propanediol
were not substrates of glycerol kinase. In general,
glycerol kinases from different species displayed only
small differences in activity with a given substrate.
Kin etic Ch ir a l Resolu tion s. To demonstrate the
utility of glycerol kinase for the kinetic chiral resolution
of analogues of glycerol, we subjected four compounds
representing a two 100-fold range of reactivity (vrel
)
atoms of each diol, respectively. All were as they were
expected to be. Upon phosphorylation, the primary
carbon of each 1,2-diol moiety displayed a downfield shift
in resonance of 1.2-4.1 ppm, relative to the unphospho-
rylated compound. Concurrently, the secondary carbon
of each 1,2-diol moiety displayed an upfield shift in
resonance of 1.1-1.6 ppm. In every case, the two-bond
coupling constant between phosphorus and the primary
0
.01-2.0% of that of glycerol) to enzymatic phosphory-
lation. 1,2,4-Butanetriol, 3-chloropropane-1,2-diol, 3-flu-
oropropane-1,2-diol, and 3-butene-1,2-diol were the sub-
strates chosen for study. Issues of concern were how to
maximize the effective catalytic activity of glycerol kinase
with relatively unreactive substrates and how to recover
both enantiomers of each compound in the free diol (or
triol) form, in high chemical yield, and with high enan-
tiomeric purity.
2
carbon ( J CP) of the diol was approximately 5 Hz while
the three-bond coupling constant between phosphorus
An initial substrate concentration of 200 mM racemic
material was used for the resolutions. Catalytic ATP was
regenerated using PEP and pyruvate kinase28 in order
to minimize the expense due to ATP and in order to
prevent inhibition of glycerol kinase by the accumulation
3
and the secondary carbon ( J CP) was approximately 7 Hz.
1
Enantiomeric purities were determined by H NMR of
the derivatives formed by peracylation of each diol or triol
with (R)-(+)-R-methoxy-R-(trifluoromethyl)phenylacetic
1
acid (MTPA). If H NMR showed no signal due to a
5
of ADP. No inhibition of glycerol kinase by PEP was
diastereomeric impurity, the NMR sample was spiked
with 0.5 mol % of the (+)-MTPA derivative of the
appropriate racemic diol or triol. By this calibration, it
was demonstrated in every case that a 0.25% diastere-
omeric impurityscorresponding to an ee for the diol or
triol of 99.5%scould be detected. Small diastereomeric
impurities in resolutions of 1,2,4-butanetriol were like-
wise calibrated by adding known quantities of the (+)-
MTPA derivative of the racemic triol.
observed.29 Both glycerol kinase and pyruvate kinase
were stable in solution and retained at least 50% of their
original activity during the course of resolutions. To
minimize autoxidation of the enzymes, reaction solutions
were sparged with nitrogen just before adding enzymes,
and reactions were run in sealed flasks. The progress of
each resolution was monitored by assaying for PEP and
pyruvate at the beginning and periodically throughout
the reaction.
3
-Chloro-1,2-propanediol was first resolved using gly-
When assays for PEP and pyruvate indicated that the
progress of the resolution had leveled off, the reaction
30
cerol kinase from C. mycoderma (Scheme 1). Although
a slight excess of PEP was used to ensure that all of the
reactive D enantiomer was consumed, enzymatic assay
indicated that the resolution proceeded to only 47%
-
mixture was applied to a column of Dowex-1 (HCO
3
).
The phosphate ester and carboxylate anions were bound
to the anion exchange column, and the unreacted sub-
strate was eluted from the column with 50% aqueous
ethanol. Carboxylate anions were eluted from the col-
umn with 65 mM ammonium bicarbonate in 50% aqueous
ethanol, and the diol or triol phosphate was eluted with
conversion. Unreacted L-3-chloro-1,2-propanediol was
31
isolated in 42% yield but in only 70% ee.
The D
enantiomer was isolated in 36% overall yield and >99.5%
ee after dephosphorylation.
When 1,2,4-butanetriol was treated similarly with
glycerol kinase from C. mycoderma, the reaction pro-
ceeded to 53% conversion. The unreactive L enantiomer
was recovered in 48% yield and >99.5% ee, while the D
enantiomer was recovered in 24% yield and only 92% ee.
Partial transformation of the L enantiomer may have
occurred through phosphorylation of the C-4 hydroxyl
group, either with or without stereoselectivity for the L
enantiomer. Stereoselectivity might result from the fact
that the topology of L-1,2,4-butanetriol bound with its C-4
hydroxyl group oriented toward the ATP binding site of
glycerol kinase would resemble that of glycerol produc-
tively bound in the same active site.
2
00 mM aqueous ammonium bicarbonate. Hydrolysis of
the phosphate ester was effected by either acid phos-
phatase (EC 3.1.3.2) or alkaline phosphatase (EC 3.1.3.1),
thereby allowing the dephosphorylation of base or acid
sensitive compounds, respectively. Both phosphatase
enzymes exhibited significant inhibition by ammonium
cation. However, removal of ammonium by cation ex-
change allowed the phosphatase reactions to proceed
uneventfully.
Glycerol kinase phosphorylated the primary hydroxyl
group of the 1,2-diol moiety of each substrate. The
regiochemistry of each reaction was demonstrated by the
changes in 13C NMR chemical shift induced by phospho-
rylation and by the two- and three-bond coupling con-
stants between phosphorus and the R- and â-carbon
To minimize the phosphorylation of the L enantiomer,
a two-stage resolution procedure was developed, in which
(
30) Until recently, glycerol kinase from C. mycoderma was available
(28) Crans, D. C.; Kazlauskas, R. J .; Hirschbein, B. L.; Wong, C.-
from Biozyme Labratories for $10/kilounit.
H.; Abril, O.; Whitesides, G. M. Methods Enzymol. 1987, 136, 263-
(31) Yields are reported based on the mass of the original racemate.
The maximum theoretical yield for a resolution is 50%. Thus, a 42%
yield corresponds to 84% of the theoretical maximum for a single
enantiomer.
2
80.
(29) Chenault, H. K.; Mandes, R. F.; Hornberger, K. R. J . Org. Chem.
1
997, 62, 331-336.

4
042 J . Org. Chem., Vol. 63, No. 12, 1998
Chenault et al.
Ta ble 2. Tw o-Sta ge Resolu tion s of 1,2,4-Bu ta n etr iol
Usin g Glycer ol Kin a se fr om Va r iou s Sp eciesa
b
%
yield (% ee)
2
nd %
unitsc t,d h conversion
e
D
L
species
C. mycoderma
S. canus
Cellulomonas sp. 210
E. coli
Arthrobacter sp.
44
435
48
12
12
41
20
3.6
14.8
9.7
13.2
18.5
33 (95) 21 (>99.5)
28 (92) 47 (91)
28 (78) 27 (97)
31 (91) 40 (91)
30 (83) 43 (57)
176
221
aResolutions were performed on 2 mmol of racemic substrate.
Yields are expressed as a percent of the mass of the original
b
racemate. Maximum theoretical yield for a pure enantiomer is
c
5
0%. Units of glycerol kinase used for the first stage of the
d
resolution. Time for the first stage of the resolution to reach 42.5%
conversion of the racemate, on the basis of assay of PEP consumed.
Additional percent of original racemate consumed during the
e
second stage of the resolution, on the basis of assay of PEP
consumed.
the amount of PEP used was limited to 42.5 mol % of
the racemic substrate. When all of the PEP was con-
sumed, products were separated by anion exchange
chromatography as described earlier. Unreacted mate-
rial enriched in the L enantiomer was resubmitted to
resolution conditions in order to consume the remaining
D enantiomer. The two-stage resolution procedure im-
proved the enantiomeric purity of the D-1,2,4-butanetriol
produced to 95% ee. When the resolution was allowed
to proceed to 48% conversion with glycerol kinase from
Cellulomonas sp., however, and the unreacted enanti-
omer was resubmitted to resolution conditions, D- and
L-1,2,4-butanetriols were obtained in 43 and 37% yields
and in 93 and 60% ee, respectively.
At this point, glycerol kinases from different sources
were examined to see if they differed substantially in
their enantioselectivities with 1,2,4-butanetriol as sub-
strate. All enzymes examined were commercially avail-
able and came from C. mycoderma, S. canus, E. coli,
Cellulomonas sp., and Arthrobacter sp. The results are
shown in Table 2. Glycerol kinase from S. canus was
selected for further study because of its good enantiose-
lectivity, relatively broad substrate specificity (Table 1),
and ready availability.32
F igu r e 1. (a) Effect of temperature on the initial rate of
phosphorylation of 3-chloro-1,2-propanediol catalyzed by glyc-
erol kinase from S. canus. (b) Effect of temperature on the
stability of glycerol kinase from S. canus. Residual activity
with 3-chloro-1,2-propanediol at 25 °C was measured after the
enzyme had been incubated at 25 °C (b), 35 °C (9), 40 °C (2),
and 45 °C ([).
the problem of hydrolysis, the procedure for isolating
products was modified. Instead of desalting and evapo-
rating aqueous solutions of diol products, free diols were
recovered from aqueous solution by continuous liquid-
liquid extraction using ethyl acetate. Thus, after sepa-
rating reactive and unreactive enantiomers by anion
exchange chromatography, the solution containing the
unreacted L enantiomer was extracted for 24 h with ethyl
acetate. The D enantiomer was eluted from the anion
exchange column, dephosphorylated with phosphatase,
and then similarly extracted from solution with ethyl
acetate.
An attempt to recover unreacted L-3-chloro-1,2-pro-
panediol directly from the resolution reaction mixture by
continuous liquid-liquid extraction led to migration of
the phosphate ester of D-3-chloro-1,2-propanediol 1-phos-
phate, giving 20% conversion to 3-chloro-1,2-propanediol
2-phosphate. Although the regiochemistry of the phos-
phate group is irrelevant since it is eventually removed,
this complication and the fact that yields were not
improved by the new procedure led us to choose anion
exchange, followed by liquid-liquid extraction, as the
method for separating and recovering enantiomeric prod-
ucts from a resolution.
To utilize the enzyme’s catalytic activity most ef-
ficiently, we investigated the effect of temperature on the
activity and stability of glycerol kinase from S. canus
(Figure 1). At 45 °C, the activity of glycerol kinase with
3
-chloro-1,2-propanediol was 15 times greater than that
observed at 25 °C. However, glycerol kinase also exhib-
ited a sharp decrease in stability at temperatures of 40
°
C and greater. At 35 °C, the enzymatic activity was 5
times greater than that at 25 °C, but the stability was
essentially the same as that seen at 25 °C. Furthermore,
the enantioselectivity of the enzyme remained outstand-
ing at 35 °C. Resolution of 3-chloro-1,2-propanediol at
either 25 or 35 °C yielded both enantiomers in >99.5%
ee.
While studying the effect of temperature on enantio-
meric purity, we found that 3-chloro-1,2-propanediol
hydrolyzed to form glycerol in the presence of mixed-bed
and strongly acidic ion-exchange resins. Although the
enantioselectivities for the reactions discussed above
were excellent, the yields of resolved enantiomers were
quite low (11-20% for D and 30-38% for L). To avoid
When the optimized resolution procedure was applied
to the resolution of 3-chloro-1,2-propanediol (47% initial
conversion), the yield of the D enantiomer was noticeably
improved by the use of continuous liquid-liquid extrac-
tion (Table 3), and both enantiomers were recovered in
excellent enantiomeric purity (>99.5% ee). Similarly,
(
32) Glycerol kinase from S. canus is available from Genzyme for
$
29/kilounit.

Kinetic Chiral Resolutions of 1,2-Diols
J . Org. Chem., Vol. 63, No. 12, 1998 4043
Ta ble 3. Kin etic Ch ir a l Resolu tion s Usin g Glycer ol
Sch em e 3
Kin a se fr om Str eptom yces Ca n u s
D
L
scale,
mmol yield
%
%
ee
%
%
ee
a
yielda
substrate
-chloro-1,2-propanediol 18
3
3
3
41
34
36
>99.5
>99.5
>99.5
38
30
38
>99.5
>99.5
99
-fluoro-1,2-propanediol
-butene-1,2-diol
2.3
2.8
1
Ta ble 4.
H NMR Da ta for (+)-MTP A P er ester s of
a
Yields are expressed as a percent of the mass of the original
En a n tiom er ic 1,2-Diols
racemate. Maximum theoretical yield for a pure enantiomer is
δ, ppm
5
0%.
diol
configuration
H-1,1′
H-3,3′
Sch em e 2a
3
3
3
1
-chloro-1,2-propanediol
-fluoro-1,2-propanediol
-butene-1,2-diol
D (S)a
L (R)
D (S)a
L (R)
D (R)a
L (S)
4.91, 4.65 3.89, 3.78
4.77, 4.57 3.94, 3.85
4.92, 4.62 4.72, 4.59
4.80, 4.53 4.79, 4.66
4.80, 4.49 5.85
4.73, 4.45 5.96
4.78, 4.50 1.96-1.91
4.72, 4.40 2.07-1.92
,2,4-butanetriol
D (R)a
L (S)
a
Enantiomer that reacts preferentially with glycerol kinase.
Absolu te Con figu r a tion of P r od u cts. 1H NMR of
a
Reagents and conditions: (a) glycerol kinase, 2.9% ATP, PEP,
the (R)-(+)-MTPA peresters of the resolved diols and
triols verified in every case that the enzymatically
produced phosphate esters were stereochemically analo-
gous to (R)-glycerol 1-phosphate.37 Thus, glycerol kinase
catalyzed the stereoselective phosphorylation of (S)-D-3-
chloro-1,2-propanediol, (S)-D-3-fluoro-1,2-propanediol, (R)-
D-1,2,4-butanetriol, and (R)-D-3-butene-1,2-diol. The ab-
solute configurations of the products were determined
from the NMR signals of the protons attached adjacent
to each chiral secondary carbinol center. Correlations
between absolute configuration and relative chemical
shifts were made using an empirical model that assumes
the structure shown in Scheme 3 as the predominant
+
pyruvate kinase; (b) Dowex 50W (H ), then (CH3O)2C(CH3)2/
CH3OH, then KOH; (c) alkaline phosphatase.
resolutions of 3-fluoro-1,2-propanediol and 3-butene-1,2-
diol gave both enantiomers of each compound in good
chemical yield and excellent enantiomeric purity.
Desym m etr iza tion of Glycer ol. The use of enzymes
to desymmetrize meso substrates is a particularly power-
ful tool in organic synthesis.33 With an aim at desym-
metrizing 2-substituted 1,3-propanediols, we first inves-
tigated the natural reaction catalyzed by glycerol kinase,
the desymmetrization of glycerol. When enzymatically
prepared (R)-glycerol 1-phosphate (1) was converted to
its free acid and dissolved in a mixture of methanol and
3
7
conformation in (+)-MTPA esters. In this conformation,
1
protons on substituent R are deshielded by the methoxy
2
,2-dimethoxypropane (Scheme 2), the acidic phosphoryl
2
group, and protons on substituent R are shielded by the
group catalyzed the conversion of the diol into (S)-2,3-
O-isopropylideneglycerol 1-phosphate (2). Compound 2
was unstable when isolated as its free acid but could be
isolated in good yield after the pH of the reaction mixture
had been adjusted to 10. Alkaline phosphatase catalyzed
the dephosphorylation of 2 to give (S)-1,2-O-isopropy-
lideneglycerol (3) in 83% overall yield and >99.5% ee.
This method compares favorably with the preparation of
other enantiomerically enriched derivatives of glycerol
by lipase-catalyzed resolutions34 and with the reduction
of enantiomerically pure 2,3-O-isopropylideneglyceral-
phenyl group. All of the bis- and tris-(+)-MTPA esters
studied in this work complied with this empirical spec-
troscopic model (Table 4). Additional (+)-MTPA ester
groups at C-1 or C-4 appear not to perturb the chemical
shift behavior of the protons adjacent to C-2.
Con clu sion
Glycerol kinase catalyzes the kinetic chiral resolution
of three- and four-carbon 1,2-diols, providing both enan-
tiomers in excellent enantiomeric purity and with pre-
dictable absolute configuration. Product recovery by
anion exchange, treatment with phosphatase and con-
tinuous liquid-liquid extraction is both convenient and
potentially scalable. In certain cases, resolutions using
glycerol kinase may be preferable to lipase-catalyzed
processes since the latter often proceed with only modest
enantioselectivity and require adsorption chromatogra-
phy to separate reactive and unreactive enantiomers.
Although most unnatural substrates display relatively
low specific activities with glycerol kinase, elevated
reaction temperatures can be used to increases reaction
rates without detriment to the enzyme’s enantioselectiv-
ity.
dehyde obtained from D-mannitol1
4,35
or ascorbic acid.
Attempted desymmetrization of 2-chloro-1,3-propanediol
14,36
(100 mM) using glycerol kinase from S. canus led only
to the hydrolysis of ATP. Even when the initial concen-
tration of ATP was increased to 50 mM, no phosphory-
lated diol was observed by 31P NMR. All of the ATP was
hydrolyzed to ADP. Although the pyruvate kinase in
solution was not irreversibly inactivated, it failed to
catalyze phosphoryl transfer from PEP to the accumu-
lated ADP.
(
33) (a) J ones, J . B. Tetrahedron 1986, 42, 3351-3403. (b) Schoffers,
E.; Golebiowski, A.; J ohnson, C. R. Tetrahedron 1996, 52, 3769-3826.
34) (a) Theil, F.; Weidner, J .; Ballschuh, S.; Kunath, A.; Schick, H.
(
J . Org. Chem. 1994, 59, 388-393. (b) Wang, Y. F.; Wong, C.-H. J . Org.
Chem. 1988, 53, 3127-3129.
(
(
35) Hirth, G.; Walther, W. Helv. Chim. Acta 1985, 68, 1863-1871.
36) (a) J ung, M. E.; Shaw, T. J . J . Am. Chem. Soc. 1980, 102, 6304-
(37) Dale, J . A.; Mosher, H. S. J . Am. Chem. Soc. 1973, 95, 512-
519.
6
311. (b) Morgenlie, S. Carbohydr. Res. 1982, 107, 137-141.

4
044 J . Org. Chem., Vol. 63, No. 12, 1998
Chenault et al.
Exp er im en ta l Section
removed and assayed for glycerol kinase activity at 25 °C using
the continuous spectrophotometric assay.
Assa y for P EP . To a final volume of 2.98 mL of 0.10 M
sodium phosphate buffer, pH 7.6, containing 10 units of lactate
Gen er a l P r oced u r es. Glycerol kinase was purchased as
an ammonium sulfate suspension from Genzyme (S. canus,
Arthrobacter sp., Cellulomonas sp.), Sigma (E. coli), and
Biozyme (C. mycoderma) and was used without further
purification. PEP was either purchased as the cyclohexylam-
monium salt from Genzyme or prepared as the potassium salt
according to a published procedure.3 Glycerol kinase showed
no dependence of activity or stability on the salt form of PEP.
NADH was purchased from Genzyme. ATP (disodium salt),
acid phosphatase (potato, Type IV-S), and alkaline phos-
phatase (bovine intestinal mucosa, Type I-S) were purchased
from Sigma. Pyruvate kinase (rabbit muscle, PK3) and lactate
dehydrogenase (rabbit muscle, LDHR2) were purchased from
Biozyme Laboratories and were used without additional
purification. Units of enzymatic activity refer to activity
measured with the natural substrate of each enzyme. (R)-
dehydrogenase, 4.9 mM MgCl
and 4.3 mM ADP was added 100 µL of the sample to be tested
for PEP content. The absorbance at 334 nm was recorded (A ),
2
, 9.1 mM KCl, 0.2 mM NADH,
o
and 1 unit (100 µL) of pyruvate kinase was added. After a
few minutes, the final absorbance (A) was recorded, and the
8
concentration of PEP was calculated from ∆A ) A - A using
o
-1
-1
ꢀ
334 ) 6.18 mM cm as the molar absorptivity of NADH.
Assa y for P yr u va te. To a final volume of 2.98 mL of 0.10
M sodium phosphate buffer, pH 7.6, containing 0.2 mM NADH
was added 100 µL of the sample to be tested for pyruvate
o
content. The absorbance at 334 nm was recorded (A ), and 1
unit (100 µL) of lactate dehydrogenase was added. After a
few minutes, the final absorbance (A) was recorded, and the
concentration of pyruvate was calculated from ∆A ) A - A
o
(
+)-MTPA was purchased from Aldrich.
-1
-1
using ꢀ334 ) 6.18 mM cm as the molar absorptivity of
Unless otherwise noted, H and 13C spectra were acquired
1
NADH.
at nominal resonance frequencies of 400 and 101 MHz,
respectively, using CDCl as the solvent and residual CHCl
δ 7.24) as the internal reference. Spectra obtained in D
were referenced internally to sodium 3-(trimethylsilyl)pro-
Gen er a l P r oced u r e for On e-Sta ge Resolu tion s. Race-
mic substrate (0.200-2.00 g, 185 mM) was combined with ATP
3
3
(
2
O
(5.4 mM), MgCl
2
2 3
‚6H O (10 mM), KCl (11 mM), NaN (1.5 mM),
2
-mercaptoethanol (10 mM), and PEP (111 mM) in deionized
3
1
panoate-2,2,3,3-d
NMR spectra were acquired at a nominal resonance frequency
of 162 MHz using D O as solvent and were referenced
externally to 85% H PO (δ 0.00).
4
(TSP, δ 0.00) unless otherwise noted.
P
water. Before the final volume was set, the solution was
adjusted to pH 9.5 using aqueous potasssium hydroxide, and
the solution was sparged with nitrogen. Glycerol kinase (about
2
3
4
0
.5 units of activity measured with the unnatural substrate
Con tin u ou s Sp ectr op h otom etr ic Assa y of Glycer ol
Kin a se Act ivit y. To a final volume of 3.00 mL of 0.10 M
sodium phosphate buffer, pH 7.6, containing 2.9 units of
pyruvate kinase, 0.13 units of lactate dehydrogenase, 10 mM
substrate (20 mM if substrate was racemic), 3.0 mM ATP, 14
per mmol of racemate) and pyruvate kinase (about 15 units
per mmol of racemic substrate) were added. Ammonium
sulfate from the suspension of glycerol kinase lowered the pH
of the reaction to 8.0. Periodically, the reaction was assayed
for PEP and pyruvate.
When assays for PEP and pyruvate indicated that conver-
sion had reached 50% or had leveled off, the reaction mixture
was diluted with an equivalent volume of ethanol and applied
mM MgCl
2
, 1 mM KCl, and 0.2 mM NADH, at 25 °C, was
added 25 µL of a solution of glycerol kinase (either 174 or 0.174
-1
units mL ). The absorbance at 334 nm was recorded continu-
ously for 10 min, and the activity of glycerol kinase was
-
to a column of Dowex-2 × 8-100 (HCO
3
) resin that was twice
-
1
-1
calculated using ꢀ334 ) 6.18 mM cm as the molar absorp-
tivity of NADH. If no activity was observed with an unnatural
substrate, glycerol was added to ensure that the glycerol
kinase was active. Calibration by varying the amount of
glycerol kinase used demonstrated that the assay was linear
the volume of the original reaction. The column was rinsed
with 2.5 column volumes of 50% aqueous ethanol to remove
the unphosphorylated substrate, eluted with 4 column volumes
of 65 mM ammonium bicarbonate in 50% aqueous ethanol to
remove carboxylate anions, and then eluted with 25 column
volumes of 200 mM aqueous ammonium bicarbonate to remove
the phosphorylated substrate. Cations were removed from the
fraction containing unreacted substrate by adding Dowex
-
3
in its response over a range of activity from 2.2 × 10 to 85
-
3
-1
×
10 µmol min
.
ADP En d -P oin t Assa y of Glycer ol Kin a se Activity. To
a final volume of 500 µL of 0.10 M sodium phosphate buffer,
pH 7.6, containing 10 mM substrate (20 mM if substrate was
50W×8 (H
+
) resin and filtering. The solvent was removed by
rotary evaporation at 40 °C.
racemic), 4.7 mM ATP, 6 mM MgCl
2
-
, and 10 mM KCl was
Fractions containing phosphorylated product were combined
and concentrated by rotary evaporation at 40 °C. The residue
was dissolved in enough water to make a solution ap-
proximately 100 mM, and the pH was adjusted to 4.8 with
aqueous hydrogen chloride. The solution was sparged with
nitrogen, and acid phosphatase (18 units per mmol of phos-
phate ester) was added. The progress of the reaction was
1
added 25 µg (25 µL of a 1.0 mg mL solution) or 1.0 mg of
glycerol kinase. Aliquots (50 µL) were removed periodically
and assayed for ADP as follows: To a final volume of 2.290
mL of 0.10 M sodium phosphate buffer, pH 7.6, containing 10
units of lactate dehydrogenase, 0.9 mM PEP, 33 mM MgCl
18 mM KCl, and 0.2 mM NADH was added 50 µL of the
sample to be tested for ADP content. The absorbance at 334
nm was recorded (A
2
,
1
1
monitored by examining aliquots by H NMR. After the
o
), and 1 unit (20 µL) of pyruvate kinase
reaction was complete (∼48 h), the solution was desalted with
Dowex MR-3 resin and freed of solvent by rotary evaporation
at 40 °C.
was added. After a few minutes, the final absorbance (A) was
recorded, and the concentration of ADP was calculated from
-
1
-1
∆
A ) A
o
- A using ꢀ334 ) 6.18 mM cm as the molar
Deter m in a tion of En a n tiom er ic P u r ity. Enantiomeric
absorptivity of NADH. The assay for ADP was calibrated
using known amounts of ADP and was found to be accurate
purities were determined by
1
H NMR analysis of the products
formed by peracylation of the resolved diols and triols with
-
3
-1
37
over the range of 3.5 × 10 -3.5 × 10 µmol of ADP.
(R)-(+)-MTPA.
To 25.8 mg (0.11 mmol) of (R)-(+)-MTPA,
Tem p er a tu r e-Dep en d en t Activity a n d Sta bility of
Glycer ol Kin a se. Temperature-dependent activity of glycerol
kinase was measured using the continuous spectrophotometric
assay. Reactions were initiated by adding 25 µL of a solution
of glycerol kinase (0 °C) to a 3.00-mL reaction volume previ-
ously equilibrated at the appropriate temperature. Temper-
ature-dependent stability of glycerol kinase was measured by
incubating glycerol kinase at the appropriate temperature in
deoxygenated 100 mM phosphate buffer, pH 7.6, containing 1
mM 2-mercaptoethanol. Periodically, 25-µL portions were
24.8 mg (0.12 mmol) of dicyclohexylcarbodiimide, and 1.2 mg
(0.01 mmol) of 4-(dimethylamino)pyridine in 750 µL of dry
ether was added 0.05 mmol of diol or 0.03 mmol of triol. The
reaction mixture was stirred overnight, diluted with 10 mL of
ether, and filtered through glass wool. The ether was washed
successively with water, 5% acetic acid, water, and brine. The
ether was dried with MgSO and concentrated under reduced
4
pressure. If necessary, the crude product was dissolved in
1
acetone and filtered through a small plug of silica gel. If H
NMR analysis revealed signals due to the (+)-MTPA derivative
of only one enantiomer of diol or triol, the NMR sample was
spiked with 0.5 mol % of the (+)-MTPA derivative of the
appropriate racemic diol or triol. By this calibration, it was
(38) Hirschbein, B. L.; Mazenod, F. P.; Whitesides, G. M. J . Org.
Chem. 1982, 47, 3765-3766.

Kinetic Chiral Resolutions of 1,2-Diols
J . Org. Chem., Vol. 63, No. 12, 1998 4045
demonstrated in every case that a 0.25% diastereomeric
(R)-Glycer ol 1-P h osp h a te (1). In a 125-mL Erlenmeyer
impurityscorresponding to an ee for the diol or triol of
flask were combined 1.00 g (10.9 mmol) of glycerol, 60 mg
9
9.5%scould be detected.
Gen er a l P r oced u r e for Tw o-Sta ge Resolu tion s. Race-
(0.109 mmol) of ATP, 44 mg (0.218 mmol) of MgCl
mg (0.545 mmol) of KCl, 3.5 mg (0.054 mmol) of NaN
2
‚6H
2
O, 40
, 3.80
3
mic substrate was phosphorylated using glycerol kinase from
S. canus according to the General Procedure for One-Stage
Resolutions, except that only 79-87 mM PEP was used. When
µL (0.054 mmol) of 2-mercaptoethanol, 2.07 g (11.0 mmol) of
PEP, and 45 mL of deionized water. The solution was adjusted
to pH 8.0 with aqueous potasssium hydroxide, and the final
volume was adjusted to 50 mL. The solution was sparged with
nitrogen, and glycerol kinase (35 units) and pyruvate kinase
(29 units) were added. Periodically, the reaction was assayed
for PEP and pyruvate.
all of the PEP had been consumed, the reaction was diluted
-
and chromatographed on Dowex-2 × 8-100 (HCO
3
) resin as
described above. The fractions containing unphosphorylated
substrate were resubjected to enzymatic phosphorylation
conditions, except using only 11-28 mM PEP. When assays
for PEP and pyruvate indicated that conversion had leveled
off, the unreacted 1,2-diol was recovered by desalting and
evaporating the solvent (isolation method A) or by continuous
liquid-liquid extraction (isolation method B).
When all of the PEP had been consumed, the reaction
mixture was diluted with 40 mL of ethanol and applied to a
-
1
00-mL column of Dowex-2 × 8-100 (HCO
3
) resin. The
column was rinsed with 100 mL of 50% aqueous ethanol,
eluted with 400 mL of 65 mM ammonium bicarbonate in 50%
aqueous ethanol to remove carboxylate anions, and then eluted
with 500 mL of 400 mM aqueous ammonium bicarbonate to
recover glycerol 1-phosphate. The glycerol 1-phosphate solu-
Fractions from the original phosphorylation reaction con-
taining phosphorylated substrate were combined and adjusted
+
to pH e 3 using Dowex-50W × 8-100 (H ). The solution was
filtered, and the phosphate ester was hydrolyzed using either
alkaline phosphatase (hydrolysis method A) or acid phos-
phatase (hydrolysis method B).
+
tion was adjusted to pH 2 by addition of Dowex-50W × 8 (H ),
filtered, and concentrated by rotary evaporation at 40 °C to
yield 1.86 g (100%) of 1 as the free acid. 1H NMR showed this
Isola tion Meth od A. Dowex MR-3 resin (2 mol equiv of
exchangeable ions) was added to the aqueous solution of 1,2-
diol or triol obtained from the second stage of resolution or
the hydrolysis of phosphate ester. The mixture was stirred
with a stir bar for 30 min, filtered, and concentrated by rotary
evaporation at 40 °C to yield the free 1,2-diol.
Isola tion Meth od B. To the 12-mL aqueous solution of
diol or triol, obtained from the second stage of resolution or
from the hydrolysis of phosphate ester, was added 2 g of
sodium chloride. The solution was extracted with 35 mL of
ethyl acetate for 24 h, using a continuous liquid-liquid
extractor. The ethyl acetate was dried (MgSO ) and concen-
4
trated by rotary evaporation to yield the enantiomerically
enriched product.
Resolu tion of 1,2,4-Bu ta n etr iol. Resolution of 1,2,4-
butanetriol at 25 °C according to the General Procedure for
Two-Stage Resolutions (42.5% initial conversion, isolation
method A, and hydrolysis method A) gave D- and L-1,2,4-
butanetriol as reported in Table 2. Resolution of 1,2,4-
butanetriol (24 mmol) with glycerol kinase from Cellulomonas
sp. (48% initial conversion) gave D- and L-1,2,4-butanetriol in
material to be a 9:1 mixture of glycerol phosphate and pyruvic
acid. It was thus 95% pure by weight.
(
R)-2,3-O-Isop r op ylid en eglycer ol 1-P h osp h a te (2). To
1
.86 g of 1 (free acid, 95% pure by weight) in 15 mL of dry
methanol was added in portions 30 mL of dimethoxypropane.
After 60 h, 1H NMR analysis of an aliquot indicated 89%
conversion. Additional portions of dimethoxypropane failed
to further the conversion. Aqueous potassium hydroxide was
added to the solution to raise the pH to 10 (as indicated by
pH meter). Evaporation of the solvent under reduced pressure
1
gave crude 2: H NMR (D
6
2
2
O) δ 4.20 (m, 1H), 3.94 (dd, J )
.8, 8.6 Hz, 1H), 3.62 (dd, J ) 6.3, 8.6 Hz, 1H), 3.59-3.48 (m,
H), 1.25 (s, 3H), 1.18 (s, 3H).
(S)-1,2-O-Isop r op ylid en eglycer ol (3). To the crude 2
prepared above was added 110 mL of H
2
O and 22 mg (0.11
mmol) of MgCl O. The solution was sparged with nitro-
2
‚6H
2
gen, and alkaline phosphatase (64 units) was added. The
1
reaction was monitored by H NMR analysis of 100-µL
aliquots. After 24 h, the reaction was complete and was
transferred to a continuous liquid-liquid extractor, where it
4
3 and 37% yield and 93 and 60% ee, respectively.
was extracted with CHCl
3
for 13 h. The chloroform was dried
1
Resolu tion of 3-Ch lor o-1,2-p r op a n ed iol. Resolution of
-chloro-1,2-propanediol (18.1 mmol) at 25 °C according to the
(MgSO ) and evaporated to give 1.18 g (87% from 1): H NMR
4
3
δ 4.16-4.11 (m, 1H), 3.95 (dd, J ) 6.6, 8.2 Hz, 1H), 3.68 (dd,
J ) 6.5, 8.2 Hz, 1H), 3.60 (dd, J ) 4.0, 11.7 Hz, 1H), 3.50 (dd,
J ) 5.4, 11.6 Hz, 1H), 2.77 (br s, 1H), 1.34 (s, 3H), 1.28 (d, J
) 0.5 Hz, 3H); 13C NMR δ 109.2, 76.1, 65.6, 62.8, 26.5, 25.1.
General Procedure for Two-Stage Resolutions (218 units, 47%
initial conversion in 24 h, isolation method A, and hydrolysis
method B) gave D- and L-3-chloro-1,2-propanediol as reported
in Table 3. Resolution of 3-chloro-1,2-propanediol at 35 °C
gave essentially identical results.
Su p p or tin g In for m a tion Ava ila ble: Selected 1H, 13C,
Resolu tion of 3-F lu or o-1,2-p r op a n ed iol. Resolution of
31
and P NMR spectra of 1,2,4-butanetriol, 3-chloro-1,2-pro-
3
-fluoro-1,2-propanediol (2.26 mmol) at 35 °C according to the
panediol, 3-fluoro-1,2-propanediol, 3-butene-1,2-diol, their
General Procedure for Two-Stage Resolutions (436 units, 47%
initial conversion in 56 h, isolation method A, and hydrolysis
method B), except using 50 mM ATP, gave D- and L-3-fluoro-
1
-phosphate esters, and their (+)-MTPA perester derivatives,
1 13 31
with peak assignments. Selected H, C, and P NMR spectra
of 1, 2, 3, and the (+)-MTPA ester of 3, with peak assignments
1
,2-propanediol as reported in Table 3.
(3 pages). This material is contained in libraries on microfiche,
Resolu tion of 3-Bu ten e-1,2-d iol. Resolution of 3-butene-
,2-diol (2.84 mmol) at 35 °C according to the General
immediately follows this article in the microfilm version of the
journal, and can be ordered from the ACS; see any current
masthead page for ordering information.
1
Procedure for Two-Stage Resolutions (522 units, 47% initial
conversion in 25 h, isolation method B, and hydrolysis method
B) gave D- and L-butene-1,2-diol as reported in Table 3.
J O980122Y