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TECHNICAL FIELD OF THE INVENTION
The present invention relates to ketoaldonic acids having formed
stereogenic centers of R configuration, particularly octulosonic and
nonulosonic acids, and methods for synthesizing such sugars using sialic
acid aldolase.
BACKGROUND OF THE INVENTION
A major synthetic value of enzyme catalysis is its usually predictable
stereoselectivity. See, e.g., Whitesides et al., Angew. Chem. Int. Ed.
Engl., 24:617 (1985); Jones, Tetrahedron, 42:3351 (1986); Yamada et al.,
Angew. Chem. Int. Ed. Engl., 27:622 (1988); Wong, C-H., Science, 244:1145
(1989); Ohno et al., Org. React., 37:1 (1989); Chen et al., Angew. Chem.
Int. Ed. Engl., 28:695 (1989).
A change of stereoselectivity, however, may occur, though very unusual,
with different substrate structures, temperatures or solvents. See. e.g.,
Mohr et al., Helv. Chim. Acta, 66:2501 (1983); Sabbioni et al., J. Chem.
Soc. Chem. Commun., 236 (1984); Ohno et al., J. Am. Chem. Soc., 103:2405
(1983); Wang et al., J. Org. Chem., 53:3127 (1988); Lalonde et al., J. Am.
Chem. Soc., 103:2405 (1981); Wang et al., J. Org. Chem., 53:2323 (1988);
Pham et al., J. Am. Chem. Soc., 111:1935 (1989); Keinan et al., J. Am.
Chem. Soc., 108:162 (1986); Sakurai et al., J. Am. Chem. Soc., 110:7236
(1988); Fitzpatrick et al., J. Am. Chem. Soc., 113:3166 (1991). These
selectivity changes are often not very significant, with some exceptions
where the enantioselectivity is inverted.
In the case of enzymatic aldol reactions, the diastereofacial selectivity
for the aldehyde component is often consistent and completely controlled
by the enzyme as documented by numerous reactions catalyzed by
fructose-1,6-diphosphate (FDP) aldolase or N-acetylneuraminic acid (or
sialic acid) aldolase (EC 4.1.3.3). In most cases, the "D" isomer of an
.alpha.-substituted aldehyde reacts faster than the "L" isomer, both with
si-facial selectivity. The Cram-Felkin mode of attack on the "D" aldehyde
is therefore proposed for the transition state of the FDP aldolase
reaction and the anti-Cram-Felkin mode for the sialic acid aldolase
reaction. See. e.g., Toone et al., Tetrahedron, 45:5365 (1989); Bednarski
et al., J. Am. Chem. Soc., 111:627 (1989); Straub et al., J. Org. Chem.,
55:3926 (1990); Durrwachter et al., J. Org. Chem., 53:4175 (1988); von der
Osten et al., J. Am. Chem. Soc., 111:3924 (1989); Kajimoto et al., J. Am.
Chem. Soc., 113:6187 (1991); Auge et al., New J. Chem., 12:733 (1988).
Because of the stereoselectivity of enzymes such as aldolases that
participate in the metabolism of carbohydrates, it is extremely difficult
to design and make new carbohydrates that can be used to study
carbohydrate metabolism. There is a need for such synthetic compounds for
use as experimental tools in elucidating the molecular character of the
numerous and varied pathways involved in carbohydrate anabolism and
catabolism.
Of particular relevance to the present invention is the sugar,
N-acetylneuraminic acid (NeuAc) or sialic acid. NeuAc is an integral
component of most cells and is believed to play a major role in imparting
electrical charge characteristics to such cells. Further, NeuAc-like
compounds such as the eight and nine-carbon sugar moieties KDO and KDN are
major constituents of non-mammalian tissues.
N-Acetylneuraminic Acid (NeuAc) aldolase, also commonly referred to as
sialic acid aldolase is a type I aldolase known to form an enamine
intermediate with pyruvate, which reversibly reacts with the second
substrate N-acetylmannosamine to give NeuAc. See, e.g., Deijl et al.,
Biochem. Biophys. Res. Commun., 111:668 (1983); and Shukla et al., Anal.
Biochem., 158:158 (1986).
NeuAc aldolase is known to accept many aldoses as acceptor substrates. In
all previously known aldol condensation reactions with such acceptor
substrates, the eneamine intermediate approaches the si face of the
incoming aldehyde substrate to form a new stereogenic center of S
configuration. Anti-Cram-Felkin attack is generally observed for good
chiral aldehyde substrates and Cram-Felkin attack is observed for weak
substrates. In both cases, a si-facial selectivity was observed. See.
e.g., Auge et al., New J. Chem., 12:733 (1988); and Auge et al.,
Tetrahedron, 46:201 (1990).
Based on such current knowledge concerning aldolase stereoselectivity,
therefore, NeuAc aldolase is considered to be useful only for the
production of D-sugars having S configuration. As is disclosed
hereinafter, NeuAc aldolase has now unexpectedly been found to be capable
of the production of certain ketoaldonic acids having a formed stereogenic
center of R configuration.
BRIEF SUMMARY OF THE INVENTION
In one aspect, the present invention contemplates ketoaldonic acids, and
particularly octulosonic or nonulosonic acids, having a formed stereogenic
center of R configuration. A contemplated ketoaldonic acid is a sialic
acid aldolase-catalyzed condensate of pyruvate and an acceptor substrate
aldose for that enzyme. The ketoaldonic acid contains a stereogenic center
of the R configuration other than present in the acceptor substrate
aldose. Exemplary acceptor substrate aldoses include D-gulose and a five
or six carbon L-configured acceptor substrate aldose other than
L-arabinose, which form an octulosonic or nonulosonic acid. In another
aspect, the present invention contemplates a compound having the Formulae
I-VIII, below, in which compounds of Formulae V-VIII, are particularly
preferred.
##STR1##
Although L-arabinose forms an octulosonic acid with a new S rather than R
stereogenic center in the above sialic acid aldolase-catalyzed
condensation with pyruvate, the product of that reaction,
3-deoxy-L-manno-octulosonic acid (L-KDO), a compound of Formula IX, below
is new and unexpectedly produced.
##STR2##
One aspect contemplates a process for preparing a ketoaldonic acid having a
new stereogenic center of the R configuration, relative to the aldose
starting material. This process comprises the steps of:
(a) admixing in an aqueous solvent (i) pyruvate (typically in excess), (ii)
a catalytic amount of sialic acid aldolase and (iii) an acceptor substrate
aldose for that enzyme, such as D-gulose or a five or six carbon
L-configured acceptor substrate aldose other than L-arabinose, to form a
reaction mixture; and
(b) maintaining the reaction mixture for a time period and under biological
reaction conditions sufficient for condensation of the pyruvate with the
acceptor substrate aldose and the formation of a ketoaldonic acid product.
That product is preferably recovered. Use of D-gulose or a five or six
carbon L-configured acceptor substrate aldose forms an octulosonic or
nonulosonic acid.
In another process aspect, the present invention contemplates a process for
synthesizing a compound of Formulae I-VIII comprising the steps of:
(a) admixing pyruvate (typically in excess), in the presence of a catalytic
amount of sialic acid (NeuAc) aldolase, with an acceptor substrate
L-rhamnose, L-mannose, L-talose, D-gulose, 2-deoxy-L-glucose,
2-deoxy-L-rhamnose, N-acetyl-L-mannosamine or 2-azido-2-deoxy-L-mannose,
respectively, (the latter four aldoses being preferred) to form a reaction
mixture; and
(b) maintaining the reaction mixture for a time period and under biological
reaction conditions sufficient for condensation of the pyruvate with the
acceptor substrate and formation of a compound of Formulae I-VIII, above.
In a preferred embodiment, the synthetic method further comprises
recovering the synthesized compound of Formulae I-VIII.
In another embodiment, the invention contemplates an enhanced process for
synthesizing any ketoaldonic acid such as an octulosonic or nonulosonic
acid like sialic acid. In accordance with this process, an excess of
pyruvate, e.g. about 2 to about 10 fold excess, and an acceptor substrate
aldose for sialic acid aldolase (EC 4.1.3.3) and a catalytic amount of
that aldolase are admixed in an aqueous solvent to form a reaction
mixture. That reaction mixture is maintained for a time period and under
biological reaction conditions sufficient for the condensation of the
pyruvate with the acceptor substrate aldose to form a ketoaldonic acid
such as an octulosonic or nonulosonic acid where a five or six carbon
acceptor substrate aldose is used.
The enzyme pyruvate decarboxylase is then added and the resulting
composition is maintained as above until the excess pryuvate is
decomposed. This addition preferably occurs after denaturation of sialic
acid aldolase as by acidification. The added pyruvate decarboyxlase can be
added as the purified enzyme or as whole acid-free baker's yeast cells.
The ketoaldonic acid is thereafter recovered by standard procedures that
include a separation from the yeast cells, where used.
DETAILED DESCRIPTION OF THE INVENTION
A. Compounds
The present invention contemplates a ketoaldonic acid such as octulosonic
and nonulosonic acids. A contemplated ketoaldonic acid is a sialic acid
aldolase-catalyzed condensate of pyruvate and an acceptor substrate aldose
for that enzyme. The ketoaldonic acid contains a stereogenic center of the
R configuration other than present in the acceptor substrate aldose.
Exemplary acceptor substrate aldoses include D-gulose and a five or six
carbon L-configured acceptor substrate aldose other than L-arabinose,
which form an octulosonic or nonulosonic acid. Exemplary octulosonic and
nonulosonic acid compounds have the Formulae I, II, III, IV, V, VI, VII or
VIII, below, with compounds of Formulae V-VIII being preferred.
##STR3##
Formula I defines 3,9-dideoxy-L-glycero-L-galactononulosonic acid. Given
that 3,9-dideoxy-D-glycero-D-galactononulosonic acid defines D-9-deoxy
KDN, the compound of Formula I can also be referred to as L-9-deoxy KDN.
Formula II defines 3-deoxy-L-glycero-L-galactononulosonic acid, which can
also be referred to as L-KDN.
The compounds of Formulae I-VIII have a .sup.5 C.sub.2 conformation as
evidenced by the adjacent transaxial coupling of protons at the carbon
atoms at positions 3, 4 and 5. Further, the compounds of Formulae I-VIII
have a formed stereogenic center of R configuration.
The compounds of Formulae I-VIII synthesized in accordance with the method
described herein have a formed stereogenic center of R configuration that
is created via the re attack of pyruvate on the acceptor substrate. This
re attack and resulting R configuration are surprising and unexpected in
view of the published literature. In all previously known aldol
condensation reactions using NeuAc aldolase (EC 4.1.3.3), the attack is on
the si face of the acceptor substrate and the resulting condensation
product has a formed stereogenic center of S configuration. See. e.g.,
Auge et al., New J. Chem., 12:733 (1988); Auge et al., Tetrahedron, 46:201
(1990); and Kim et al., J. Am. Chem. Soc., 110:6481 (1988).
Thus, where N-acetyl D-mannosamine (D-ManNAc), D-mannose (Man),
4-deoxy-D-Man, 2-deoxy-2-phenyl-D-Man, 6-O-Ac-D-ManNAc, 6-O-Ac-D-Man,
2-deoxy-D-glucose, 6-deoxy-6-N.sub.3 -D-ManNAc, 6-deoxy-6-F-D-ManNAc,
4,6-dideoxy-4,6-F.sub.2 -D-talose, D-glucose (D-Glc), D-altrose,
2-deoxy-D-galactose, D-glucosamine (GlcNAc), D-lyxose, D-talose,
D-arabinose, L-arabinose or 2-deoxy-ribose was reacted with pyruvate and a
catalytic amount of NeuAc aldolase, the resulting condensation products
were all found to have formed stereogenic centers of S configuration
resulting from a si facial attack. Wong, C-H., Microbial Aldolases in
Enzymes in Carbohydrate Synthesis ed. by Bednarski and Simon, American
Chemical Society, ACS Symposium Series No. 466 (1991).
The re attack and resulting R configuration where L-rhamnose, L-mannose,
L-talose, D-gulose, 2-deoxy-L-glucose, 2-deoxy-L-rhamnose,
N-acetyl-L-mannosamine and 2-azido-2-deoxy-L-mannose were used as the
acceptor substrate are even more surprising and unexpected because such
reversal of stereoselectivity was not observed with all L-isomeric
acceptor substrates. Where L-glucose or L-fucose were reacted with
pyruvate in the presence of NeuAc aldolase, no aldol condensation product
was formed. Wong, C. -H., Microbial Aldolases in Enzymes in Carbohydrate
Synthesis ed. by Bednarski and Simon, American Chemical Society, ACS
Symposium Series No. 466 (1991).
Another new and useful compound that has a newly formed S rather than R
stereogenic center is L-KDO, a compound of Formula IX, below.
##STR4##
B. Synthetic Process
Another aspect of the present invention contemplates an aldol condensation
process of synthesizing a ketoaldonic acid such as an octulosonic or
nonulosonic acid that are exemplified by the compounds of Formulae I-VIII.
The formed ketoaldonic acid such as an octulosonic or nonulosonic acid
contains a new stereogenic center relative to the starting reactants;
i.e., not present in the acceptor substrate aldose reactant, and that new
stereogenic center has the R configuration. This process comprises the
steps of admixing in an aqueous solvent pyruvate (typically in excess), a
catalytic amount of sialic acid aldolase and an acceptor substrate aldose
for that enzyme such as D-gulose or a five or six carbon L-configured
acceptor substrate aldose other than L-arabinose to form a reaction
mixture. The reaction mixture is maintained for a time period and under
biological reaction conditions sufficient to condense the pyruvate and
acceptor substrate aldose and form a desired octulosonic or nonulosonic
acid.
In accordance with another aldol condensation process, pyruvate (typically
in excess) is admixed in an aqueous solvent in the presence of a catalytic
amount of sialic acid (NeuAc) aldolase, with an acceptor substrate as
named before to form a reaction mixture. The reaction mixture is
maintained for a time period and under biological reaction conditions
sufficient to condense the pyruvate and acceptor substrate and form a
compound of Formulae I, II, III, IV, V, VI, VII or VIII, as appropriate to
the before-noted acceptor substrate, with formation of a compound of
Formulae V-VIII being preferred.
The structure of the acceptor substrate dictates the structure of the
synthesized aldol condensation product. Where the acceptor substrate is
L-rhamnose, the compound of Formula I is synthesized. Where the acceptor
substrate is L-mannose, the compound of Formula II is synthesized. Where
the acceptor substrate is L-talose, the compound of Formula III is
synthesized. Where the acceptor substrate is D-gulose, the compound of
Formula IV is synthesized. With 2-deoxy-L-glucose as acceptor substrate,
the compound of Formula V is synthesized in a 5:1 ratio to the si face
adduct (axial 3-hydroxy group). 2-Deoxy-L-rhamnose is the acceptor
substrate for the compound of Formula VI. Where N-acetyl-L-mannosamine is
the acceptor substrate, a compound of Formula VII is synthesized. Where
2-azido-2-deoxy-L-mannose is the acceptor substrate, a compound of Formula
VIII is formed in a 4.5:1 ratio to the si face adduct (axial 3-hydroxy
group).
Pyruvate is readily available from commercial sources (Sigma Chemical Co.,
St. Louis, Mo.). A preferred formulation of pyruvate is sodium pyruvate.
Pyruvate is typically used in an amount in excess of the one mole required
for the reaction to drive the reaction to completion. A 2- to about
10-fold excess is usually used.
L-Mannose, L-rhamnose, L-talose and D-gulose are also available from Sigma
Chemical Co. 2-Deoxy-L-glucose (Compound 5), 2-deoxy-L-rhamnose (Compound
6), N-acetyl-L-mannosamine (Compound 11) and 2-azido-2-deoxy-L-mannose
(Compound 9a) are synthesized as discussed hereinafter.
Highly stable NeuAc aldolase in a free or immobilized form is readily
available. See. e.g., Auge et al., New J. Chem., 12:733 (1988); Auge et
al., Tetrahedron, 46:201 (1990); and Kim et al., J. Am. Chem. Soc.,
110:6481 (1988).
As used herein, the phrase "catalytic amount" means that amount of NeuAc
aldolase at least sufficient to catalyze, in a non-rate limiting manner,
the condensation of pyruvate and acceptor substrate to product. More than
a catalytic amount can be used.
The catalytic amount of NeuAc aldolase varies according to the specific
activity of NeuAc aldolase
The reaction time varies with the temperature and the activity of the NeuAc
aldolase. Where the NeuAc aldolase has an activity of about 10 Units, the
temperature is about 37.degree. C., and the concentration of acceptor
substrate is about 1 mM, the reaction time is about 48 hours (See Examples
1A and 1B hereafter).
The synthetic method of the present invention can further include
recovering a synthesized (formed) ketonaldonic acid such as a compound of
Formulae I-VIII. Recovering comprises isolating the synthesized compound
from the reaction mixture. Means for isolating a synthesized ketoaldonic
acid such as a compound of Formulae I-VIII include gel filtration, column
chromatography, paper chromatography, affinity chromatography, extraction,
crystallization, precipitation and the like.
In a preferred embodiment, isolation is accomplished by applying a reaction
mixture containing about 1 mM acceptor substrate to an anion exchange
chromatography column of Dowex.RTM. 1.times.8-100 (HCOO.sup.- or
HCO.sub.3.sup.- form; 30.times.2 or 20.times.2.5 cm) and eluting a
compound of Formulae I-VIII with formic acid (0.2M) or bicarbonate
(0.fwdarw.0.2M), respectively. Product-containing fractions are then
pooled, freeze-dried and deionized with Dowex.RTM. W-X8 [H.sup.+ ], and
freeze dried again. The pure compounds are finally obtained by bio-gel
chromatography. Where such an embodiment is used for isolation, a compound
of Formula I can typically be recovered with a yield of about 80 percent
(See Example 1A).
The reaction rate of the method of the present invention is within a factor
of about 10 of the reaction rate of NeuAc aldolase-catalyzed condensation
of pyruvate with acceptor substrates having an enantiomeric configuration
(i.e., D-rhamnose, D-mannose, D-talose, L-gulose, 2-deoxy-D-glucose,
2-deoxy-D-(Units/mg), the concentration of acceptor substrate as well as
biological reaction conditions such as temperature, time and pH value.
Means for determining the catalytic amount of NeuAc aldolase under
preselected substrate concentrations and biological reaction conditions
are well known to those of skill in the art. Typical amounts range from
about 5 to about 20 Units (U) per millimole (mmol) of acceptor substrate,
with about 10 to about 15 U/mmol typically being used.
Each ingredient is admixed with each of the other ingredients in a suitable
aqueous solvent to form a reaction mixture. The reaction mixture is
maintained under biological reaction conditions (temperature, pH, solvent
osmolality, ionic composition and ambient pressure) for a period of time
sufficient to condense the substrate acceptor and pyruvate to form a
compound of Formula I, II, III, IV, V, VI, VII or VIII. A compound of
Formula IX can be similarly prepared using L-arabinose as the acceptor
substrate aldol.
Temperature can range from about 15.degree. C. to about 40.degree. C.
Preferably, temperature is from about 20.degree. C. to about 40.degree. C.
and, more preferably from about 25.degree. C. to about 37.degree. C.
The pH value of the solvent and for maintenance can range from about 6.0 to
about 11.0. Preferably, the pH value is from about 6.0 to about 8.5 and,
more preferably from about 7.0 to about 7.5. The pH value is maintained by
buffers in the aqueous solvent. A preferred buffer is potassium phosphate.
The aqueous solvent preferably further comprises an anti-oxidant. A
preferred anti-oxidant is a sulfur-containing reducing agent such as a
mercaptan (thiol). Exemplary mercaptans are mercaptoethanol and
dithiothreitol. rhamnose, N-acetyl-D-mannosamine or
2-azido-2-deoxy-D-mannose). The substantial similarity of the reaction
rates with D- and L-configured acceptor substrates is surprising and
unexpected. With aldolases other than NeuAc aldolase (i.e.,
fructose-1,6-diphosphate aldolase), the reaction rate is markedly faster
with D-configured acceptor substrates than with L-configured acceptor
substrates. See. e.g., Toone et al., Tetrahedron, 45:5365 (1989);
Bednarski et al., J. Am. Chem. Soc., 111:627 (1989); Straub et al., J.
Org. Chem., 55:3926 (1990); Durrwachter et al., J. Org. Chem., 53:4175
(1988); von der Osten et al., J. Am. Chem. Soc., 111:3924 (1989); Kajimoto
et al., J. Am. Chem. Soc., 113:6187 (1991).
An improved process for the synthesis of a ketoaldonic acid such as an
octulosonic or nonulosonic acid is contemplated in another embodiment of
the invention. This improved process is useful regardless of which
enantiomer is prepared; i.e., for any ketoaldonic acid.
Here, an excess of pyruvate, typically about a 2- to about 10-fold excess,
is admixed in an aqueous solvent with a catalytic amount of sialic acid
aldolase and an acceptor substrate for that enzyme to form a reaction
mixture. Specific exemplary D- and L-acceptor substrates are noted before.
The reaction mixture is maintained for a time period and under biological
reaction conditions sufficient to condense the pyruvate and acceptor
substrate to form an octulosonic or nonulosonic acid.
The reaction conditions utilized in this embodiment are as discussed
previously. Additional aldose acceptor substrates for sialic acid aldolase
are discussed before and are discussed in Wong, C-H., Microbial aldolases
in Enzymes in Carbohydrate
1. 3-Deoxy-D-glycero-L-altro-nonulosonic acid (Compound of Formula III)
[a].sup.25.sub.D +32.1.degree. (c 0.41, H.sub.2 O); .sup.1 H NMR (D.sub.2
O, HOD=4.80 ppm) .delta.3.98 (1H, ddd, J.sub.8-9a =7.0 Hz, J.sub.8-9a =5
Hz, J.sub.8-7 =2.5 Hz, H-8), 3.95 (1H, ddd, J.sub.4-3ax =12.5 Hz,
J.sub.4-5 =9.0 Hz, J.sub.4-3eq =5.0 Hz, H-4), 3.93 (1H, dd, J.sub.7-6 =3.5
Hz, J.sub.7-8 =2.5 Hz, H-7), 3.85 (1H, dd, J.sub.6-5 =9.5 Hz, J.sub.6-7
3.5 Hz, H-6), 3.69 (1H, dd, J.sub.9b-9a =11.5 Hz, J.sub.9b-8 =5.0 Hz,
H-9b), 3.65 (1H, dd, J.sub.9a-9b =11.5 Hz, J.sub.9a-8 =7 Hz, H-9a), 3.56
(1H, t, J.sub.5-6 =J.sub.5-4 =9.5 Hz, H-5), 2.21 (1H, dd, J.sub.3eq-3ax
=12.5 Hz, H-3ax). .sup.13 C NMR (D.sub.2 O+CD.sub.3 CN) .delta.176.9,
96.7, 74.4, 96.7, 74.4, 72.2, 71.6, 71.1, 69.4, 63.2, 39.3 HRMS (FAB)
calcd for C.sub.9 H.sub.16 O.sub.9 Na (M+Na.sup.+) 291.0692, found
291.0698.
2. 3,5-Dideoxy-L-glycero-L-galacto-nonulosonic acid (Compound of Formula V)
[a].sup.25.sub.D +35.80.degree. (c 0.27, H.sub.2 O); .sup.1 H NMR (D.sub.2
O) .delta.4.13 (1H, m, H-4) 4.10 (1H, dt, J.sub.6-5ax =12.0 Hz,
J.sub.6-5eq =J.sub.6-7 =2.0 Hz, H-6), 3.77 (1H, dd, J.sub.9a-9b =12.0 Hz,
J.sub.9a-8 =3.0 Hz, H-9a), 3.72 (1H, ddd, J.sub.8-7 =9.0 Hz, J.sub.8-9a
=3.0 Hz, H-8), 3.56 (1H, dd, J.sub.9b-9a =12.0 Hz, J.sub.9b-8 =6.5 Hz,
H-9b), 3.41 (1H, dd, J.sub.7-8 =3.5 Hz, J.sub.7-6 =1.5 Hz, H-7), 2.03 (1H,
ddd, J.sub.3eq-3ax =12.5 Hz, J.sub.3eq-4 =4.5 Hz, J.sub.3eq-5eq =1.5 Hz,
H-3eq), 1.82 (1H, b dt, J.sub.5eq-5ax =12.0 Hz, J.sub.5eq-6 =J.sub.5eq-4
=2.0 Hz, H-5eq), 1.56 (1H, dt, J.sub.5ax-4 =11.5 Hz, J.sub.5ax-6
=J.sub.5ax-5eq =12.0 Hz, H-5ax) 1.49 (1H, t, J.sub.3ax-3eq =J.sub.3ax-4
=12.0 Hz, H-3ax). .sup.13 C NMR (D.sub.2 O+CD.sub.3 CN) .delta.177.8,
97.4, 73.2, 71.3, 68.6, 64.5, 63.5, 40.3, 35.7. HRMS (FAB) calcd for
C.sub.9 H.sub.16 O.sub.8 Na (M+Na.sup.+) 275.0743, found 275.0751.
3. 3,5,9-Trideoxy-L-glycero-L-galacto-nonulosonic acid (Compound of Formula
VI)
[a].sup.25.sub.D +22.1.degree. (c 0.19, H.sub.2 O) .sup.1 H NMR (D.sub.2 O)
.delta.4.12 (1H, ddt, J.sub.4-3eq =5.0 Hz, J.sub.4-5eq =2.5 Hz,
J.sub.4-3ax =J.sub.4-5ax =12.0 Hz, H-4), 4.07 (1H, dt, J.sub.6-5ax =12.0
Hz, J.sub.6-5eq =J.sub.6-7 =2.5 Hz, H-6), Synthesis, ed. by Bednarski and
Simon, American Chemical Society, ACS Symposium Series No. 466 (1991).
Non-substrates are also discussed. Whether an aldose is an acceptor
substrate for this enzyme can be readily ascertained by admixture of
excess pyruvate, the enzyme and potential acceptor substrate aldose as
discussed herein, followed by maintenance as discussed herein, e.g. 2-3
days. Analysis of the reaction mixture as by thin layer chromatography
indicates whether an octulosonic or nonulosonic acid has been formed.
After the ketoaldonic acid has formed, a catalytic amount of pyruvate
decarboxylase is admixed to the aqueous solvent medium and the resulting
admixture is maintained as before, but using a pH value of about 5.5 to
about 6.5, until the pyruvate has decomposed. This step is utilized
because it has been found that the excess pyruvate utilized in the
condensation reaction interferes with recovery of the ketoaldonic acid
product. Thus, for example, in previously reported procedures for the
isolation of enzymatically produced sialic acid [Kim et al., J. Am. Chem.
Soc., 110:6481 (1988) and Liu et al., J. Am. Chem. Soc., 114:3901 (1992)]
a repetitive extraction of pyruvic acid with ethyl acetate under acidic
conditions was used. Under those conditions, the pyruvate exists mainly as
the hydrated form in the aqueous phase where its presence makes isolation
of sialic acid difficult.
The pyruvate decarboxylase (EC 4.1.1.1) is preferably admixed after
denaturation of the sialic acid aldolase. That enzyme is conveniently
denatured by adjusting the solution pH value to about 2 and maintaining
the pH value for about one hour.
The pyruvate decarboxylase can be provided as a purified enzyme as is
available from Sigma Chemical Co. at $80.00 per 100 Units. That enzyme can
also be provided by culturing baker's yeast cells. Baker's yeast cells are
much less costly, e.g. $16.00 per 500 g from Sigma. The baker's yeast
cells must be acid-free, which can be accomplished by washing the cells as
described hereinafter.
After the excess pyruvate has been decomposed, the ketoaldonic acid such as
octulosonic or nonulosonic acid is recovered by usual techniques such as
by ion exchange chromatography or crystallization. Where baker's yeast
cells are used as the source of the pyruvate decarboxylase, the cells are
removed as by centrifugation prior to use of ion exchange or other
techniques. Exemplary procedures for recovery of the octulosonic or
nonulosonic acids are illustrated hereinafter.
The following Examples illustrate particular embodiments of the present
invention and are not limiting of the specification and claims in any way.
EXAMPLE 1
Syntheses
A. Synthesis of 3,9-Dideoxy-L-glycero-L-galactononulosonic acid (L-9-deoxy
KDN) (Compound of Formula I)
A 0.1 M solution of L-rhamnose (1 mmol) in a 0.05 M potassium phosphate
buffer, pH 7.2, containing 0.01 M dithiothreitol, sodium pyruvate (3
equivalent) and 10 U of NeuAc aldolase was incubated at 37.degree. C.
(total volume=10 mL) for two days. The reaction was monitored by TLC
(PrOH/water=7:3 v/v).
The title product was isolated by anion exchange chromatography on
Dowex.RTM. 1.times.8-100 (HCOO.sup.- form; 30.times.2 cm) using a gradient
of formic acid (0.2M) as eluant. Fractions containing the product were
pooled and freeze-dried. Yield 200 mg (80 percent).
.sup.1 H-NMR (500 MHz, D.sub.2 O) .delta.1.08 (d, J.sub.8,CH3 =6.5
CH.sub.3), 1.62 (dd, J.sub.3ax,3eq =13.3 Hz, J.sub.3ax,4 =11.5 Hz,
H.sub.3ax) 2.06 (dd, J.sub.3eq,4 =6.7 Hz, H.sub.3eq), 3.39 (t, J.sub.4,5
=J.sub.5,6 =9.5 Hz, H-5), 3.675 (dd, J.sub.6,7 =0.8 Hz, J.sub.7,8 =8.2 Hz,
H-7), 3.66 (dd, H-8), 3.8 (dd, H-6), 3.76-3.83 (ddd, H-4). .sup.13 C-NMR
(125 MHz, reference CH.sub.3 CN 1.6), 175.3800 (C-1), 96.1195 (C-2),
72.7438 (C-8), 72.3426 (C-6), 70.9565 (C-5), 69.3967 (C-7), 67.4548 (C-4),
39.3253 (C-3), 19.8777 (CH.sub.3); [a].sub.D.sup.20 +60.degree. (c 1.2,
water); HRMS for C.sub.9 H.sub.15 O.sub.8 calcd. 253.0923, found 253.0923.
B. Synthesis of 3-Deoxy-L-glycero-L-galactononulosonic acid (L-KDN)
(Compound of Formula II)
A 0.1 M solution of L-mannose (1 mmol) in a 0.05 M potassium phosphate
buffer, pH 7.2, containing 0.01 M dithiothreitol, sodium pyruvate (3
equivalent) and 10 U of NeuAc aldolase was incubated at 37.degree. C.
(total volume=10 mL) for two days. The reaction was monitored by TLC
(PrOH/water=7:3 v/v).
The title product was isolated by anion exchange chromatography on
Dowex.RTM. 1.times.8.times.100 (HCOO.sup.- form; 30.times.2 cm) using a
gradient of formic acid (0.2M) as eluant. Fractions containing the product
were pooled and freeze-dried. Yield 200 mg (80 percent).
The physical data (.sup.1 H, .sup.13 C-NMR and HRMS) were identical to the
reported values of D-KDN except for the specific rotation
[[.alpha.].sub.D.sup.20 -60.degree. (c 1.2, H.sub.2 O)].
C. Synthesis of Compounds of Formulae III-IX
The compounds of Formulae III-IX were synthesized in accordance with the
procedures of A and B, above. 3.86 (1H, qd, J.sub.8-7 =J.sub.8-9 =6.5 Hz,
H-8), 3.32 (1H, dd, J.sub.7-8 =6.5 Hz, J.sub.7-6 =3.0 Hz, H-7), 2.06 (1H,
ddd, J.sub.3eq-3ax =12.0 Hz, J.sub.3 eq-4=5.0 Hz, J.sub.3eq-5 =2.0 Hz,
H-3eq), 1.85 (1H, dt, J.sub.5eq-5ax =12.0 Hz, J.sub.5eq-6 =J.sub.5eq-4
=2.5 Hz, H-5eq), 1.53 (1H, q, J.sub.5ax-4 =J.sub.5ax-5 =J.sub.5ax-5eq 12.0
Hz, H.sub.5ax), 1.51 (1H, t, J.sub.3ax-3eq =J.sub.3ax-4 =12.0 Hz, H-3ax),
1.18 (d, J.sub.9-8 =6.5 Hz, H-9). .sup.13 C NMR (D.sub.2 O+CD.sub.3 CN)
177.8 97.3, 77.2, 69.3, 67.5, 64.6 40.3, 35.8, 18.6. HRMS (FAB) calcd for
C.sub.9 H.sub.16 O.sub.7 Na (M+Na.sup.+) 259.0794, found 259.0799.
4. 5-Acetamido-3,5-dideoxy-L-glycero-.beta.-L-galacto-2-nonulosonic acid
(N-Acetyl-L-neuraminic acid) (Compound of Formula VII)
[a].sup.25.sub.D +27.6.degree. (c 0.17, H.sub.2 O [(lit..sup.9 for d-NeuAc:
[a].sup.21.sub.D -29.degree. (H.sub.2 O)]; .sup.1 H NMR (D.sub.2 O)
.delta.4.03 (1H, ddd, J.sub.4-3ax =12.0 Hz, J.sub.4-5 =10.0 Hz,
J.sub.4-3eq =5.5 Hz, H-4), 4.01 (1H, dd, J.sub.6-5 =10.0 Hz, J.sub.6-7
=1.0 Hz, H-6), 3.90 (1H, t, J.sub.5-6 =J.sub.5-4 =10.0 Hz, H-5), 3.82 (1H,
dd, J.sub.9a-9b =12.0 Hz, J.sub.9a-8 =2.5 Hz, H-9a), 3.73 (1H, ddd,
J.sub.8-7 =9.0 Hz, J.sub.8-9b =6.0 Hz, J.sub.8-9a =2.5 Hz, H.sub.8), 3.60
(1H, dd, J.sub.9b-9a =12.0 Hz, J.sub.9b-8 =6.0 Hz, H-9b), 3.51 (1H, dd,
J.sub.7-8 =9.0 Hz, J.sub.7-6 =1.0 Hz, H-7), 2.26 (1H, dd, J.sub.3eq-3ax
=12.0 Hz, J.sub.3eq-4 =5.5 Hz, H-3eq), 2.03 (3H, s, acetyl), 1.84 (1H, t,
J.sub.3ax-3eq =J.sub.3ax-4 =12.0 Hz, H-3ax). .sup.13 C NMR (D.sub.2
O+CD.sub.3 CN) .delta.175.3 174.1, 95.9, 70.8, 70.6, 68.7, 67.2, 63.6,
52.5, 39.4, 22.5. Its .sup.1 H and .sup.13 C NMR spectra were identical
with those of an authentic D-NeuAc from Pfanstiehl Co. HRMS (FAB) calcd
for C.sub.11 H.sub.18 NO.sub.9 Na (M+Na.sup.+) 331.2572, found 331.2579.
5. 3-Deoxy-L-manno-octulosonic Acid (L-KDO)(Compound of Formula IX)
The specific rotation and spectral data were obtained after L-KDO had been
converted to its ammonium salt: [.alpha.].sup.25.sub.D -37.2.degree. (c
0.68, H.sub.2 O) [for D-KDO:[.alpha.].sup.27.sub.D +42.3.degree. (c 1.7,
H.sub.2 O) Unger, Adv. Charbohydr. Chem. Biochem., 381:323 (1981);
authentic sample from Sigma [.alpha.].sup.25.sub.D +40.1.degree. (c 2.1,
H.sub.2 O)]. Since KDO has an axial 5-OH group, it is known that it exists
as a mixture of pyranose and furanose forms, and readily cyclizes to the
corresponding lactone. The .sup.1 H and .sup.13 C NMR data of the
predominant form are: .sup.1 H MMR (D.sub.2 O) .delta.4.03 (1H, ddd,
J.sub.4-3ax =13.0 Hz, J.sub.4-3eq =5.5 Hz, J.sub.4-5 =3.0 Hz, H-4), 4.00
(1H, m, H-5) 3.87 (1H, dt, J.sub.7-8b =3.0 Hz, J.sub.7-6 J.sub.7-8a =5.5
Hz, H-7), 3.84 (1H, m, H-6), 3.78 (1H, dd, J.sub.8b-8a =12.0 Hz,
J.sub.8b-7 =3.0 Hz, H-8b), 3.60 (1H, dd, J.sub.8a-8b =12.0 Hz, J.sub.8a-7
=5.5 Hz, H-8a), 2.00 (1H, t, J.sub.3ax-3eq =J.sub.3ax-4 =13.0 Hz, H-3ax),
1.86 (1H, dd, J.sub.3eq-3ax =13.0 Hz, J.sub.3eq-4 =5.5 Hz, H-3eq). .sup.13
C NMR (D.sub.2 O+CD.sub.3 CN) .delta.177.3, 96.8, 72.0, 71.3, 67.0, 66.6,
63.4, 34.0. .sup.1 H and .sup.13 C NMR spectra were identical with those
of D-KDO from Sigma. HRMS (FAB) calcd for C.sub.8 H.sub.14 O.sub.8
Na(M+Na+) 261.0586, found 261.0591.
D. Benzyl 2-deoxy-.beta.-D-glucopyranoside (Compound 1)
2-deoxy-D-glucose (Compound 1a, 2.5 g, 15.2 mmol) was dissolved in benzyl
alcohol (20 mL), and then Dowex.RTM. 50W-X8 (H.sup.+ form, 3.5 g) was
added. The reaction solution was stirred at 60.degree. C. for 24 hours.
Then the resin was filtered off, and the product was purified by flash
column chromatography with CHCl.sub.3 --MeOH (15:1) to give Compound 1
(2.67 g, 10.5 mmol) in 69 percent yield.
.sup.1 H NMR (CD.sub.3 OD) .delta.7.33 (5H, m), 4.98 (1H, br d, J.sub.1-2ax
=3.5 Hz, H-1), 4.70 (1H, d, J=12.0 Hz, benzyl), 4.46 (1H, d, J=12.0 Hz,
benzyl), 3.88 (1H, ddd, J.sub.3-2ax =12.0 Hz, J.sub.3-4 =9.5 Hz,
J.sub.3-2eq =5.0 Hz, H-3), 3.78 (1H, dd, J.sub.6a-6b =12.0 Hz, J.sub.6a-5
=2.5 Hz, H-6a), 3.59 (1H, ddd, J.sub.5-4 =9.5 Hz, J.sub.5-6b =6.0 Hz,
J.sub.5-6a =2.5 Hz, H-5), 3.58 (1H, dd, J.sub.6b-6a =12.0 Hz, J.sub.6b-5
6.0 Hz, H-6b), 3.25 (1H, t, J.sub.4-3 =J.sub.4.5 =9.5 Hz, H-4), 2.09 (1H,
dd, J.sub.2eq-2ax =13.0 Hz, J.sub.2eq-3 =5.0 Hz, H-2eq), 1.63 (1H, ddd,
J.sub.2ax-2eq =13.0 Hz, J.sub.2ax-3 =12.0 Hz, J.sub.2ax-1 3.5 Hz, H-2ax).
.sup.13 C NMR (CD.sub.3 OD) .delta.139.4, 129.3, 129.0, 138.6, 97.9, 74.3,
73.1, 69.9, 69.7, 62.8, 38.8. HRMS (FAB) calcd for C.sub.13 H.sub.18
O.sub.5 Na (M+Na.sup.+) 277.1052, found 277.1041.
E. Benzyl 6-bromo-2,6-dideoxy-.beta.-D-glucopyranoside (Compound 2)
A solution of triphenylphosphine (4.5 g, 17.3 mmol, 2.2 eq) in pyridine (30
mL) was added dropwise to a cooled solution of Compound 1 (2.0 g, 7.9
mmol) and CBr.sub.4 (3.1 g, 9.4 mmol) in pyridine (30 mL) at zero degrees
C. The mixture was then heated at 50.degree. C. for 10 hours. After
cooling, methanol (5 mL) was added dropwise, and the mixture was
concentrated. The product was purified by flash column chromatography with
CHCl.sub.3 --MeOH (32:1) to give 1.82 g (yield 73 percent) of Compound 2.
.sup.1 H NMR (CD.sub.3 OD): 7.20 (5H, m), 4.87 (1H, br d, J.sub.1-2ax =3.5
Hz, H-1), 4.63 (1H, d, J=12.0 Hz, benzyl), 4.38 (1H, d, J=12.0 Hz,
benzyl), 3.77 (1H, ddd, J.sub.3-2ax =11.5 Hz, J.sub.3-4 =9.0 Hz,
J.sub.3-2eq =5.0 Hz, H-3), 3.67 (1H, dd, J.sub.6a-6b =10.5 Hz, J.sub.6a-5
=2.0 Hz, H-6a), 3.64 (1H, ddd, J.sub.5-4 =9.0 Hz, J.sub.5-6b =6.5 Hz,
J.sub.5-6a =2.0 Hz, H-5), 3.46 (1H, dd, J.sub.6b-6a =10.5 Hz, J.sub.6b-5
=6.5 Hz, H-6b), 3.13 (1H, t, J.sub.4-3 =J.sub.4-5 =9.0 Hz, H-4), 2.01 (1H,
dd, J.sub.2eq-2ax =13.0 Hz, J.sub.2ax-3 =5.0 Hz, H-2eq), 1.55 (1H, ddd,
J.sub.2ax-2eq =13.0 Hz, J.sub.2ax-3 =11.5 Hz, J.sub.2 ax-1=3.5 Hz, H-2ax).
.sup.13 C NMR (CD.sub.3 OD) .delta.139.0, 129.3, 129.1, 128.7, 97.7, 75.3,
73.1, 69.8, 69.7, 38.7, 34.6. HRMS (FAB) calcd for C.sub.13 H.sub.17
O.sub.4 NaBr (M+Na.sup.+) 339.0208, found 339.0218.
F. Benzyl 2,6-dideoxy-.beta.-D-glucopyranoside (Compound 3)
A solution of Bu.sub.3 SnH (1.0 g, 3.6 mmol, 1 mL) in toluene (10 mL) was
added dropwise to a gentle refluxing solution of Compound 2 (0.76 g, 2.40
mmol) in toluene (15 mL) over 10 minutes, and then the mixture was
refluxed for 10 hours. After cooling, the mixture was concentrated, and
the residue was chromatographed on a flash column with CHCl.sub.3 --MeOH
(29:1) to give crude Compound 3 which was acetylated with Ac.sub.2 O (10
mL), pyridine (10 mL) and catalytic amount of N,N-dimethylaminopyridine
(DMAP). The product was purified by flash column chromatography with
toluene-EtOAc (15:1) to give the corresponding peracetate, which was
treated with NaOMe (2 mL, 1N solution) in MeOH (20 mL) for 0.5 hours at
room temperature. The mixture was neutralized by adding Dowex.RTM.
50W-X8[H.sup.+ ], then the resin was filtered off, and the filtrate was
concentrated to give Compound 3.
.sup.1 H NMR (CDCl.sub.3) .delta.7.28 (5H, m), 4.89 (1H, br d, J.sub.1-2ax
=3.5 Hz, H-1), 4.62 (1H, d, J=12.0 Hz, benzyl), 4.43 (1H, d, J=12.0 Hz,
benzyl), 3.83 (1H, ddd, J.sub.3-2ax =11.5 Hz, J.sub.3-4 =9.0 Hz,
J.sub.3-2eq =5.0 Hz, H-3), 3.64 (1H, dq, J.sub.5-4 =9.0 Hz, J.sub.5-6 =6.0
Hz, H-5), 2.97 (1H, t, J.sub.4-3 =J.sub.4-5 =9.0 Hz, H-4), 2.09 (1H, dd,
J.sub.2e-2ax =13.0 Hz, J.sub.2eq-3 =5.0 Hz, H-2eq), 1.63 (1H, ddd,
J.sub.2ax-2eq =13.0 Hz, J.sub.2ax-3 =11.5 Hz, J.sub.2ax-1 =3.5 Hz, H-2ax),
1.25 (3H, d, J.sub.6-5 =6.0 Hz, 6-CH.sub.3). .sup.13 C NMR (CDCl.sub.3)
.delta.137.6, 128.4, 127.9, 127.7, 96.5, 77.9, 69.1, 68.8, 67.8, 37.7,
17.8. HRMS (FAB) calcd for C.sub.13 H.sub.18 O.sub.4 Na (M+Na.sup.+)
261.1103, found 261.1116.
G. 2.6-Dideoxy-D-plucopyranose (Compound 4)
A solution of Compound 3 (0.56 g, 2.3 mmol) in 60 percent aqueous acetic
acid (AcOH) (25 mL) was hydrogenated over 100 mg of 10 percent palladium
on charcoal under atmospheric pressure. After 12 hours, the solution was
filtered and the filtrate was concentrated in vacuo. By flash column
chromatography with CHCl.sub.1 --MeOH (10:1), 0.29 g (2.0 mmol) of
Compound 4 was obtained (85 percent yield).
.beta.-isomer: .sup.1 H NMR (CD.sub.3 OD) .delta.5.12 (1H, br d,
J.sub.1-2ax 3.5 Hz, H-1), 3.75 (1H, ddd, J.sub.3-2ax =12.0 Hz, J.sub.3-4
=9.0 Hz, J.sub.3-2 =5.0 Hz, H-3), 3.18 (1H, dq, J.sub.5-4 =9.0 Hz,
J.sub.5-6 =6.5 Hz, H-5), 2.85 (1H, t, J.sub.4-3 =J.sub.4-5 =9.0 Hz, H-4),
1.96 (1H, dd, J.sub.2eq-2ax =13.0 Hz, J.sub.2eq-3 =5.0 Hz, H-2eq), 1.50
(1H, ddd, J.sub.2ax-2eq =13.0 Hz, J.sub.2ax-3 =12.0 Hz, J.sub.2ax-1 =3.5
Hz, H-2ax), 1.14 (3H, d, J.sub.6-5 =6.5 Hz, 6-CH.sub.3). .sup.13 C NMR
(CD.sub.3 OD) .delta.92.6, 79.2, 73.2, 68.7, 39.8, 18.3. HRMS (FAB) calcd
for C.sub.6 H.sub.12 O.sub.4 Na (M+Na.sup.+) 171.0633, found 171.0640.
H. 2-Deoxy-L-alucopyranose (Compound 5)
A solution of triacetyl L-glucal (Compound 5a, 1.0 g, 3.7 mmol) and NaOMe
(2 mL, 1N solution) in MeOH (15 mL) was stirred for 0.5 hours at room
temperature. After the mixture was neutralized by adding Dowex.RTM.
50W-X8[H.sup.+ ], the resin was filtered off, and the filtrate was
concentrated.
The crude product was dissolved in a diluted H.sub.2 SO.sub.4 solution (pH
1). After 24 hours, the reaction solution was neutralized with 1N NaOH,
and then freeze-dried. The product was purified by flash column
chromatography with CHCl.sub.3 --MeOH (5:1) to give Compound 5 (0.39 g,
overall yield for these two steps: 65 percent).
.sup.1 H NMR (CD.sub.3 OD) for the a-isomer: .delta.4.78 (1H, dd,
J.sub.1-2ax =10.0 Hz, J.sub.1-2eq =2.0 Hz, H-1), 3.85 (1H, dd, J.sub.6a-6b
=12.5 Hz, J.sub.6a-5 =2.0 Hz, H-6a), 3.67 (1H, dd, J.sub.6b-6a =12.5 Hz,
J.sub.6b-5 =6.0 Hz, H-6b), 3.54 (1H, ddd, J.sub.3-2ax =12.0 Hz, J.sub.3-4
=9.0 Hz, J.sub.3-2eq =5.0 Hz, H-3), 3.22 (1H, ddd, J.sub.5-4 =9.0 Hz,
J.sub.5-6b =6.0 Hz, J.sub.5-6a =2.0 Hz, H-5), 3.15 (1H, t, J.sub.4-3
=J.sub.4-5 =9.0 Hz, H-4), 2.13 (1H, dd, J.sub.2eq-2ax =12.5 Hz,
J.sub.2eq-3 =5.0 Hz, J.sub.2eq-1 =2.0 Hz, H-2eq), 1.46 (1H, ddd,
J.sub.2ax-2eq =12.5 Hz, J.sub.2ax-3 =12.0 Hz, J.sub.2ax-1 =10.0 Hz,
H-2ax). .sup.13 C NMR (CD.sub.3 OD) .delta.95.1, 78.1, 73.0, 72.6, 63.0,
41.8. HRMS (FAB) calcd for C.sub.6 H.sub.12 O.sub.5 Na (M+Na.sup.+)
187.0582, found 187.0582.
I. 2,6-Dideoxy-L-glucopyrannose (2-deoxy-L-Rhamnose; Compound 6)
The reaction procedure was similar to that described above (from Compound
5a to Compound 5). The final product was purified by flash column
chromatography with CHCl.sub.3 --MeOH (9:1) to give Compound 6 in 67
percent overall yield from triacetyl L-rhamnal, Compound 6a.
The data of .sup.1 H and .sup.13 C NMR are identical with those of Compound
4, which is an enantiomer of Compound 6. HRMS (FAB) calcd for C.sub.6
H.sub.12 O.sub.4 Na (M+Na.sup.+) 171.0633, found 171.0640.
J. 2-Azido-2-deoxy-3,4,6-O-triacetyl-.beta.-L-mannopyranosvl nitrate
(Compound 7)
A solution of Compound 5a (1.50 g, 5.5 mmol) in CH.sub.3 CN (30 mL) was
added dropwise to a solid mixture of NaN.sub.3 (0.54 g, 8.3 mmol, 1.5 eq)
and ammonium cerium nitrate (CAN, 9.1 g, 16.5 mmol, 3 eq) at about
-20.degree. C. The suspension was stirred vigorously under N.sub.2. After
seven hours, the starting material disappeared on TLC, the solution was
poured into ice-water, and extracted with EtOAc. The combined extracts
were successively washed with water, brine, dried over anhydrous
MgSO.sub.4 and concentrated in vacuo. The compound was chromatographed on
a flash silica gel column with EtOAc-toluene (1:12) to give 1.57 g of the
azido compound. From .sup.1 H NMR spectrum, three isomers were observed,
(gluco and manno types which contained their .alpha., .beta. isomers). The
major manno-type product was characterized:
.sup.1 H NMR (CDCl.sub.3) .delta.6.22 (1H, d, J.sub.1-2 =2.0 Hz, H-1), 5.40
(1H, t, J.sub.4-5 =J.sub.4-3 =9.5 Hz, H-4), 5.25 (1H, dd, J.sub.3-4 =9.5
Hz, J.sub.3-2 =4.0 Hz, H-3), 4.28 (1H, dd, J.sub.6a-6b =13.0 Hz,
J.sub.6a-5 =5.0 Hz, H-6a), 4.21 (1H, dd, J.sub.2-3 =4.0 Hz, J.sub.2-1 =2.0
Hz H-2), 4.11 (1H, ddd, J.sub.5-4 =9.5 Hz, J.sub.5-6a =5.0 Hz, J.sub.5-6b
=2.5 Hz, H-5), 4.11 (1H, dd, J.sub.6b-6a =13.0 Hz, J.sub.6b-5 2.5 Hz,
H-6b), 2.12 (3H, s, acetyl), 2.09 (3H, s, acetyl), 2.07 (3H, s, acetyl).
.sup.13 C NMR (CDCl.sub.3) .delta.170.4, 169.6, 169.2, 97.0, 71.0, 70.2,
64.6, 61.2, 58.6, 20.4, 20.3, 20.2. HRMS (FAB) calcd for C.sub.12 H.sub.16
N.sub.4 O.sub.10 Na (M+Na.sup.+) 399.0674, found 399.0670.
K. 2-Azido-2-deoxy-1,3,4,6-tetra-O-acetyl-.beta.-L-glucopyranose (Compound
8) and 2-Azido-2-deoxy-1,3,4,6-tetra-O-acetyl-.beta.-L-mannopyranose
(Compound 9)
The mixture mentioned above was used subsequently without further
purification. Two equivalents of NaOAc (0.60 g, 7.3 mmol) were added to a
solution of Compound 7 (1.37 g, 3.6 mmol) in acetic acid (30 mL), and the
reaction was heated at 100.degree. C. overnight (about 18 hours). After
cooling, the reaction mixture was diluted with EtOAc (30 mL), then
successively washed with water, aqueous NaHCO.sub.3, brine, dried over
anhydrous MgSO.sub.4 and concentrated. The residue was chromatographed on
a flash column with EtOAc-toluene (1:5) to give two products. The less
polar isomer was identified as gluco-type Compound 8 (0.78 g), and the
more polar isomer was identified as manno-type Compound 9 (0.75 g).
Compound 8: .sup.1 H NMR (CDCl.sub.3) .delta.6.30 (1H, d, J.sub.1-2 =3.5
Hz, H-1), 5.46 (1H, t, J.sub.3-2 =J.sub.3-4 =10.0 Hz, H-3), 5.12 (1H, t,
J.sub.4-5 =J.sub.4-3 =10.0 Hz, H-4), 4.30 (1H, dd, J.sub.6a-6b =12.5 Hz,
J.sub.6a-5 =4.0 Hz, H-6a), 4.08 (1H, ddd, J.sub.5-4 =10.0 Hz, J.sub.5-6a
=4.0 Hz, J.sub.5-6b =2.5 Hz, H-5), 4.06 (1H, dd, J.sub.6-6a =12.5 Hz,
J.sub.6b-5 =2.5 Hz, H-6b), 3.68 (1H, dd, J.sub.2-3 =10.0 Hz, J.sub.2-1
=3.5 Hz, H-2), 2.20 (3H, s, acetyl), 2.11 (3H, s, acetyl), 2.08 (3H, s,
acetyl), 2.05 (3H, s, acetyl). .sup.13 C NMR (CDCl.sub.3) .delta.170.4,
170.0, 169.4, 168.4, 89.8, 70.6, 69.6, 67.6, 61.3, 60.2, 20.8, 20.6, 20.6,
20.4. HRMS (FAB) calcd for C.sub.14 H.sub.19 N.sub.3 O.sub.9 (M+Na.sup.+)
396.1019, found 396.1023.
The manno-type isomer Compound 9 was further purified by recrystallization
(prisms from diethyl ether): mp 130-131.degree. .degree. C.,
[a].sup.25.sub.D -82.1.degree. (c 1.12, CHCl.sub.3); .sup.1 H NMR
(CDCl.sub.3) .delta.6.11 (1H, d, J.sub.1-2 =2.0 Hz, H-1), 5.39 (1H, t,
J.sub.4-5 =J.sub.4-3 =9.5 Hz, H-4), 5.36 (1H, dd, J.sub.3-4 =9.5 Hz,
J.sub.3-2 =3.0 Hz, H-3), 4.24 (1H, dd, J.sub.6a-6b =12.5 Hz, J.sub.6a-5
=4.5 Hz, H-6a), 4.07 (1H, dd, J.sub.6b-6a =12.5 Hz, J.sub.6b-5 =2.5 Hz, | | |
