Note: Descriptions are shown in the official language in which they were submitted.
11aS~47
--1--
- P.C. 6122A
PREPARATION OF 2-KETOGULONIC ACID
This invention relates to a process for preparing
2-ketogulonic acid, its alkyl esters and salts by the
selective reduction of 2,5-diketogluconic acid, alkyl
esters or salts thereof. 2-ketogulonic acid is useful
as an intermediate for the preparation of ascorbic acid.
The complete reduction of 2,5-diketogluconic acid
with an excess of sodium borohydride has been reported
as part of a structural determination of the acid, see
Agr. Biol. Chem. 28, 819 (1964), J. Biol. Chem. 204, 34
(1953) and Antonie Van Leeuwenhoeck 37, 185 (1971). The
catalytic reduction of 2,5-diketogluconic acid using a
Raney nickel catalyst and hydrogen gives a low yield of
a mixture of 2-ketogluconic acid and 2 ketogulonic acid,
with 2-ketogluconic acid being the major product, Agr.
Biol. Chem. 28, 819 (1964). Applicant's United States
Patent No. 4,159,990 discloses a process for the ,~
reduction of a 2,5-diketogluconate with one equivalent
of an alkali metal borohydride to form a mixture of
2-ketogulonic acid and 2-ketogluconic acid.
It has now been found that a 2,5-diketogluconate
can be reduced with greater regioselectivity and
stereoselectivity, thereby giving higher yields of the
desired 2-ketogulonic acid for subsequent conversion to
ascorbic acid, by use of an amine-borane reducing agent
at a pH between about 2 and 7. Thus, for example
yields of 2-ketogulonic acid and 2-ketogluconic
114~147 `
--2--
acid of 94% or higher, with about 96% of the product
mixture being the desired 2-ketogulonic acid, may be
obtained by this process. These higher yields can
be obtained without conducting the reduction reaction
in the presence of a boron-complexing agent as is
required for optimum yields in the alkali metal
borohydride reduction. It has also been found that
2-ketogulonic acid formed by this process is not subject
to further reduction i.e. reduction of the 2-keto group,
at a reasonable rate even in the presence of excess
amine-borane reducing agent. The 2-keto group of
2-ketogulonic acid is, however, rapidly reduced by an
alkali metal borohydride, resulting in less than optimum
yields of 2-ketogulonic acid when formed by the alkali
metal borohydride reduction of a 2,5-diketogluconate
and precluding the use of excess alkali metal boro-
hydride to increase rates of reduction and conversions of
the 2,5-diketogluconate starting material. Further, the
2,5-diketogluconate is most stable under the acidic condi-
tions employed in the present amine-borane reduction.
The present invention relates to a process for
preparing a 2-ketogulonate which comprises reducing a
2,5-diketogluconate with an amine-borane of the formula
RlR2HN.BH3 or with pyridine-borane in solution at a pH
between about 2 and 7 at a temperature between about
-20 to 70C, wherein Rl and R2 are each selected from
hydrogen and alkyl of 1 to 4 carbon atoms; and said
2,5-diketogluconate is selected from 2,5-diketogluconic
acid, a normal alkyl ester of said acid wherein said
alkyl group is of 1 to 4 carbon atoms, and the salt
1~41;~ 7
--3--
of said acid having a counterion selected from an
alkali metal, an alkaline earth metal, ammonium and
tetraalkyl-ammonium having from 1 to 4 carbon atoms
in each alkyl group.
Preferred amine-borane reducing agents of the
formula RlR2HN.BH3 include ammonia-borane, methylamine-
borane, dimethylamine-borane, and t-butylamine-borane.
Pyridine-borane is also a preferred amine-borane.
The reaction is preferably conducted at temperatures
in the range 0-25C, preferably at a pH in the range
4 to 6. Preferred 2,5-diketogluconate starting materials
include 2,5-diketogluconic acid, sodium 2,5-diketoglucon-
ate, calcium 2,5-diketogluconate and methyl 2,5-diketo-
gluconate.
The present process provides for the regioselective
and stereoselective reduction of a 2,5-diketogluconate
with an amine-borane. The reaction product is pre-
dominantly a 2-ketogulonate, with only minor amounts,
about 2 to 12%, of a 2-ketogluconate being formed.
The reaction product is therefore suitable for the
preparation of ascorbic acid by means known in the
art, for example the base catalyzed lactonization of
the lower alkyl esters of 2-ketogulonic acid. If
desired, the minor amounts of 2-ketogluconic acid present
in the reaction product may be converted to erythorbic
acid by similar methods, either separately or concurrently
with the conversion of the 2-ketogulonate to ascorbic
acid.
The 2,5-diketogluconate used as the starting material
in the present invention may be either 2,5-diketogluconic
acid or salts of the acid. Suitable salts include
those having as counterions an alkali metal, an alkaline
earth metal, ammonium and tetra-alkylammonium where the
~40147
--4--
alkyl groups have from 1 to 4 carbon atoms. Also useful
as starting materials for the present process are the
normal alkyl esters of 2,5-diketogluconic acid wherein
the alkyl group is of 1 to 4 carbon atoms. As used in
the specification and claims hereof, the terms 2,5-
diketogluconate, 2-ketogulonate and 2-ketogluconate
include the free acids and suitable alkyl esters and
salts thereof as previously described. The 2,5-diketo-
gluconic acid and salts thereof may be produced by any
means known in the art. Generally, the 2,5-diketo-
gluconate is produced as the calcium salt in aqueous
solution by fermentation using methods well known in
the fermentation industry and may be used directly in
this form as the starting material for the present
process. The 2,5-diketogluconate can also be produced
by fermentation in the presence of other ions such as
sodium and the resulting sodium 2,5-diketogluconate may
likewise be used directly in the present process. In an
alternative method, the 2,5-diketogluconate is prepared
in the conventional way as the calcium 2,5-diketoglucon-
ate and converted to the desired compound by addition of
a salt effective to precipitate calcium and leave the
2,5-diketogluconate in solution with the desired
counterion. Thus for example sodium or ammonium 2,5-
diketogluconate can be produced by addition of sodium orammonium carbonate, respectively, to a solution of
calcium 2,5-diketogluconate produced by fermentation.
Calcium is precipitated as calcium carbonate leaving the
2,5-diketogluconate in solution with sodium or ammonium
counterions. The free acid may also be neutralized with
an appropriate hydroxide or other salt. If desired, the
2,5-diketogluconate can be isolated, purified and
redissolved.
The normal alkyl esters of 2,5-diketogluconic acid
wherein alkyl is of 1 to 4 carbon atoms may be prepared
by heating a solution of 2,5-diketogluconic acid or a
~401~7
--5--
suitable salt thereof in the appropriate normal alkanol
at 50C to 100C in the presence of a catalytic amount
of a strong acid, such as concentrated sulfuric acid,
hydrochloric acid, p-toluene sulfonic acid and the like,
to form the corresponding alkyl 2,5-diketogluconate-5,5-
dialkyl acetal. Salts of 2,5-diketogluconic acid
suitable for preparation of the esters by this means
include the alkali metal, alkaline earth metal, ammonium
and tetraalkyl ammonium salts, wherein each alkyl group
of the tetraalkyl ammonium ion has from 1 to 4 carbon
atoms. The acetal is then hydrolyzed with aqueous acid
at a temperature between about -10C and 30C to afford
the desired alkyl ester of 2,5-diketogluconic acid.
Suitable acids include aqueous hydrochloric acid,
trifluoroacetic acid, sulfuric acid, sulfonic acid ion
exchange resins and the like.
When an alkali metal 2,5-diketogluconate is utilized
as the starting material in the present amine-borane
reduction process the sodium salt is preferred. A
preferred alkaline earth 2,5-diketog~uconate is the
calcium salt. When tetraalkyl ammonium salts are
employed, the tetramethylammonium salt is preferred. A
preferred alkyl ester starting material is methyl 2,5-
diketogluconate.
The reduction of the 2,5-diketogluconate is
effected by contacting a solution of the 2,5-diketo-
glucon~te with an effective amount of an amine-borane of
the formula RlR2HN-BH3 wherein Rl and R2 are as
previously defined, or with pyridine-borane. Pre-
ferably, the reaction is effected in aqueous solution,
optionally contain ng organic cosolvents such as,
but not limited to, alkanols of 1 to 4 carbon atoms,
alkanediols of 2 to 4 carbon atoms, and the like.
Methanol is a preferred cosolvent. The concentration
114(~i~7
--6--
of the 2,5-diketogluconate is not critical but is
preferably between about 5 and 20 weight percent. The
concentration of the 2,5-diketogluconate formed by
fermentation is generally within this range and thereby
provides a suitable aqueous solution of the starting
material for use in the present process. When an
alkyl ester is utilized as starting material the
reaction may be conducted in anhydrous solvents, such
as alkanols, especially methanol. In all cases, it
is not necessary that all the 2,5-diketogluconate
be dissolved in the solvent, provided a substantial
part of the material of the starting material is in
solution.
The amine-boranes useful as reducing agents in
the present process are well known in the art and are
generally commercially available, see for example
C. F. Lane, Aldrichimica 6, 51 (1973). If desired,
they can be prepared by known methods, for example
by the reaction of diborane with an appropriate
amine of the formula RlR2NH to form the amine-borane
RlR2HN.BH3, the reaction generally being conducted at
temperatures of about 0C or below.
The amount of amine-borane employed in the re-
duction reaction will determine the amount of 2,5-
diketogluconate starting material present in thereaction mixture that will be converted to the desired
reaction product. Preferably, sufficient amine-borane
will be employed to convert all of the 2,5-diketo-
gluconate starting material present in the reaction
mixture, since this will give optimum yields of the
desired 2-ketogulonate suitable for subsequent con-
version to ascorbic acid. However, if desired
lesser amounts of the amine-borane reducing agent
can be used to achieve lower conversions i.e. re-
duction of only a part of the 2,5-diketogluconate
present in the reaction mixture. Unreacted 2,5-diketo-
11~V1L~7
-7-
gluconate may then be recycled and subjected to
further reduction reactions. It is intended that
the specification and claims hereof be understood
to include the above methods of practicing the
present invention, as well as other procedures for
carrying out the reduction which will be evident to
those skilled in the art such as, but not limited to,
conducting the reduction as either a batch or continuous
process.
It will be understood that one mole of an amine-
borane contains three equivalents of hydride ion.
Thus, high yields of the desired 2-ketogulonate can
be formed by employing between about 0.30 to about
0.40 moles, preferably about 0.33 moles, of an amine-
lS borane per mole of 2,5-diketogluconate starting material
present in the reaction mixture. However, since the
2-keto group of the product 2-ketogulonate is only
very slowly reduced by excess amine-borane reducing
agent, especially when Rl and R2 are both other than
hydrogen, higher rates of reduction of the 5-keto
group can be achieved, if desired, by use of relatively
larger amounts of reducing agent, for example up to
about 2 to 3 moles of amine-borane per mole of 2,5-
diketogluconate and the use of such an excess ensures
complete conversion of the 2,5-diketogluconate starting
material in the reaction mixture. The amine-borane
reducing agent can be added to the solution of the
2,5-diketogluconate either in one batch at the start
of the reaction or in portions during the course
of the reaction and may be added either as a solid or as
a solution.
During the reduction of the 2,5-diketogluconate
with the amine-borane the pH of the solution should
be maintained at between about 2 and 7, preferably
between about 4 and 6. To maintain the pH in the
above range, an acid such as a mineral acid, for
1140147
--8--
example hydrochloric acid, sulfuric acid, phosphoric
acid and the like, or an crganic acid such as a lower
alkyl carboxylic acid, for example a Cl to C6 alkyl
carboxylic acid, may be added to the reaction mixture.
The pH of an aqueous solution of sodium or calcium 2,5-
diketogluconate produced by fermentation is usually
lower than 5 and such a solution is therefore suitable
for use in the present reduction process.
The time necessary to complete the reduction will
depend on the temperature and the amounts of reagents
employed. However, generally reaction times will be
relatively short with the reaction being substantially
complete in periods of about 15 minutes to about 3 hours.
On completion of the selective reduction any unreacted
2,5-diketogluconate can be recycled for further reaction
or it can be effectively removed from the reaction
mixture by heating with an acid or base followed by
filtration.
The 2-ketogulonate formed in the above process can
be isolated, together with lesser amounts of the 2-keto-
gluconate, by filtering the reaction mixture and
adjusting the filtrate to a pH between about 1.5 and 2
by addition of an acid such as concentrated sulfuric
acid and filtering off and discarding any precipitate
that is formed. The desired product can be collected
by removing the water or water-organic cosolvent,
for example by freeze drying or heating under
reduced pressure. The ratio of 2-ketogulonic acid
to 2-ketogluconic acid in the mixture can be deter-
mined by gas-liquid chromatography of the pertrimethyl-
silylated methyl esters using a five foot OV-210 (Ohio
Valley Specialty Co.) column at 135C. However, other
methods of analysis, for example liquid chromatography
or thin layer chromatography, may be employed. The
2-ketogulonic acid formed in the present reduction process
1140147
g
can readily be converted to ascorbic acid by means known
in the art. The small amounts of 2-ketogluconate in the
reaction mixture may be separated, for example by
chromatography, and the 2-ketogulonate converted to
ascorbic acid. However, the mixture of 2-ketogulonate
containing small amounts of 2-ketogluconate can be used
directly in the subsequent reaction, the 2-ketogluconate
being converted to erythorbic acid, which can be
separated from the ascorbic acid formed. Thus for example,
the mixture of the 2-keto acids can be converted to the
methyl esters by refluxing in methanol in the presence
of an acid catalyst such as hydrochloric acid or a
sulfonic ion e~change resin for about 3 to 24 hours.
Other esters can be formed in this manner using the
appropriate alcohol. The esters are formed directly
when an alkyl ester of 2,5-diketogluconic acid is the
starting material for the selective reduction. The
mixture of methyl esters can be separated and is then
refluxed in methanol in the presence of a base, such as
sodium bicarbonate, in an inert atmosphere. On cooling,
sodium ascorbate and sodium erythorbate precipitate out.
The crude salts are collected by filtration, mixed with
water~and deionized with a cation exchange resin such as
Dowex~0 ~Dow Chemical Co.). The water is removed and
ascorbic acid and erythrobic acid are recrystallized
from methanol-water to give ascorbic acid containing
small amounts of erythorbic acid. If desired, ascorbic
acid may be obtained by recrystallization from, for
example, a 4:1 methanol-water solution. Other suitable
solvents or cosolvents can be employed if desired. If
desired, the methyl esters of 2-ketogulonic acid and 2-
ketogluconic acid can be separated and converted to
ascorbic acid and erythorbic acid respectively using the
same conditions as described above for the mixture of esters.
`frRde ~r,~
1140147
--10--
Ascorbic acid can also be prepared selectively from
the 2-ketogulonate containing small amounts of the 2-
ketogluconate obtained by the present reduction process
by heating in a suitable organic solvent, such as benzene,
toluene, xylene and the like, at about 50C to 130C,
preferably 60C to 90C, in the presence of an acid
selected from hydrochloric acid, hydrobromic acid,
sulfuric acid and sulfonic ion exchange resins, although
other similar acids may be used. A preferred acid is
hydrochloric acid. After heating for a period of about
3 to 12 hours, depending on the reaction employed,
lactonization of the 2-ketogulonate to ascorbic acid is
substantially complete. In this process, erythorbic acid
is not produced from the small amounts of 2-ketogluconate
present and this method thereby affords a simple method
of selectively forming ascorbic acid from the reaction
product of the present amine-borane reduction.
The present invention is illustrated by the
following examples. It should be understood, however,
that the invention is not limited to the specific details
of these examples.
EXAMPLE 1
To 100 ml of a 15% (w/v~ aqueous solution of 2,5-
diketogluconic acid at a temperature of 6-8C and pH
3.0 was added 4.6 g (1.07 mole~ of borane-dimethylamine
``~ complex. After 1 hour h.p.l.c. analysis (Aminex~A~21
resin in formate form, ammonium formate buffer at pH
5.0) indicated complete reduction had occurred, where-
upon 30 ml of acetone was added and the solution
slowly poured into a slurry of 150 ml of Dowex~50 ion
exchange resin (hydrogen form). After hydrogen evolution
had ceased, the resin was removed by filtration, solvent
removed via rotary evaporation and the residue placed
in 200 ml of anhydrous methanol. Amberlyst 15 ion
exchange resin catalyst (20 ml, hydrogen form) was
added and the methanol/trimethylborate azeotrope was
1~40~47
--11--
distilled off at atmospheric pressure with concomitant
esterification of the 2-ketogulonic acid. The methanol
solution was filtered and reduced to 30 ml whereupon
crystallization proceeded affording on isolation 6.9 g
of methyl 2-keto~ulonate (mp 152-154C, lit 153-154C).
The mother liquor was evaporated in vacuo to a solid
containing 3.5 g of methyl-2-ketogulonate and methyl-
2-ketogluconate in a ratio of 77:23 (by glpc analysis
of the per-silylated methyl esters on a 5 ft OV-210
column at 135C), corresponding to an overall reduction
stereoselectivity of 92:8, 2-ketogulonic:2-ketogluconic
acids.
EXAMPLE 2
The procedure of Example 1 was repeated maintaining
~1 15 the pH at 3.5 with 6N HCl. Analysis by hlpc (Dowe ~50
ion exchange resin in the calcium form, 0.01 M CaC12
buffer at pH 8) after 3 hours at 0C showed both 2-
ketogulonate and 2-ketogluconate in a ratio of 94:6.
The reaction was treated as described in Example 1
affording after esterification a 96:4 mixture (by glpc
analysis) of methyl 2-ketogulonate and methyl 2-keto-
gluconate.
EXAMPLES 3-23
The reduction of sodium 2,5-diketogluconate was
effected with a number of amine-boranes, under different
temperature and pH conditions, by the following procedure:
To a stirred solution of 10.5% (w/v) sodium 2,5-diketo-
gluconate at specified temperature and pH is added the
solid amine-borane in one portion. Reactions are
followed for completeness by hplc analysis (Aminex~25
resin in formate form, ammonium formate buffer at pH
5.3) and yields determined by hplc analysis with internal
standard based on hydride equivalents used. Ratios
of 2-ketogulonic and 2-ketogluconic acids in Examples
-12-
3 through 20 were determined by conversion as described
in Example 1 to the corresponding methyl esters and
glpc analysis of their corresponding pertrimethylsilylated
methyl esters. (5 ft OV-210 column at 135C). Ratios
of 2-ketogulonic and 2-ketogluconic acids in Examples
21 through 23 were determined by conversion to
ascorbic and erythorbic acids and analysis of their
corresponding pertrimethylsilylated methyl esters by
glpc analysis as previously described.
The results obtained were as follows:
1~4(~4'7
--13--
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