U.S. patent application number 10/838551 was filed with the patent office on 2004-10-21 for synthesis of 1,2,3,4-tetrahydroxybenzenes from biomass-derived carbon.
Invention is credited to Frost, John W., Hansen, Chad A..
Application Number | 20040209337 10/838551 |
Document ID | / |
Family ID | 23049395 |
Filed Date | 2004-10-21 |
United States Patent
Application |
20040209337 |
Kind Code |
A1 |
Frost, John W. ; et
al. |
October 21, 2004 |
Synthesis of 1,2,3,4-tetrahydroxybenzenes from biomass-derived
carbon
Abstract
A bioengineered synthesis scheme for the production of
1,2,3,4-tetrahydroxybenzene from a carbon source is provided.
Methods of producing 1,2,3,4-tetrahydroxybenzene acid from a carbon
source based on the synthesis scheme are also provided. Methods are
also provided for converting 1,2,3,4-tetrahydroxybenzene to
1,2,3-trihydroxybenzene by catalytic hydrogenation.
Inventors: |
Frost, John W.; (Okemos,
MI) ; Hansen, Chad A.; (East Lansing, MI) |
Correspondence
Address: |
HARNESS, DICKEY & PIERCE, P.L.C.
P.O. BOX 828
BLOOMFIELD HILLS
MI
48303
US
|
Family ID: |
23049395 |
Appl. No.: |
10/838551 |
Filed: |
May 4, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10838551 |
May 4, 2004 |
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09937243 |
May 7, 2002 |
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6750049 |
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09937243 |
May 7, 2002 |
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PCT/US00/06808 |
Mar 16, 2000 |
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09937243 |
May 7, 2002 |
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09274732 |
Mar 23, 1999 |
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Current U.S.
Class: |
435/156 ;
568/763 |
Current CPC
Class: |
C12P 7/22 20130101; C12P
7/26 20130101; C12P 7/02 20130101 |
Class at
Publication: |
435/156 ;
568/763 |
International
Class: |
C12P 007/22; C07C
039/10 |
Goverment Interests
[0002] Work on this invention was sponsored in part by the National
Science Foundation Grant No. CHE963368. The Government may have
certain rights in the invention.
Claims
We claim:
1. A method for the production of 1,2,3,4-tetrahydroxybenzene,
comprising: a) incubating, in the presence of a carbon source, a
microbe comprising a genome, a DNA encoding a naturally occurring
myo-inositol-1-phosphate synthase and a DNA encoding a naturally
occurring inositol dehydrogenase, to produce myo-2-inosose; and b)
converting the myo-2-inosose to 1,2,3,4-tetrahydroxybenzene by acid
catalyzed dehydration.
2. The method of claim 1, wherein the DNA encoding the naturally
occurring myo-inositol-1-phosphate synthase comprises an INO1
gene.
3. The method of claim 2, wherein the INO1 gene comprises a
Saccharomyces cerevisiae INO1.
4. The method of claim 1, wherein the DNA encoding the naturally
occurring inositol dehydrogenase comprises an iolG gene.
5. The method of claim 1, wherein the DNA encoding the naturally
occurring inositol dehydrogenase comprises a Bacillus subtilis
iolG.
6. The method of claim 1, wherein the DNA encoding the naturally
occurring myo-inositol-1-phosphate synthase and the DNA encoding
the naturally occurring inositol dehydrogenase are comprised by
plasmid pAD2.28A.
7. The method of claim 1, wherein the microbe further comprises a
DNA encoding a naturally occurring myo-2-inosose dehydratase.
8. The method of claim 7, wherein the DNA encoding a naturally
occurring myo-2-inosose dehydratase is a recombinant DNA encoding a
naturally occurring myo-2-inosose dehydratase.
9. The method of claim 1, wherein the microbe further comprises a
DNA encoding a naturally occurring inositol monophosphatase.
10. The method of claim 9, wherein the DNA encoding a naturally
occurring inositol monophosphatase is a recombinant DNA encoding a
naturally occurring inositol monophosphatase.
11. The method of claim 1, wherein the DNA encoding a naturally
occurring myo-inositol-1-phosphate synthase is a recombinant DNA
encoding a naturally occurring myo-inositol-1-phosphate
synthase.
12. The method of claim 1, wherein the DNA encoding a naturally
occurring inositol dehydrogenase is a recombinant DNA encoding a
naturally occurring inositol dehydrogenase.
13. The method of claim 1, wherein the microbe is an Escherichia
coli.
14. The method of claim 1, wherein the Escherichia coli is
Escherichia coli JWF1/pAD2.28A.
15. The method of claim 1, wherein the genome comprises the DNA
encoding a naturally occurring myo-inositol-1-phosphate
synthase.
16. The method of claim 1, wherein the genome comprises the DNA
encoding a naturally occurring inositol dehydrogenase.
17. The method of claim 7, wherein the genome comprises the DNA
encoding a naturally occurring myo-2-inosose dehydratase.
18. The method of claim 9, wherein the genome comprises the DNA
encoding a naturally occurring inositol monophosphatase.
19. The method of claim 1, wherein the carbon source comprises
glucose.
20. A microbe comprising a genome, a recombinant DNA encoding a
naturally occurring myo-inositol-1-phosphate synthase and a
recombinant DNA encoding a naturally occurring inositol
dehydrogenase.
21. The microbe of claim 20, wherein the DNA encoding the naturally
occurring myo-inositol-1-phosphate synthase comprises an INO1
gene.
22. The microbe of claim 21, wherein the INO1 gene comprises a
Saccharomyces cerevisiae INO1.
23. The microbe of claim 20, wherein the DNA encoding the naturally
occurring inositol dehydrogenase comprises an iolG gene.
24. The microbe of claim 20, wherein the DNA encoding the naturally
occurring inositol dehydrogenase comprises a Bacillus subtilis
iolG.
25. The microbe of claim 20, wherein the DNA encoding the naturally
occurring myo-inositol-1-phosphate synthase and the DNA encoding
the naturally occurring inositol dehydrogenase are comprised by
plasmid pAD2.28A.
26. The microbe of claim 20, wherein the microbe further comprises
a DNA encoding a naturally occurring myo-2-inosose dehydratase.
27. The microbe of claim 20, wherein the microbe further comprises
a DNA encoding a naturally occurring inositol monophosphatase.
28. The microbe of claim 20, wherein the DNA encoding a naturally
occurring myo-inositol-1-phosphate synthase is a recombinant DNA
encoding a naturally occurring myo-inositol-1-phosphate
synthase.
29. The microbe of claim 20, wherein the DNA encoding a naturally
occurring inositol dehydrogenase is a recombinant DNA encoding a
naturally occurring inositol dehydrogenase.
30. The microbe of claim 20, wherein the microbe is an Escherichia
coli.
31. The microbe of claim 20, wherein the Escherichia coli is
Escherichia coli JWF1/pAD2.28A.
32. The microbe of claim 20, wherein the genome comprises the DNA
encoding a naturally occurring myo-inositol-1-phosphate
synthase.
33. The microbe of claim 20, wherein the genome comprises the DNA
encoding a naturally occurring inositol dehydrogenase.
34. The microbe of claim 26, wherein the genome comprises the DNA
encoding a naturally occurring myo-2-inosose dehydratase.
35. The microbe of claim 27, wherein the genome comprises the DNA
encoding a naturally occurring inositol monophosphatase.
36. A method for the production of 1,2,3,4-tetrahydroxybenzene,
comprising: a) incubating, in the presence of a carbon source, a
microbe comprising a genome, a DNA encoding a naturally occurring
myo-inositol-1-phosphate synthase, a DNA encoding a naturally
occurring inositol dehydrogenase and a DNA encoding a naturally
occurring myo-2-inosose dehydratase to produce
D-2,3-diketo-4-deoxy-epi-inositol; and b) converting the
D-2,3-diketo-4-deoxy-epi-inositol to 1,2,3,4-tetrahydroxybenzene by
acid catalyzed dehydration.
37. The method of claim 36, wherein the DNA encoding the naturally
occurring myo-inositol-1-phosphate synthase comprises an INO1
gene.
38. The method of claim 36, wherein the INO1 gene comprises a
Saccharomyces cerevisiae INO1.
39. The method of claim 36, wherein the DNA encoding the naturally
occurring inositol dehydrogenase comprises an iolG gene.
40. The method of claim 36, wherein the DNA encoding the naturally
occurring inositol dehydrogenase comprises a Bacillus subtilis
iolG.
41. The method of claim 36, wherein the DNA encoding the naturally
occurring myo-inositol-1-phosphate synthase and the DNA encoding
the naturally occurring inositol dehydrogenase are comprised by
plasmid pAD2.28A.
42. The method of claim 36, wherein the DNA encoding a naturally
occurring myo-2-inosose dehydratase is a recombinant DNA encoding a
naturally occurring myo-2-inosose dehydratase.
43. The method of claim 36, wherein the microbe further comprises a
DNA encoding a naturally occurring inositol monophosphatase.
44. The method of claim 43, wherein the DNA encoding a naturally
occurring inositol monophosphatase is a recombinant DNA encoding a
naturally occurring inositol monophosphatase.
45. The method of claim 36, wherein the DNA encoding a naturally
occurring myo-inositol-1-phosphate synthase is a recombinant DNA
encoding a naturally occurring myo-inositol-1-phosphate
synthase.
46. The method of claim 36, wherein the DNA encoding a naturally
occurring inositol dehydrogenase is a recombinant DNA encoding a
naturally occurring inositol dehydrogenase.
47. The method of claim 36, wherein the microbe is an Escherichia
coli.
48. The method of claim 36, wherein the Escherichia coli is
Escherichia coli JWF1/pAD2.28A.
49. The method of claim 36, wherein the genome comprises the DNA
encoding a naturally occurring myo-inositol-1-phosphate
synthase.
50. The method of claim 36, wherein the genome comprises the DNA
encoding a naturally occurring inositol dehydrogenase.
51. The method of claim 36, wherein the genome comprises the DNA
encoding a naturally occurring myo-2-inosose dehydratase.
52. The method of claim 43, wherein the genome comprises the DNA
encoding a naturally occurring inositol monophosphatase.
53. The method of claim 36, wherein the carbon source comprises
glucose.
54. A fermentation composition comprising a microbe which comprises
a recombinant DNA encoding a naturally occurring
myo-inositol-1-phosphate synthase and a recombinant DNA encoding a
naturally occurring inositol dehydrogenase.
55. The fermentation composition of claim 54, wherein the DNA
encoding the naturally occurring myo-inositol-1-phosphate synthase
comprises an INO1 gene.
56. The fermentation composition of claim 55, wherein the INO1 gene
comprises a Saccharomyces cerevisiae INO1.
57. The fermentation composition of claim 54, wherein the DNA
encoding the naturally occurring inositol dehydrogenase comprises
an iolG gene.
58. The fermentation composition of claim 54, wherein the DNA
encoding the naturally occurring inositol dehydrogenase comprises a
Bacillus subtilis iolG.
59. The fermentation composition of claim 54, wherein the DNA
encoding the naturally occurring myo-inositol-1-phosphate synthase
and the DNA encoding the naturally occurring inositol dehydrogenase
are comprised by plasmid pAD2.28A.
60. The fermentation composition of claim 54, wherein the microbe
further comprises a DNA encoding a naturally occurring
myo-2-inosose dehydratase.
61. The fermentation composition of claim 60, wherein the DNA
encoding a naturally occurring myo-2-inosose dehydratase is a
recombinant DNA encoding a naturally occurring myo-2-inosose
dehydratase.
62. The fermentation composition of claim 54, wherein the microbe
further comprises a DNA encoding a naturally occurring inositol
monophosphatase.
63. The fermentation composition of claim 62, wherein the DNA
encoding a naturally occurring inositol monophosphatase is a
recombinant DNA encoding a naturally occurring inositol
monophosphatase.
64. The fermentation composition of claim 54, wherein the microbe
is an Escherichia coli.
65. The fermentation composition of claim 54, wherein the
Escherichia coli is Escherichia coli JWF1/pAD2.28A.
66. The fermentation composition of claim 54, wherein the genome
comprises the DNA encoding a naturally occurring
myo-inositol-1-phosphate synthase.
67. The fermentation composition of claim 54, wherein the genome
comprises the DNA encoding a naturally occurring inositol
dehydrogenase.
68. The fermentation composition of claim 62, wherein the genome
comprises at least one recombinant DNA encoding a naturally
occurring enzyme selected from the group consisting of inositol
dehydrogenase, inositol monophosphatase and myo-2-inosose
dehydratase.
69. The fermentation composition of claim 54, wherein the carbon
source comprises glucose.
Description
RELATED APPLICATIONS
[0001] The present application is a divisional of U.S. application
Ser. No. 09/937,243, filed Sep. 21, 2001, which is a national phase
continuation of PCT international publication WO 00/56911 filed
Mar. 16, 2000, which is a Continuation-in-Part of U.S. application
Ser. No. 09/274,732 filed Mar. 23, 1999, which is hereby expressly
incorporated by reference.
FIELD OF THE INVENTION
[0003] The present invention is related to the production of
1,2,3,4-tetrahydroxybenzene and more specifically, to methods of
producing 1,2,3,4-tetrahydroxybenzene from the bioconversion of a
carbon source.
BACKGROUND OF THE INVENTION
[0004] Polyhydroxy benzenes and quinones possessing the oxygenation
pattern of 1,2,3,4-tetrahydroxybenzene 1 (FIG. 1) often display
biological activity. Aurantiogliocladin 2 and fumigatin 3 (FIG. 1)
are antibiotics. Vischer, E. B., J. Chem. Soc. 815 (1953); Baker,
W. et al., J. Chem. Soc. 820 (1953); Baker, W. et al., J. Chem.
Soc. 670 (1941). Coenzyme Q.sub.n=10 4 (FIG. 1) is an essential
antioxidant in humans protecting low density lipoproteins from
atherosclerosis-related oxidative modification. Ingold, K. U. et
al., PNAS (USA) 90:45 (1993); Stocker, R. et al., PNAS (USA)
88:1646 (1991); Steinberg, D., Circulation 84:1420 (1991).
Dillapiole 5 (FIG. 1) is a pyrethrin synergist and is responsible
for the sedative effect of Perilla frutescens leaves. Honda, G. et
al., Chem. Pharm. Bull. 36:3153 (1988); Tomar, S. S. et al., Agric.
Biol. Chem. 50:2115 (1986).
[0005] The current method of preparing 1,2,3,4-tetrahydroxybenzene
uses pyrogallol as the synthetic starting material. Pyrogallol is
converted to aminopyrogallol using a four-step synthesis.
Aminopyrogallol is then hydrolyzed to give
1,2,3,4-tetrahydroxybenzene. Conversion of pyrogallol to
1,2,3,4-tetrahydroxybenzene requires the use of such reagents as
phosgene, solvents such as pyridine and xylene, and has a
nitroaromatic as a synthetic intermediate.
[0006] It would also be desirable to provide an improved method for
producing derivatives of 1,2,3,4-tetrahydroxybenzene. Particularly,
it would be desirable to provide a method for producing
1,2,3-trihydroxybenzene (pyrogallol). It would also be desirable if
such a method were cost efficient and employed readily available
materials. Currently, 1,2,3-trihydroxybenzene is obtained by
thermal decarboxylation of gallic acid. However, gallic acid is
isolated from natural sources such as gall nuts and tara powder and
therefore is in limited supply.
[0007] It would thus be desirable to provide an improved method for
producing 1,2,3,4-tetrahydroxybenzene. It would also be desirable
if such a method was cost-efficient, using inexpensive starting
materials. It would be further desirable if the method employed
non-toxic compounds and was environmentally benign.
SUMMARY OF THE INVENTION
[0008] A bioengineered synthesis scheme for the production of
1,2,3,4-tetrahydroxybenzene from a carbon source is provided. In
one embodiment, the bioconversion methods of the present invention
comprise the steps of microbe-catalyzed conversion of a carbon
source to myo-2-inosose followed by acid-catalyzed dehydration of
myo-2-inosose to produce 1,2,3,4-tetrahydroxybenzene. As shown in
the synthesis scheme of FIG. 2, the microbe-catalyzed conversion
step of the present invention requires four enzymes. In one
embodiment, the microbe-catalyzed conversion comprises the
conversion of a carbon source to myo-inositol by a recombinant
microbe and the subsequent conversion of myo-inositol to
myo-2-inosose catalyzed by a second microbe. In another embodiment,
the recombinant microbe is Escherichia coli designed to cause the
conversion of glucose-6-phosphate to myo-inositol-1-phosphate. In
yet another embodiment, the conversion of myo-inositol to
myo-2-inosose is catalyzed by the microbe Gluconobacter oxydans.
Acid-catalyzed dehydration of the resulting myo-2-inosose yields
1,2,3,4-tetrahydroxybenzene.
[0009] The biocatalytic synthesis of 1,2,3,4-tetrahydroxybenzene
provided herein is environmentally benign, economically attractive,
and utilizes abundant renewable sources as a starting material.
[0010] Methods are also provided for the production of derivatives
of 1,2,3,4-tetrahydroxybenzene, particularly Coenzyme Q
1,2,3-trihydroxybenzene (pyrogallol). In one embodiment
1,2,3-trihydroxybenzene is produced by reduction of
1,2,3,4-tetrahydroxybenzene. In a preferred embodiment, the
reduction is achieved by catalytic hydrogenation followed by
hydrolysis.
[0011] Additional objects, advantages, and features of the present
invention will become apparent from the following description and
appended claims, taken in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The various advantages of the present invention will become
apparent to one skilled in the art by reading the following
specification and subjoined claims and by referencing the following
drawings in which:
[0013] FIG. 1 is an illustration showing the structures of products
that can be derived from 1,2,3,4-tetrahydroxybenzene;
[0014] FIG. 2 is a schematic illustrating the bioengineered
synthesis scheme of the present invention for producing
1,2,3,4-tetrahydroxybenzene- ;
[0015] FIG. 3 is a graph showing the production of myo-inositol
(solid bars) and myo-inositol-1-phosphate (open bars) in comparison
to cell dry weight (filled circles);
[0016] FIG. 4 is a schematic illustrating the conventional
synthetic scheme for synthesizing 1,2,3,4-tetrahydroxybenzene;
and
[0017] FIG. 5 is a schematic illustrating the synthesis scheme for
converting 1,2,3,4-tetrahydroxybenzene to Coenzyme Q.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0018] A bioengineered synthesis scheme for the production of
1,2,3,4-tetrahydroxybenzene from a carbon source is provided
herein. Methods of producing 1,2,3,4-tetrahydroxybenzene from a
carbon source based on the synthesis scheme are also provided. In
one embodiment, a method is provided wherein the carbon source is
converted to myo-inositol by a recombinant microbe, the
myo-inositol is further converted to myo-2-inosose by a second
microbe, followed by acid-catalyzed dehydration of myo-2-inosose to
produce 1,2,3,4-tetrahydroxybenzene.
[0019] Novel methods are also provided for the production of
derivatives of 1,2,3,4-tetrahydroxybenzene, particularly
1,2,3-trihydroxybenzene (pyrogallol). In one embodiment
1,2,3-trihydroxybenzene is produced by reduction of
1,2,3,4-tetrahydroxybenzene. In a preferred embodiment, the
reduction is achieved by catalytic hydrogenation of
1,2,3,4-tetrahydroxybenzene followed by acid catalyzed hydrolysis
to yield 1,2,3-trihydroxybenzene. In a more preferred embodiment,
the hydrogenation is catalyzed by Rh/Al.sub.2O.sub.3. These methods
take advantage of inexpensive and abundant carbon sources as
starting materials to produce 1,2,3,4-tetrahydroxybenzene which can
then be converted to 1,2,3-trihydroxybenzene.
[0020] Although microbe-catalyzed conversion of a carbon source to
myo-inositol by a recombinant microbe followed by conversion of the
myo-inositol to myo-2-inosose by a second microbe is described in
detail herein, in an alternative embodiment, a single recombinant
microbe is employed to convert a carbon source directly to
myo-2-inosose which is then converted to
1,2,3,4-tetrahydroxybenzene by an acid-catalyzed dehydration. This
single-microbe conversion may be carried out by any type of microbe
sufficiently engineered to produce the desired outcome.
[0021] In another alternative embodiment, a recombinant microbe
catalyzes the conversion of a carbon source to
D-2,3-diketo-4-deoxy-epi-inositol which is subsequently converted
to 1,2,3,4-tetrahydroxybenzene by an acid-catalyzed dehydration.
D-2,3-Diketo-4-deoxy-epi-inositol is an intermediate in the
microbial catabolism of myo-inositol as well as a likely
intermediate in the acid-catalyzed conversion of myo-2-inosose into
1,2,3,4-tetrahydroxybenzene.
[0022] The bioconversion methods of the present invention are
carried out under conditions of time, temperature, pH, nutrient
type and concentration, aeration conditions and glucose
concentrations, to provide maximal conversion of the carbon source
to 1,2,3,4-tetrahydroxybenzene. As described in detail in Specific
Example 1, in a preferred embodiment, a fed-batch fermentor is used
to convert the carbon source to myo-inositol, followed by isolation
of myo-inositol, e.g., deionization and decolorization of the
fermentation broth and precipitation by the addition of an organic
solvent. The isolated myo-inositol is then converted to
myo-inositol to myo-2-inosose followed by isolation of the
myo-2-inosose, e.g., precipitation of the myo-2-inosose from the
culture broth. The fed-batch fermentor process and the
precipitation methods are also known to those skilled in the
art.
[0023] As used herein, the phrase "carbon source" is meant to
include biomass-derived carbon sources including, but not limited
to, xylose, arabinose, glycerol, glucose and the intermediates
(e.g., dicarboxylic acids) in the Krebs cycle, either alone or in
combination. In a preferred embodiment, the carbon source is
glucose.
[0024] In one embodiment, a recombinant E. coli microbe is employed
in the methods of the present invention. In a preferred embodiment,
the E. coli comprises a non-functional serA locus. This recombinant
E. coli, designated, JWF1, may further comprise a plasmid carrying
an INO1 gene insert and a serA gene insert. The INO1 gene encodes
myo-inositol-1-phosphate synthase which converts
glucose-6-phosphate to myo-inositol-1-phosphate. In a preferred
embodiment, the INO1 gene is from Saccharomyces cerevisiae.
Overexpression of myo-inositol-1-phosphate synthase will increase
carbon flow into the myo-inositol pathway. This recombinant microbe
is capable of converting glucose to myo-inositol.
[0025] In another embodiment, the myo-inositol produced by the
first recombinant microbe, is converted to myo-2-inosose by a
second microbe. This second microbe can either be a recombinant
microbe or a naturally occurring microbe. A recombinant microbe
comprises a plasmid carrying the iolG gene insert. The iolG gene
insert encodes the enzyme inositol dehydrogenase, which catalyzes
the conversion of myo-inositol to myo-2-inosose. Alternatively, the
iolG gene insert is inserted directly into the genome of the
recombinant microbe. Preferably, the iolG gene is isolated from
Bacillus subtilis. In a preferred embodiment, the second microbe is
a naturally occurring microbe that express inositol dehydrogenase
activity. Examples of such microbes include, but are not limited
to, Bacillus subtilis and Gluconobacter oxydans. In a preferred
embodiment, the second microbe is G. oxydans, which converts
myo-inositol to myo-2-inosose without loss of the myo-2-inosose
product to further catabolism.
[0026] In a preferred embodiment, the recombinant E. coli comprises
plasmid pAD1.88A carrying an INO1 gene insert and a serA gene
insert. As described above, the INO1 gene insert encodes
myo-inositol-1-phosphate synthase which converts
glucose-6-phosphate to myo-inositol-1-phosphate, thus increasing
the carbon flow into the myo-inositol pathway. Due to a mutation in
the E. coli genomic serA locus required for L-serine biosynthesis,
growth in minimal salts medium and plasmid maintenance follows from
expression of plasmid-localized serA. The serA insert thus allows
microbial growth in minimal salts medium, distinguishing the
microbes containing the plasmid from non-plasmid containing
microbes.
[0027] In an alternative embodiment, the recombinant E. coli
comprises a plasmid carrying an INO1 gene insert, an iolG gene
insert and a serA gene insert. As described above, the iolG gene
insert encodes inositol dehydrogenase which catalyzes the
conversion of myo-inositol to myo-2-inosose. In a preferred
embodiment, the plasmid also carries the gene insert for inositol
monophosphatase. While not wishing to be bound by theory,
hydrolysis of myo-inositol-1-phosphate to produce myo-inositol can
occur in the cytosol or periplasm. If a cytoplasmic phosphatase
hydrolyzes myo-inositol-1-phosphate, plasmid-localized INO1 and
iolG will lead to myo-2-inosose synthesis. Periplasmic phosphatase
activity would result in periplasmic production of myo-inositol
while inositol dehydrogenase expression is localized in the
cytoplasm. Transport of myo-inositol from the periplasm into the
cytoplasm is unlikely in E. coli given that this microbe does not
catabolize myo-inositol. To correct for periplasmic phosphatase
activity, plasmid-localization of genes encoding mammalian inositol
monophosphatase, which have been cloned, sequenced, and
successfully expressed in E. coli, would be desirable. Diehl, R. E.
et al., J. Biol. Chem. 265:5946 (1990); McAllister, G. et al.,
Biochem. J. 284:749 (1992). Because of the specificity of this
enzyme for myo-inositol-1-phosphate, a molecule which is not a
normal metabolite in E. coli, cytoplasmic expression of the cDNA
encoding inositol monophosphatase in E. coli should not be
problematic. An E. coli comprising a plasmid carrying both the INO1
gene insert and the iolG gene insert and the gene for inositol
monophosphatase can convert glucose directly to myo-2-inosose. The
myo-2-inosose can then be converted to 1,2,3,4-tetrahydroxybenzene
by an acid-catalyzed dehydration.
[0028] In another embodiment, the recombinant E. coli, designated
JWF1, comprises a plasmid carrying an INO1 gene insert, an iolG
gene insert, and a serA gene insert. This recombinant microbe is
capable of converting glucose to myo-2-inosose. In a preferred
embodiment, the recombinant E. coli comprises plasmid pAD2.28A
carrying an INO1 gene insert, an iolG gene insert and a serA gene
insert. Examples of these recombinant microbes, E. coli
JWF1/pAD1.88A and JWF1/pAD2.28A, are described in Specific Examples
1 and 4, respectively, and have been deposited with the American
Type Culture Collection (ATCC), 10801 University Boulevard,
Manassas, Va. 20110-2209, under the terms of the Budapest Treaty,
and accorded the ATCC designation numbers 207153 and 207154,
respectively. The deposit will be maintained in the ATCC
depository, which is a public depository, for a period of 30 years,
or 5 years after the most recent request, or for the effective life
of a patent, whichever is longer, and will be replaced if the
deposit becomes depleted or non-viable during that period. Samples
of the deposit will become available to the public and all
restrictions imposed on access to the deposit will be removed upon
grant of a patent on this application.
[0029] In yet another embodiment, a recombinant E. coli is employed
to convert glucose to D-2,3-diketo-4-deoxy-epi-inositol. Such a
recombinant E. coli comprises a plasmid carrying the INO1 gene
insert, the iolG gene insert and the gene insert encoding for the
enzyme, myo-2-inosose dehydratase. The plasmid may further comprise
the gene insert for inositol monophosphatase. Myo-2-inosose
dehydratase catalyzes the conversion of myo-2-inosose to
D-2,3-diketo-4-deoxy-epi-inositol which can undergo an
acid-catalyzed dehydration to yield 1,2,3,4-tetrahydroxybenzene.
While not wishing to be bound by theory,
D-2,3-diketo-4-deoxy-epi-inositol is likely the first intermediate
in the acid-catalyzed conversion of myo-2-inosose to
1,2,3,4-tetrahydroxybenzene- . Acid-catalyzed aromatization of
D-2,3-diketo-4-deoxy-epi-inositol would eliminate a step and may
lead to higher yields of 1,2,3,4-tetrahydroxyben- zene as compared
to the acid-catalyzed dehydration of myo-2-inosose.
[0030] It will be appreciated that the INO1 gene, iolG gene, serA
gene and the genes encoding inositol dehydrogenase, inositol
monophosphatase and/or myo-2-inosose dehydratase, can be inserted
directly into the E. coli genome. Such a recombinant E. coli would
not require a plasmid to produce significant amounts of
myo-inositol, myo-2-inosose or
D-2,3-diketo-4-deoxy-epi-inositol.
[0031] Although E. coli is specifically described herein as the
microbe for carrying out the methods of the present invention, it
will be appreciated that any microorganism such as the common types
cited in the literature and known to those skilled in the art, may
be employed, provided the microorganism can be altered to effect
the desired conversion (e.g., carbon source to myo-inositol, carbon
source to myo-2-inosose, carbon source to
D-2,3-diketo-4-deoxy-epi-inositol, myo-inositol to myo-2-inosose,
etc.). Thus it is envisaged that many types of fungi, bacteria and
yeasts will work in the methods of the present invention. Such
microorganisms may be developed, for example, through selection,
mutation, and/or genetic transformation processes with the
characteristic and necessary capability of converting one
constituent of the synthesis scheme of the present invention to
another. Methods for such development are well known to the skilled
practitioner.
[0032] In order to carry out the bioconversion methods of the
present invention, a solution containing a carbon source is
contacted with the recombinant or wild-type microbe to form a
bioconversion mixture which is maintained under appropriate
conditions to promote the conversion of the carbon source to the
desired constituent, e.g., myo-inositol or myo-2-inosose. In a
preferred embodiment, the bioconversion mixture is maintained at a
temperature of about 30.degree. C. to about 37.degree. C. and a pH
of about 6.5 to about 7.5. It is preferred that the bioconversion
mixture also contain other substances necessary to promote
viability of the microbes such as mineral salts, buffers,
cofactors, nutrient substances and the like. The more general
requirements for the maintenance of viability of microorganisms are
well known and specific requirements for maintaining the viability
of specific organisms are also well known as documented in the
literature, or are otherwise easily determined by those skilled in
the art.
[0033] In another embodiment, the myo-inositol produced in the
fermentation broth of the first bioconversion with the first
microbe can be isolated before being utilized in the second
bioconversion. The isolation can be a total isolation to provide
pure myo-inositol as described in Specific Example 1. The isolation
can also be a partial isolation where the fermentation broth is
deproteinized and decolorized before use in the second
bioconversion. Such deproteinization and decolorization are well
known to those skilled in the art. Further purification of the
myo-inositol can be obtained by concentration of the
deproteinized/decolorized fermentation broth to a volume where the
myo-inositol concentration is greater than 50 g/L. At
concentrations greater than 50 g/L, the myo-inositol can be
precipitated out by the addition of methanol. By way of
non-limiting example, myo-inositol can be isolated from the
fermentation broth by the following four steps: ultrafiltration to
remove cells and proteins; decolorization by activated charcoal;
concentration of the fermentation broth to give a myo-inositol
concentration greater than 50 g/L; and precipitation of
myo-inositol by the addition of methanol. The myo-inositol
precipitate can be washed, resuspended in buffer or water, and used
in the second bioconversion to myo-2-inosose.
[0034] Novel methods for converting 1,2,3,4-tetrahydroxybenzene to
1,2,3-trihydroxybenzene are also provided. In one embodiment, a
method is provided wherein 1,2,3,4-tetrahydroxybenzene is reduced
to 1,2,3-trihydroxybenzene.
[0035] In one embodiment, the 1,2,3,4-tetrahydroxybenzene is
converted to 1,2,3-trihydroxybenzene by hydrogenation in the
presence of a catalyst followed by acid catalyzed hydrolysis. In a
preferred embodiment, the catalyst is Rh/Al.sub.2O.sub.3. The
amount of catalyst required and the conditions required for
hydrogenation (e.g., pressure, time) are well known to the skilled
practitioner.
[0036] In another embodiment, the hydrogenation reaction is carried
out in an aqueous 1,2,3,4-tetrahydroxybenzene solution. In a
preferred embodiment, the aqueous 1,2,3,4-tetrahydroxybenzene
solution will be free of any compounds known to quench the
hydrogenolysis catalyst. In a more preferred embodiment, the
aqueous 1,2,3,4-tetrahydroxybenzene solution is comprised of
isolated 1,2,3,4-tetrahydroxybenzene and water.
[0037] In order to more fully demonstrate the advantages arising
from the present invention, the following examples are set forth.
It is to be understood that the following is by way of example only
and is not intended as a limitation on the scope of the
invention.
SPECIFIC EXAMPLE 1
Synthesis of 1,2,3,4-Tetrahydroxybenzene from Glucose
[0038] I. Results
[0039] A synthetic route (FIG. 2) has now been elaborated which
provides convenient access to 1,2,3,4-tetrahydroxybenzene via
myo-inositol intermediacy. The general utility of this route is
demonstrated by a concise synthesis of coenzyme Q.sub.n=3 4. While
the shikimate pathway and polyketide biosynthesis have
traditionally provided biocatalytic access to aromatic chemicals,
syntheses of 1,2,3,4-tetrahydroxybenzene 1 and coenzyme Q.sub.n=3 4
are distinguished by the recruitment of myo-inositol
biosynthesis.
[0040] Synthesis of myo-inositol by E. coli JWF1/pAD1.88A begins
with D-glucose uptake and conversion to D-glucose-6-phosphate
catalyzed by the E. coli phosphotransferase system (Postma, P. W.
et al., In Escherichia coli and Salmonella, 2nd ed., Neidhardt, F.
C. et al., Eds., ASM: Washington, Vol. 1, p. 1149 (1996)) where
phosphoenolpyruvate is the source of the transferred phosphoryl
group. D-Glucose-6-phosphate then undergoes cyclization to
myo-inositol 1-phosphate catalyzed by myo-inositol-1-phosphate
synthase. This enzyme activity, which results from expression of
the Saccharomyces cerevisiae INO1 gene (Dean-Johnson, M. et al., J.
Biol. Chem. 264:1274 (1989)) on plasmid pAD1.88A, varied
significantly (0.022, 0.043, 0.018, and 0.009 .mu.mol/min/mg at 18
h, 30 h, 42 h, and 54 h, respectively) over the course of the
fermentation.
[0041] E. coli JWF1/pAD1.88A synthesized 21 g/L myo-inositol (solid
bars, FIG. 3) and 4 g/L myo-inositol-1-phosphate (open bars, FIG.
3) in 11% combined yield (mol/mol) from D-glucose under fed-batch
fermentor conditions. Both myo-inositol and
myo-inositol-1-phosphate accumulated in the culture supernatant. In
eucaryotes, hydrolysis of myo-inositol-1-phosphate to myo-inositol
is catalyzed by the enzyme inositol monophosphatase. McAllister, G.
et al., Biochem. J. 284:749 (1992). Phosphoester hydrolysis was
fortuitously catalyzed in E. coli JWF1/pAD1.88A by unidentified
cytosolic or periplasmic phosphatase activity.
[0042] Oxidation of myo-inositol to myo-2-inosose, the next step in
the conversion of D-glucose into 1,2,3,4-tetrahydroxybenzene 1, is
the first catabolic step when myo-inositol is used as a sole source
of carbon for growth and metabolism by microbes such as Bacillus
subtilis. Yoshida, K.-I. et al., J. Bacteriol. 179:4591 (1997).
myo-Inositol can also be oxidized by Gluconobacter oxydans without
loss of product myo-2-inosose to catabolism. Postemak, T., Bioch.
Prep. 2:57 (1952). Accordingly, incubation of G. oxydans ATCC 621
in medium containing microbe-synthesized myo-inositol led to the
formation of myo-2-inosose (Scheme 1) in 95% isolated yield.
[0043] Inososes have been thought to be stable under acidic
conditions and reactive under basic conditions with reported
aromatizations resulting from successive .beta.-eliminations being
dominated by formation of 1,2,3,5-tetrahydroxybenzene. Postemak,
T., The Cyclitols, Holden-Day: San Francisco, Chap. 8 (1965);
Angyal, S. J. et al., Carbohydr. Res. 76:121 (1979). However, it
was observed that myo-2-inosose was reactive under acidic
conditions with no apparent formation of 1,2,3,5-tetrahydroxybenze-
ne. Refluxing G. oxydans-produced myo-2-inosose for 9 h in
degassed, aqueous 0.5 M H.sub.2SO.sub.4 under argon cleanly
afforded 1,2,3,4-tetrahydroxybenzene in 66% isolated yield.
II. Materials And Methods
[0044] General. .sup.1H NMR spectra were recorded on a 300 MHz
spectrometer. Chemical shifts for .sup.1H NMR spectra are reported
(in parts per million) relative to internal tetramethylsilane
(Me.sub.4Si, .delta.=0.0 ppm) with CDCl.sub.3 as solvent, to sodium
3-(trimethylsilyl)propionate-2,2,3,3-d.sub.4 (TSP, .delta.=0.0 ppm)
when D.sub.2O was the solvent, and to acetone (CHD.sub.2COCD.sub.3,
.delta.=2.04 ppm) with d.sub.6-acetone. .sup.13C NMR spectra were
recorded at 75 MHz. Chemical shifts for .sup.13C NMR spectra are
reported (in parts per million) relative to CDCl.sub.3
(.delta.=77.0 ppm), relative to CD.sub.3COCD.sub.3 (.delta.=29.8
ppm), and relative to internal CH.sub.3OH (.delta.=49.0 ppm) or
internal CH.sub.3CN (.delta.=1.4 ppm) in D.sub.2O. FAB mass spectra
were performed by University of South Carolina (Columbia, S.C.).
Elemental analyses were performed by Atlantic Microlab Inc.
(Norcross, Ga.). Melting points were uncorrected and were
determined using a Mel-Temp II melting point apparatus.
[0045] Radial chromatography was carried out with a Harrison
Associates Chromatotron using 1, 2 or 4 mm layers of silica gel 60
PF.sub.254 containing gypsum (E. Merck). Silica gel 60 (40-63 .mu.m
E. Merck) was used for flash chromatography. Analytical thin-layer
chromatography (TLC) utilized precoated plates of silica gel 60
F-254 (0.25 mm, E. Merck or Whatman). TLC plates were visualized by
immersion in anisaldehyde stain (by volume: 93% ethanol, 3.5%
sulfuric acid, 1% acetic acid and 2.5% anisaldehyde) followed by
heating. Dimethylformamide, N-methylformanilide and acetone were
dried and stored over activated Linde 4 .ANG. molecular sieves
under Ar. Tetrahydrofuran and diethyl ether were distilled under
nitrogen from sodium benzophenone ketyl. n-Hexanes and TMEDA were
distilled from sodium under Ar and stored over activated Linde 4
.ANG. molecular sieves under Ar. Organic solutions of products were
dried over MgSO.sub.4.
[0046] For .sup.1H NMR quantitation of solute concentrations during
microbial synthesis of myo-inositol and myo-2-inosose, solutions
were concentrated to dryness under reduced pressure, concentrated
to dryness one additional time from D.sub.2O, and then redissolved
in D.sub.2O containing a known concentration of TSP purchased from
Lancaster Synthesis Inc. Concentrations were determined by
comparison of integrals corresponding to each compound with the
integral corresponding to TSP (.delta.=0.00 ppm) in the .sup.1H
NMR. Protein concentrations were determined using the Bradford
dye-binding procedure (Bradford, M. M., Anal. Biochem. 72:248
(1979)) by comparison with a standard curve prepared with bovine
serum albumin. Protein assay solution was purchased from Bio-Rad.
E. coli DH5.alpha. is available from Gibco BRL.
[0047] Culture Medium. All culture solutions were prepared in
distilled, deionized water. LB medium (1 L) contained Bacto
tryptone (10 g), Bacto yeast extract (5 g), and NaCl (10 g). M9
salts (1 L) contained Na.sub.2HPO.sub.4 (6 g), KH.sub.2PO.sub.4 (3
g), NaCl (0.5 g) and NH.sub.4Cl (1 g). M9 minimal medium (1 L)
consisted of 1 L of M9 salts containing D-glucose (10 g),
MgSO.sub.4 (0.12 g), and thiamine hydrochloride (0.001 g).
Ampicillin was added (0.05 g/L) where indicated. Solutions of M9
salts, MgSO.sub.4, and glucose were autoclaved individually and
then mixed. Ampicillin was sterilized through a 0.22 .mu.m
membrane. Solid medium was prepared by addition of 1.5% (w/v) Difco
agar to medium.
[0048] Fermentation medium (1 L) contained K.sub.2HPO.sub.4 (7.5
g), ammonium iron(III) citrate (0.3 g), citric acid monohydrate
(2.1 g), and concentrated H.sub.2SO.sub.4 (1.2 mL). The
fermentation medium was adjusted to pH 7.0 by addition of
concentrated NH.sub.4OH before autoclaving. The following
supplements were added immediately prior to initiation of the
fermentation: D-glucose (20 g), MgSO.sub.4 (0.24 g), and trace
minerals including (NH.sub.4).sub.6(Mo.sub.7O.sub.24).4H.sub.2O
(0.0037 g), ZnSO.sub.4.7H.sub.2O (0.0029 g), H.sub.3BO.sub.3
(0.0247 g), CuSO.sub.4.5H.sub.2O (0.0025 g), and
MnCl.sub.2.4H.sub.2O (0.0158 g). D-Glucose and MgSO.sub.4 were
autoclaved separately while trace minerals were sterilized through
0.22 .mu.m membranes prior to addition to the medium.
[0049] Genetic Manipulations. Standard procedures were used for the
construction, purification, and analysis of plasmid DNA. Sambrook,
J. et al., Molecular Cloning A Laboratory Manual, Cold Spring
Harbor Laboratory Press: New York (1989). E. coli DH5.alpha. served
as the host strain for all plasmid constructions. The INO1 open
reading frame was amplified from pJH318 (Hirsch, J. P. et al., Mol.
Cell. Biol. 6:3320 (1986)) using PCR. Inclusion of EcoRI
recognition sequences facilitated localization of INO1 into the
EcoRI site in pJF118EH (Furste, J. P. et al., Gene 48:119 (1986))
to afford pAD1.45A. Transcription of INO1 in pAD1.45A utilized the
vector-encoded tac promoter (P.sub.tac) which was regulated by
vector-encoded expression of lacl.sup.q. Digestion of pD2625 with
DraI and EcoRV liberated a 1.9 kb serA fragment that was
subsequently ligated into the SmaI site of pAD1.45A to provide
pAD1.88A.
[0050] E. coli JWF1 was prepared by homologous recombination of a
non-functional serA gene into E. coli RB791(W3110 lacl.sup.q).
Localization of the 1.9 kb serA fragment obtained from pD2625 into
pMAK705 (Hamilton, C. M. et al., J. Bacteriol. 171:4617 (1989))
provided pLZ1.68A. Linearization of pLZ1.68A at the unique BamHI
site internal to serA followed by treatment with Klenow fragment
and dNTP's and relegation afforded pLZ1.71A. Homologous
recombination (Ohta, K. et al., Appl. Environ. Microbiol. 57:893
(1991)) of the resulting non-functional serA locus of pLZ1.71A into
RB791 afforded JWF1.
[0051] Myo-Inositol-1-phosphate Synthase Activity. Partial
purification of cellular lysate was required to quantify
myo-inositol-1-phosphate synthase activity over background cellular
phosphatase activity. Cells were collected from 30 mL of
fermentation broth by centrifugation at 2000 g for 6 min at
4.degree. C. Cells were resuspended in 10 mL of resuspension buffer
consisting of Tris.HCl (20 mM), pH 7.4, NH.sub.4Cl (10 mM),
2-mercaptoethanol (.beta.-ME, 10 mM), phenylmethylsulphonyl
fluoride (PMSF, 2 mM), and EDTA (1 mM). Resuspended cells were
frozen at -80.degree. C. for up to 4 days until purification was
carried out.
[0052] Thawed cells were lysed by two passages through a French
press at 2000 psi. Cellular debris was removed by centrifugation at
30000 g for 30 min at 4.degree. C. Clarified cellular lysate
containing approximately 200 mg of protein was loaded onto a DEAE
cellulose column (5.times.25 cm) at 4.degree. C. The column was
eluted with a step gradient of NH.sub.4Cl in the following buffer
(Buffer A): Tris.HCl (20 mM), pH 7.4, .beta.-ME (10 mM), PMSF (1
mM), and EDTA (1 mM). The step gradient consisted of 20 mL of
Buffer A with NH.sub.4Cl (10 mM), 45 mL of Buffer A with NH.sub.4Cl
(90 mM), and 100 mL of Buffer A with NH.sub.4Cl (150 mM). Fractions
(9 mL) were collected throughout the step gradient. Fractions 10-18
were collected and concentrated to less than 5 mL using an Amicon
Ultrafiltration Stirred Cell equipped with a PM10 membrane.
Concentrated protein (1.5-2.0 mg) was used to measure
myo-inositol-1-phosphate synthase activity. The
myo-inositol-1-phosphate synthase activity was measured as
previously reported (Migaud, M. E. et al., J. Am. Chem. Soc.
118:495 (1996)) except that the assay solution contained Tris.HCl
(20 mM), pH 7.4, NH.sub.4Cl (2 mM), and DTT (0.2 mM).
[0053] myo-Inositol. Fermentations employed a 2.0 L working
capacity B. Braun MD2 culture vessel. Utilities were supplied by a
B. Braun Biostat MD controlled by a Dell Optiplex Gs.sup.+ 5166
personal computer equipped with B. Braun MFCS/Win software.
Temperature, pH, and glucose feeding were controlled with PID
control loops. Temperature was maintained at 33.degree. C. pH was
maintained at 7.0 by addition of concentrated NH.sub.4OH or 2 N
H.sub.2SO.sub.4. Dissolved oxygen (D.O.) was measured using a
Mettler-Toledo 12 mm sterilizable O.sub.2 sensor fitted with an
Ingold A-type O.sub.2 permeable membrane. D.O. was maintained at
10% air saturation. Antifoam (Sigma 204) was added manually as
needed.
[0054] Inoculants were started by introduction of a single colony
of JWF1/pAD1.88A into 100 mL M9 medium containing ampicillin. The
culture was grown at 37.degree. C. with agitation at 250 rpm for 15
h and then transferred to the fermentation vessel. The initial
glucose concentration in the fermentation medium was 20 g/L. Three
staged methods were used to maintain D.O. levels at 10% air
saturation during each fermentor run. With the airflow at an
initial setting of 0.06 L/L/min, D.O. concentration was maintained
by increasing impeller speed from its initial set point of 50 rpm
to its preset maximum of 940 rpm. Approximately 7 h was required
for the impeller speed to increase to 940 rpm. With the impeller
constant at 940 rpm, the mass flow controller then maintained D.O.
levels by increasing the airflow rate from 0.06 L/L/min to its
preset maximum of 1.0 L/L/min over approximately 1.5 h. At constant
impeller speed and constant airflow rate, D.O. levels were
maintained at 10% saturation for the remainder of the fermentation
by oxygen sensor-controlled glucose feeding. At the beginning of
this stage, D.O. levels fell below 10% air saturation due to
residual initial glucose in the medium. This lasted for
approximately 50 min before glucose (60% w/v) feeding started. The
PID control parameters were set to 0.0 (off) for the derivative
control (.tau..sub.D) and 999.9 s (minimum control action) for
integral control (.tau..sub.I). X.sub.p was set to 950% to achieve
a K.sub.c of 0.1.
[0055] Samples (6 mL) of fermentation broth were taken at 6 h
intervals starting at 12 h.
Isopropyl-.beta.-D-thiogalactopyranoside (4.8 mg) was added when
both the impeller speed and airflow had reached the maximum
settings, and again at 12 h and every 6 h thereafter. Cell
densities were determined by dilution of fermentation broth with
water (1:100) followed by measurement of absorption at 600 nm
(OD.sub.600). Dry cell weight (g/L) was obtained using a conversion
coefficient of 0.43 g/L/OD.sub.600. Fermentation broth was
centrifuged to remove cells. Solute concentrations in cell-free
broth were determined by .sup.1H NMR. Fermentation broth (30 mL)
was removed at designated times for assay of
myo-inositol-1-phosphate synthase activity. The final concentration
of myo-inositol at 54 h was 20.9 g/L synthesized in 8.7% yield
(mol/mol) from glucose.
[0056] The fermentation broth (950-1200 mL) was centrifuged at
18000 g for 35 min at 4.degree. C. and the cells discarded. The
resulting supernatant was acidified to pH 2.0 with concentrated
H.sub.2SO.sub.4 and then centrifuged at 18000 g for 20 min to
remove precipitated proteins. The clear yellow supernatant was
neutralized with concentrated NH.sub.4OH. The solution was
decolorized with Darco KB-B activated carbon (10 g/L) for 4 h with
agitation at 50 rpm and subsequently filtered through Whatman 2
filter paper. The filtered material was washed with an additional
200 mL of water.
[0057] The combined filtrates were applied to a column of AG1-x8
(acetate form, 5 cm.times.20 cm) at 4.degree. C. and eluted with 1
L H.sub.2O. The entire eluent (approximately 2.3 L) was then run
through a column of Dowex 50 (H.sup.+ form, 5 cm.times.20 cm) at
4.degree. C. and eluted with 500 mL H.sub.2O. The resulting
solution (approximately 2.8 L) was concentrated to 200 mL by
boiling and then concentrated to dryness under reduced pressure.
The resulting powder was dissolved in a minimal volume of H.sub.2O,
diluted with 6 volumes of MeOH, and stored at 4.degree. C. to
crystallize. Crystals were collected after a few days, washed with
MeOH, allowed to air dry overnight, and dried under vacuum to yield
white crystals (78% recovery based on inositol quantified in crude
fermentation broth). .sup.1H NMR (D.sub.2P) .delta. 4.06 (dd, J=3,
3 Hz, 1H), 3.61 (dd, J=10, 9 Hz, 2H), 3.53 (ddd, J=10, 3, 1 Hz,
2H), 3.28 (ddd, J=9, 9, 1 Hz, 1H). .sup.13C NMR (D.sub.2O) .delta.
45.3, 43.4, 43.2, 42.1.
[0058] myo-2-Inosose. Angyal, S. J. et al. Carbohydr. Res. 76:121
(1979); Posternak, T., Biochem. Prep. 2:57 (1952). A solution
containing sorbitol (1.0 g) and yeast extract (0.05 g) in 10 mL
distilled, deionized water was autoclaved for 25 min and cooled to
room temperature. After inoculation with Gluconobacter oxydans ATCC
621 the culture was incubated in an orbital shaker at 200 rpm for
24 h at 30.degree. C. This G. oxydans culture was subsequently
added to a second sterile solution containing myo-inositol (12.0 g,
66.7 mmol), D-sorbitol (0.4 g), and yeast extract (2.0 g) in 400 mL
distilled, deionized water. After incubation in an orbital shaker
at 200 rpm for 48 h at 30.degree. C., cells were removed by
centrifugation. The resulting culture supernatant was concentrated
to 75 mL, MeOH (400 mL) added, and the solution maintained at
-20.degree. C. for 12 h. Precipitate which formed was filtered,
washed with MeOH, and dried to afford myo-2-inosose as a white
powder (8.17 g, 69%). A second crop of myo-2-inosose (3.09 g, 26%)
was obtained after maintaining the filtrate at -20.degree. C. for
an additional 12 h. mp 188-192.degree. C. .sup.1H NMR (D.sub.2O):
.delta. 4.25 (d, J=10 Hz, 2H), 3.66 (dd, J=9, 9 Hz), 3.26 (m, 2H).
.sup.13C NMR (D.sub.2O): .delta. 206.0, 94.3, 76.2, 74.5, 74.1,
74.0, 73.3, 73.2.
[0059] 1,2,3,4-Tetrahydroxybenzene 1. A solution of myo-2-inosose
(11.0 g, 61.2 mmol) in 310 mL of degassed 0.5 M H.sub.2SO.sub.4 was
refluxed under Ar. After 9 h, the solution was cooled to 4.degree.
C. and then adjusted to pH 4 by addition of saturated aqueous
NaHCO.sub.3. Concentration of the reaction solution to 100 mL was
followed by continuous liquid-liquid extraction for 18 h using
t-butyl methyl ether (500 mL). Upon concentration of the organic
layer to 100 mL, a precipitate formed which was filtered, washed
with cold hexanes, and dried to afford 1 (4.72 g, 54%) as a tan
powder. Addition of hexanes (300 mL) to the filtrate followed by
filtering, washing, and drying of the resulting precipitate
afforded additional 1 (1.08 g, 12%). mp 162-164.degree. C. .sup.1H
NMR (d.sub.6-acetone):.delta. 7.24 (s, 4H), 6.20 (s, 2H). .sup.13C
NMR (d.sub.6-acetone): .delta. 139.7, 134.7, 106.2. Anal. Calcd for
C.sub.6H.sub.6O.sub.4: C, 50.71; H, 4.23. Found: C, 50.63; H, 4.32.
HRMS (FAB) calcd for C.sub.6H.sub.6O.sub.4 (M+H.sup.+): 142.0266.
Found: 142.0268.
SPECIFIC EXAMPLE 2
Chemical Synthesis of 1,2,3,4-Tetrahydroxybenzene
[0060] I. Results
[0061] Conversion of D-glucose into 1,2,3,4-tetrahydroxybenzene 1
is a three step synthesis. 1,2,3,4-Tetrahydroxybenzene 1 has
historically been obtained from pyrogallol 6 by a longer route
(FIG. 4) involving synthesis and subsequent hydrolysis of
aminopyrogallol 7. Leston, G., In Kirk-Othmer Encyclopedia of
Chemical Technology: Fourth Ed., Kroschwitz, J. I. et al. Ed.,
Wiley: New York, Vol. 19, p. 778 (1996); Einhorn, A. et al., Ber.
37:110 (1904). Due to the tedious nature of this synthesis
(Einhorn, A. et al., Ber. 37:110 (1904)), two alternate routes
(FIG. 4) were developed to obtain authentic samples of
1,2,3,4-tetrahydroxybenzene 1. Low-yielding, direct hydroxylation
of protected pyrogallol 8 or higher-yielding, indirect oxidation
via formyl 10 intermediacy yielded, respectively, quinone 9 and
phenol 11. Hydrogenation of 9 and 11 afforded products which were
identical to 1,2,3,4-tetrahydroxybenzene 1 synthesized (FIG. 2)
from D-glucose.
[0062] II. Materials and Methods
[0063] General. .sup.1H NMR spectra were recorded on a 300 MHz
spectrometer. Chemical shifts for .sup.1H NMR spectra are reported
(in parts per million) relative to internal tetramethylsilane
(Me.sub.4Si, .delta.=0.0 ppm) with CDCl.sub.3 as solvent, to sodium
3-(trimethylsilyl)propionate-2,2,3,3-d.sub.4 (TSP, .delta.=0.0 ppm)
when D.sub.2O was the solvent, and to acetone (CHD.sub.2COCD.sub.3,
.delta.=2.04 ppm) with d.sub.6-acetone. .sup.13C NMR spectra were
recorded at 75 MHz. Chemical shifts for .sup.13C NMR spectra are
reported (in parts per million) relative to CDCl.sub.3
(.delta.=77.0 ppm), relative to CD.sub.3COCD.sub.3 (.delta.=29.8
ppm), and relative to internal CH.sub.3OH (.delta.=49.0 ppm) or
internal CH.sub.3CN (.delta.=1.4 ppm) in D.sub.2O. FAB mass spectra
were performed by University of South Carolina (Columbia, S.C.).
Elemental analyses were performed by Atlantic Microlab Inc.
(Norcross, Ga.). Melting points were uncorrected and were
determined using a Mel-Temp II melting point apparatus.
[0064] Radial chromatography was carried out with a Harrison
Associates Chromatotron using 1, 2 or 4 mm layers of silica gel 60
PF.sub.254 containing gypsum (E. Merck). Silica gel 60 (40-63 .mu.m
E. Merck) was used for flash chromatography. Analytical thin-layer
chromatography (TLC) utilized precoated plates of silica gel 60
F-254 (0.25 mm, E. Merck or Whatman). TLC plates were visualized by
immersion in anisaldehyde stain (by volume: 93% ethanol, 3.5%
sulfuric acid, 1% acetic acid and 2.5% anisaldehyde) followed by
heating. Dimethylformamide, N-methylformanilide and acetone were
dried and stored over activated Linde 4 .ANG. molecular sieves
under Ar. Tetrahydrofuran and diethyl ether were distilled under
nitrogen from sodium benzophenone ketyl. n-Hexanes and TMEDA were
distilled from sodium under Ar and stored over activated Linde 4
.ANG. molecular sieves under Ar. Organic solutions of products were
dried over MgSO.sub.4.
[0065] Hydrogenation of 2,3-dibenzyloxy-1,4-benzoquinone 9. A
solution of 9 (0.18 g, 0.56 mmol) in EtOH (7.0 mL) was stirred with
10% Pd on C (0.050 g) at room temperature under H.sub.2 (1.0 atm)
for 3 h. The solution was filtered through Celite.RTM. and
concentrated to afford a tan solid (0.079 g, 99%) which was
identical by .sup.1H and .sup.13C NMR to 1 obtained from
myo-2-inosose. Hydrogenation of 2,3,4-tribenzyloxyphenol 11. A
solution of 11 (5.8 g, 14.1 mmol) in EtOH (100 mL) was stirred with
10% Pd on C (1.0 g) at room temperature under H.sub.2 (1.0 atm) for
2 h. The solution was filtered through Celite.RTM. and
concentrated. The residue was purified by flash chromatography
(MeOH/CH.sub.2Cl.sub.2, 1:9, v/v) affording a product which was
identical by .sup.1H and .sup.13C NMR to 1 obtained from
myo-2-inosose.
[0066] 1,2,3-Tribenzyloxybenzene 8. Lowe, W. et al., Arch. Pharm.
(Weinheim) 327:255 (1994). Benzyl bromide (57 mL, 0.481 mol) and
then K.sub.2CO.sub.3 (100 g, 0.725 mol) were added to a solution of
pyrogallol (20 g, 0.159 mol) in 200 mL dry, degassed acetone under
Ar. This reaction mixture was subsequently stirred for 30 min at
room temperature and then for 24 h at reflux under Ar. A solution
of NaOH (1.6 g) in MeOH (32 mL) was then added and the reaction
refluxed for an additional 30 min. After cooling to room
temperature, solids were filtered and washed with acetone. The
filtrate was concentrated and the residue recrystallized from MeOH
to afford 8 (52 g, 83%) as an off-white solid. mp 67-68.degree. C.
.sup.1H NMR (CDCl.sub.3): .delta. 7.44-7.19 (m, 15H), 6.85 (dd,
J=9, 9 Hz, 1H), 6.57 (d, J=8.2 Hz, 2H), 5.05 (s, 2H), 5.02 (s, 4H).
.sup.13C NMR (CDCl.sub.3): .delta. 152.9, 138.4, 137.8, 137.0,
128.4, 128.3, 128.0, 127.8, 127.6 (2), 127.3, 127.2, 123.5, 107.7,
75.0, 70.9.
[0067] 2,3-Dibenzyloxy-1,4-benzoquinone 9. To a solution of
1,2,3-tribenzyloxybenzene 8 (2.0 g, 5.0 mmol) in HOAc (30 mL),
K.sub.3Fe(CN).sub.6 (0.82 g, 2.5 mmol) and 30% H.sub.2O.sub.2 (1.3
g, 11.5 mmol) were added and the resulting solution stirred at room
temperature for 18 h. The solution was diluted with 50 mL
CH.sub.2Cl.sub.2 and the organic layer subsequently washed with
H.sub.2O, saturated aqueous NaHCO.sub.3 and brine. Drying and
concentration resulted in a red oil. Purification by radial
chromatography (2 mm thickness, EtOAc/hexane, 1:19, v/v) afforded 9
as a red oil. .sup.1H NMR (CDCl.sub.3): .delta. 7.36-7.32 (m, 10H),
6.58 (s, 2H), 5.20 (s, 4H). .sup.13C NMR (CDCl.sub.3): .delta.
184.1, 145.2, 136.1, 134.6, 128.5, 128.4, 128.1, 75.1. Anal. Calcd
for C.sub.20H.sub.16O.sub.4: C, 74.99; H, 5.03. Found: C, 75.04; H,
5.06. HRMS (FAB) calcd for C.sub.20H.sub.16O.sub.4 (M+H.sup.+):
320.1049. Found: 320.1059.
[0068] 2,3,4-Tribenzyloxybenzaldehyde 10. Kolonits, P. et al., Acta
Chim. Hung. 113:367 (1983). POCl.sub.3 (155 mL, 1.66 mol) was
slowly added to N-methylformanilide (175 mL, 1.4 mol) at room
temperature under Ar which resulted in formation of a yellow solid.
After 2 h, the solid was treated with a solution of
1,2,3-tribenzyloxybenzene 8 (20 g, 51 mmol) in anhydrous DMF (40
mL) and heated to 60.degree. C. After 3 h, the resulting crimson
solution was cooled to room temperature and then poured into ice
water (3 L) with vigorous stirring for 12 h. The resulting brown
precipitate was filtered, washed with hexanes (3.times.100 mL) and
finally recrystallized from MeOH to afford 10 (19.8 g, 93%) as a
white powder. mp 73-74.degree. C. .sup.1H NMR (CDCl.sub.3): .delta.
10.11 (s, 1H), 7.57 (d, J=9 Hz, 1H), 7.44-7.28 (m, 15H), 6.83 (d,
J=9 Hz, 1H), 5.21(s, 2H), 5.16 (s, 2H), 5.08 (s, 2H); .sup.13C NMR
(CDCl.sub.3): .delta. 188.8, 158.5, 155.9, 141.1, 136.9, 136.2,
135.8, 128.6, 128.5 (2), 128.3 (2), 128.2, 127.5, 124.0, 109.1,
76.8, 75.5, 70.9. Anal. Calcd for C.sub.28H.sub.24O.sub.4: C,
79.22; H, 5.70. Found: C, 79.17; H, 5.80. HRMS (FAB) calcd for
C.sub.28H.sub.24O.sub.4 (M+H.sup.+): 424.1675. Found: 424.1669.
[0069] 2,3,4-Tribenzyloxyphenol 11. Kolonits, P. et al., Acta Chim
Hung. 113:367 (1983). A solution of 30% H.sub.2O.sub.2 (6 mL, 57.8
mmol) and 85% formic acid (32 mL, 600 mmol) was added dropwise to a
solution of 2,3,4-tribenzyloxybenzaldehyde 10 (9.8 g, 23.1 mmol) in
CH.sub.2Cl.sub.2 (50 mL) over 30 min at 0.degree. C. After 1 h of
stirring at 0.degree. C., the reaction was stirred at room
temperature for 24 h. The reaction was subsequently cooled to
4.degree. C. and diluted with 10% (w/v) aqueous Na.sub.2SO.sub.3
(50 mL). The aqueous phase was washed with CH.sub.2Cl.sub.2
(3.times.40 mL). Drying and concentration afforded a brown oil
which was dissolved in a methanolic solution of NaOMe (30 mL, 0.1
N) and refluxed. After 10 min, the solution was cooled to 4.degree.
C. and acidified with 6 N HCl. MeOH was removed in vacuo. The
mixture was diluted with H.sub.2O (15 mL) followed by extraction of
the aqueous phase with benzene (3.times.40 mL). Drying and
concentration afforded 11 (9.0 g, 95%) as a brown oil. .sup.1H NMR
(CDCl.sub.3): .delta. 7.45-7.31 (m, 15H), 6.65 (d, J=9 Hz, 1H),
6.58 (d, J=9 Hz, 1H), 5.28 (s, 1H), 5.12 (s, 2H), 5.11 (s, 2H),
5.04 (s, 2H). .sup.13C NMR (CDCl.sub.3): .delta. 146.0, 144.0,
142.0, 139.6, 137.3, 137.1, 136.8, 128.4, 128.3 (2), 128.2, 127.9,
127.7, 127.4, 110.4, 109.0, 75.6, 75.3, 71.7. Anal. Calcd for
C.sub.27H.sub.24O.sub.4: C, 78.62; H, 5.87. Found: C, 78.71; H,
5.86. HRMS (FAB) calcd for C.sub.27H.sub.24O.sub.4 (M+H.sup.+):
412.1675. Found: 412.1673.
SPECIFIC EXAMPLE 3
Synthesis of Coenzyme Q from 1,2,3,4-Tetrahydroxybenzene
[0070] I. Results
[0071] Variations in strategies employed for hydroxyl protection
combined with the ease of metallation and alkylation of the
aromatic nucleus makes 1 a versatile intermediate for the synthesis
of a wide spectrum of naturally-occurring
1,2,3,4-tetrahydroxybenzene derivatives. For example,
permethylation (FIG. 5) of 1 leads to tetramethyl 12 which
undergoes facile lithiation and methylation affording 13 in high
yield. Formation of an organocuprate from 13, farnesylation, and
subsequent reaction with (NH.sub.4).sub.2Ce(NO.sub.3).sub.6 affords
coenzyme Q.sub.n=34. This four-step synthesis of coenzyme Q.sub.n
from tetrahydroxybenzene 1 is equal in length to the shortest
reported (Keinan, E. et al., J. Org. Chem. 52:3872 (1987))
synthesis of coenzyme Q.sub.n which uses p-cresol as a starting
material and substantially shorter than syntheses of coenzyme
Q.sub.n from pyrogallol, gallic acid, or vanillin. Syper, L. et
al., Tetrahedron 36:123 (1980); Sugihara, H. et al., Liebigs Ann.
Chem. 763:109 (1972); Mayer, J. et al., Meth. Enzymol. 18:182
(1971).
[0072] Only one oxygen atom in coenzyme Q.sub.n, a shikimate
pathway product, is directly derived from D-glucose. The remaining
oxygen atoms are derived from O.sub.2 via enzyme-catalyzed
hydroxylations. Trihydroxybenzenes, pyrogallol, and phloroglucinol
possess the maximum number of oxygen atoms attached to a benzene
nucleus by the shikimate pathway or polyketide biosynthesis in lieu
of enzyme-catalyzed hydroxylation. At least a dozen enzymes are
required to disassemble and reassemble the carbon atoms of
D-glucose into the benzene nucleus of coenzyme Q.sub.n,
pyrogallols, and phloroglucinols. By comparison, synthesis of
1,2,3,4-tetrahydroxybenzene 1 via myo-inositol intermediacy
requires only four enzymes and an acid-catalyzed dehydration for
all six carbon and all four oxygen atoms to be directly derived
from the carbon and oxygen atoms of D-glucose. The method of the
present invention for synthesis of 1,2,3,4-tetrahydroxybenzene 1 is
thus a useful example of enzyme and atom (Trost, B. M., In Green
Chemistry, Anastas, P. T., Williamson, T. C. Eds., Oxford: New
York, Chap. 6 (1998)) economy in organic synthesis in addition to
being a significant strategic departure from previous biocatalytic
syntheses of aromatic chemicals from D-glucose.
[0073] II. Materials And Methods
[0074] General. .sup.1H NMR spectra were recorded on a 300 MHz
spectrometer. Chemical shifts for .sup.1H NMR spectra are reported
(in parts per million) relative to internal tetramethylsilane
(Me.sub.4Si, .delta.=0.0 ppm) with CDCl.sub.3 as solvent, to sodium
3-(trimethylsilyl)propionate-2,2,3,3-d.sub.4 (TSP, .delta.=0.0 ppm)
when D.sub.2O was the solvent, and to acetone (CHD.sub.2COCD.sub.3,
.delta.=2.04 ppm) with d.sub.6-acetone. .sup.13C NMR spectra were
recorded at 75 MHz. Chemical shifts for .sup.13C NMR spectra are
reported (in parts per million) relative to CDCl.sub.3
(.delta.=77.0 ppm), relative to CD.sub.3COCD.sub.3 (.delta.=29.8
ppm), and relative to internal CH.sub.3OH (.delta.=49.0 ppm) or
internal CH.sub.3CN (.delta.=1.4 ppm) in D.sub.2O. FAB mass spectra
were performed by University of South Carolina (Columbia, S.C.).
Elemental analyses were performed by Atlantic Microlab Inc.
(Norcross, Ga.). Melting points were uncorrected and were
determined using a Mel-Temp II melting point apparatus.
[0075] Radial chromatography was carried out with a Harrison
Associates Chromatotron using 1, 2 or 4 mm layers of silica gel 60
PF.sub.254 containing gypsum (E. Merck). Silica gel 60 (40-63 .mu.m
E. Merck) was used for flash chromatography. Analytical thin-layer
chromatography (TLC) utilized precoated plates of silica gel 60
F-254 (0.25 mm, E. Merck or Whatman). TLC plates were visualized by
immersion in anisaldehyde stain (by volume: 93% ethanol, 3.5%
sulfuric acid, 1% acetic acid and 2.5% anisaldehyde) followed by
heating. Dimethylformamide, N-methylformanilide and acetone were
dried and stored over activated Linde 4 .ANG. molecular sieves
under Ar. Tetrahydrofuran and diethyl ether were distilled under
nitrogen from sodium benzophenone ketyl. n-Hexanes and TMEDA were
distilled from sodium under Ar and stored over activated Linde 4
.ANG. molecular sieves under Ar. Organic solutions of products were
dried over MgSO.sub.4.
[0076] 1,2,3,4-Tetramethoxybenzene 12. Syper, L. et al.,
Tetrahedron 36:123 (1980). A solution of
1,2,3,4-tetrahydroxybenzene 1 (8.4 g, 59 mmol) and dimethyl sulfate
(37.5 mL, 0.396 mol) in EtOH (21 mL) was added dropwise to an 8.5 M
aqueous solution of NaOH (42 mL) over 20 min at room temperature.
After 2 h, the reaction was diluted with H.sub.2O (300) mL and
cooled to -20.degree. C. for 12 h. The resulting precipitate was
filtered, washed with H.sub.2O, and then recrystallized from
hexanes to afford 12 (8.12 g, 69%) as colorless needles. mp
84-85.degree. C. .sup.1H NMR (CDCl.sub.3): .delta. 6.58 (s, 2H),
3.90 (s, 6H) 3.82 (s, 6H). .sup.13C NMR (CDCl.sub.3): .delta.
147.7, 143.3, 106.3, 61.1, 56.3. Anal. Calcd for
C.sub.10H.sub.14O.sub.4: C, 60.59; H, 7.12. Found: C, 60.44; H,
7.07.
[0077] 2,3,4,5-Tetramethoxytoluene 13. Syper, L. et al.,
Tetrahedron 36:123 (1980). To a solution of
1,2,3,4-tetramethoxybenzene 12 (4.0 g, 20.2 mmol) and TMEDA (6 mL,
38.0 mmol) in hexanes (44 mL) and THF (80 mL) at 0.degree. C. under
Ar, n-BuLi in hexane (1.6 M, 25.6 mmol) was added dropwise over a
10 min period and the reaction stirred for 30 min at 0.degree. C.
under Ar. Subsequent to dropwise addition of CH.sub.3I (20 mL, 160
mmol) over an 8 min period, the reaction was stirred for 3 h at
0.degree. C. under Ar and then quenched by addition of aqueous
NH.sub.4Cl and ether (20 mL). The organic layer was sequentially
washed with concentrated NH.sub.4OH, water, and brine. Drying and
concentration of the organic layer was followed by purification of
the residue by flash chromatography (hexanes, hexanes/EtOAc, 19:1,
v/v) to afford 13 as a clear oil (3.6 g, 83%). .sup.1H NMR
(CDCl.sub.3): .delta. 6.45 (s, 1H), 3.93 (s, 3H), 3.87 (s, 3H),
3.82 (s, 3H), 3.79 (s, 3H), 2.23 (s, 3H). .sup.13C NMR
(CDCl.sub.3): .delta. 149.0, 146.9, 145.3, 140.7, 125.7, 108.2,
61.0, 60.9, 60.5, 55.9, 15.7.
[0078] Protected coenzyme Q.sub.3 14. Keinan, E. et al., J. Org.
Chem. 52:3872 (1987). n-BuLi (1.6 M, 0.9 mL) was added dropwise
over a 15 min period to a solution of 1,2,3,4-tetramethoxytoluene
13 (0.212 g, 1 mmol) and TMEDA (0.3 mL, 1.9 mmol) in hexane (2.2
mL) at 0.degree. C. under Ar. This yellow precipitate-containing
reaction mixture was then stirred at 0.degree. C. under Ar for 30
min, diluted with THF (4 mL) and ether (11 mL), followed by
addition of CuCN (0.125 g, 1.4 mmol). After stirring for 30 min at
0.degree. C. under Ar, the temperature was reduced to -78.degree.
C., and a solution of farnesyl bromide (0.285 g, 1 mmol) in hexane
(2 mL) was dropwise added over a 30 min period. Further reaction
for 3 h at -78.degree. C. and subsequent slow warming to room
temperature was followed by addition of saturated aqueous
NH.sub.4Cl (10 mL) and ether (20 mL). Washing the organic phase
with concentrated NH.sub.4OH, water, and brine was followed by
drying and concentration. Purification of the residue by radial
chromatography (2 mm thickness, hexane/EtOAc, 9:1, v/v) afforded 14
as a clear oil (0.236 g, 57%). .sup.1H NMR (CDCl.sub.3): .delta.
5.12-5.01 (m, 3H), 3.90 (s, 6H), 3.78 (s, 6H), 3.32 (d, J=7 Hz,
2H), 2.14 (s, 3H), 2.08-1.91 (m, 8H), 1.77 (s, 3H), 1.66 (s, 3H),
1.58 (s, 6H). .sup.13C NMR (CDCl.sub.3): .delta. 147.8, 147.6,
144.9, 144.6, 135.0, 134.9, 131.2, 129.2, 125.4, 124.3, 124.1,
122.8, 61.1, 60.6, 39.7, 26.7, 26.5, 25.7, 25.6, 17.6, 16.2, 15.9,
11.7.
[0079] Coenzyme Q.sub.3 4. Keinan, E. et al., J. Org. Chem. 52:3872
(1987). A suspension maintained at 0.degree. C. resulting from
addition of pyridine-2,6-dicarboxylate (0.125 g, 0.75 mmol) to a
solution of protected coenzyme Q.sub.3 14 in CH.sub.3CN (1.4 mL)
and water (0.6 mL) at 0.degree. C. was reacted with a 0.degree. C.
solution of (NH.sub.4).sub.2Ce(NO.sub.3).sub.6 (0.411 g, 0.75 mmol)
in CH.sub.3CN (0.4 mL) and water (0.4 mL) added dropwise over a 10
min period. After 40 min at 0.degree. C., the reaction was warmed
to room temperature and stirred for 20 min. Water (10 mL) was added
to the reaction mixture and the resulting solution extracted with
CH.sub.2Cl.sub.2 (3.times.100 mL). The combined organic phases were
dried, concentrated, and purified by radial chromatography (1 mm
thickness, hexane/EtOAc, 19:1, v/v) to afford 4 (0.053 g, 46%) as
an orange oil. .sup.1H NMR (CDCl.sub.3): .delta. 5.07 (dd, J=7, 7
Hz, 1H), 5.05 (dd, J=7,7 Hz, 1H), 4.94 (dd, J=7,7 Hz, 1H), 3.99 (s,
3H), 3.98 (s, 3H), 3.18 (d, J=6.8 Hz, 2H), 2.08-1.91 (m, 8H), 2.01
(s, 3H), 1.74 (s, 3H), 1.67 (s, 3H), 1.59 (s, 3H), 1.58 (s, 3H);
.sup.13C NMR (CDCl.sub.3): .delta..sub.--8184.7, 183.9, 144.3,
144.2, 141.6, 138.8, 137.6, 135.2, 131.3, 124.3, 123.8, 118.8,
61.1, 39.7, 26.7, 26.4, 25.7, 25.3, 17.6, 16.3, 16.0, 11.9; HRMS
(FAB) calcd for C.sub.24H.sub.34O.sub.4 (M+H.sup.+): 386.2457.
Found: 386.2461
SPECIFIC EXAMPLE 4
Synthesis of Myo-2-Inosose by a Single Microbe
[0080] Myo-2-inosose (1 g/L), myo-inositol (18 g/L), and
myo-inositol-1-phosphate (3.1 g/L) were synthesized by E. coli
JWF1/pAD2.28A in 9.6% (mol/mol) yield from glucose under fed-batch
fermentation conditions as described in Specific Example 1. The
fermentation ran for 54 h with incremental addition of IPTG (0.0048
g added each time) at 7 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, 48
h, and 54 h. The myo-2-inosose can then be converted to
1,2,3,4-tetrahydroxybenzene by acid-catalyzed dehydration as
described in Specific Example 1.
[0081] Plasmid pAD2.28A carries INO1 under P.sub.tac promoter
control, iolG under P.sub.lac promoter control, lacl.sup.q, and
serA. INO1 encodes myo-inositol-1-phosphate synthase and was cloned
out of Saccharomyces cerevisiae. Inositol dehydrogenase is encoded
by the iolG locus, which was cloned out of Bacillus subtilis.
SPECIFIC EXAMPLE 5
Conversion of 1,2,3,4-Tetrahydroxybenzene to Pyrogallol
[0082] A solution consisting of 0.4 g of NaOH (10 mmol) dissolved
in 10 mL H.sub.2O was freeze-thaw degassed three times under Ar.
This solution was then added via cannula under Ar to a 250 mL Parr
bottle containing 1,2,3,4-tetrahydroxybenzene (1.42 g, 10 mmol) and
5% Rh/Al.sub.2O.sub.3 (0.25 g), which had been flushed with Ar and
then sealed with a septum. The resulting red/brown solution was
hydrogenated under 50 psi. H.sub.2 using a Parr Hydrogenator. After
12 h, the solution was filtered through Celite.RTM. and the
catalyst rinsed with 10 mL H.sub.2O. The resulting dark brown
solution was adjusted to pH=6.0 with 10% HCl and then concentrated
to a brown oil. The oil was dissolved in 50 mL 0.5 M
H.sub.2SO.sub.4 that had been degassed by aeration with Ar for 20
minutes. The solution was heated to reflux under Ar for 12 h,
cooled to room temperature, and then extracted with Et.sub.2O
(4.times.50 mL). The combined organic layers were dried over
MgSO.sub.4, filtered and concentrated to a brown oil. Kugel-Rohr
distillation of the oil under vacuum (1 mm Hg) at 90.degree. C.
afforded 0.56 g pyrogallol (4.44 mmol, 44% yield) as a white,
crystalline solid.
[0083] The foregoing discussion discloses and describes merely
exemplary embodiments of the present invention. One skilled in the
art will readily recognize from such discussion, and from the
accompanying drawings and claims, that various changes,
modifications and variations can be made therein without departing
from the spirit and scope of the invention as defined in the
following claims.
[0084] All references cited herein are incorporated by reference as
if fully set forth.
* * * * *