11590 J. Am. Chem. Soc., Vol. 118, No. 46, 1996
Richman et al.
Reaction of DHS with Hydrogen Peroxide. DHS (1 g, 5.81 mmol)
Clearly, DHS and its enediol tautomers are quite reactive with
O2 and H2O2. As for the phenolic products formed from DHS,
gallic acid is a phenolic radical scavenger and is known to form
complexes with metals.14c Protocatechuic acid is also a phenolic
radical scavenger precedented to possess antioxidant activity.15
Ultimately, the ease of synthesizing DHS may prove to be
as important as its antioxidant activity. Propyl gallate is derived
from esterification of gallic acid which is isolated from gall
nuts and tara powder whose supply has historically been
unreliable.16 R-Tocopherol is obtained by an expensive isolation
process from deodorized soybean oil17 which is in short supply.18
By contrast, suitably engineered microbes can synthesize DHS
in a single step from D-glucose.2 This starting material, in turn,
is derived from inexpensive plant starch whose supplies are
reliable and abundant. Depending on its toxicological profile
in mammals and the particular antioxidant application, DHS
could supplant expensive antioxidants such as propyl gallate19a
and TBHQ.19b The natural biogenesis of DHS may also allow
it to be used in applications previously restricted to natural
antioxidants such as R-tocopherol.
and H2O2 (2 wt%, 10 mL) were dissolved in 20 mL of 1.0 M Na1.5H1.5
-
PO4 and the solution adjusted to pH 6.7. After 36 h reaction at 40 °C,
5% of the DHS starting material remained unreacted. The crude
reaction product consisted of gallic acid (22%), protocatechuic acid
(10%) and tricarballylic acid (2%). After acidification to pH 3, the
reaction solution was extracted (3×) with EtOAc. Drying of the organic
layer and concentration gave a solid (0.33 g). Chromatography using
octadecyl-functionalized silica gel (86 mL) eluted with CH3OH/H2O
(3:7, v/v, adjusted to pH 2.8) afforded pure gallic acid (0.18 g, 18%).
Isolation of Dihydrogallic Acid. A warm filtered solution of DHS
(0.18 g, 1.06 mmol) in 4 M Nal.5H1.5PO4 (0.91 mL) was sparged with
N2 for about 3 min, capped, and then heated 44 h at 40-42 °C. After
adding 0.11 g of D2O containing 1.0 wt% maleic acid and 0.59 wt%
pivalic acid, NMR analysis indicated a 69% yield of dihydrogallic acid
and 31% yield of protocatechuic acid. Crystals of dihydrogallic acid
or, more likely, its sodium or disodium salt, separated during the NMR
acquisition. Unlike crystals of Na2HPO4 which also can separate from
this saturated solution, the crystals of dihydrogallic acid did not readily
dissolve on gentle warming but required brief heating in boiling water
to redissolve. The resulting solution was acidified by addition of 4 M
H3PO4 (0.46 mL). Extraction with EtOAc removed protocatechuic acid.
On standing, the resulting aqueous phase deposited crystals of dihy-
drogallic acid which were collected and washed once with cold water.
The hydrate of dihydrogallic acid melted 181-2 °C (dec). After
vacuum drying these crystals melted 191.5-192.5 °C (lit.6a mp 192-3
°C, dec); 1H NMR (1.2M Pi, TSP, δ ) 0 ppm) 2.70 (dd, J ) 17, 8 Hz,
2H), 2.65 (dd, J ) 17, 6 Hz, 2H), 2.94 (m, lH); 13C NMR (H2O/D2O,
1,4-dioxane, δ ) 70.0 ppm) 180.5, 179.7, 133.6, 40.8, 36.8; IR (mineral
oil mull, cm-1) 3610 (w), 3425 (m), 1706 (m), 1665 (m), 1551 (s),
1319 (s), 1238 (s), 1210 (m), 1158 (s), 1020 (m) 962 (m); HRMS (EI)
calcd. for C7H8O5 (M+) 172.0372, found 172.0368.
Experimental Section
General. Isomerization, dehydration and oxidation reactions of
1-2% aqueous solutions of DHS were monitored by 1H NMR using a
Varian VXR-300 FT-NMR Spectrometer (300 MHz) and solvent
suppression techniques.20 Presaturation used the program “Presat”
supplied with the instrumentation. For a lock signal, solutions were
diluted by addition of a weighed amount of D2O (5-12% of initial
sample on a v/wt basis) containing 0.5-1.0 wt% of maleic acid, pivalic
acid or disodium fumarate as internal integration and shift references.
The lock signal for deuterium was quite broad, usually 3-4 Hz, as
indicated by observing the dominant proton exchange peak prior to
presaturation. Standard shimming and auto shimming procedures were
inadequate under these conditions. For each sample, shimming was
optimized to produce a symmetrical exchange peak with minimized
peak width by repetitive shimming and observation of the spectrum
obtained after one transient. Presat spectra were obtained from sixteen
transients at relatively high saturation power and gain. This technique
typically produced spectra where normally sharp lines were resolved
to widths of less than 1 Hz. In these presaturated spectra, reliable
integrations were obtained excluding the region from δ 3.0-5.8.
Inorganic phosphate buffer was prepared by combining equimolar
amounts of dibasic (HPO42-) and monobasic (H2PO41-) sodium or
potassium phosphate. Octadecyl-functionalized silica gel (Aldrich) was
activated by initial elution with CH3OH followed by elution with
CH3OH/H2O (1:1, v/v).
Reaction of DHS with O2. DHS (1 g, 5.81 mmol) was dissolved
in 30 mL of 1.0 M Na1.5H1.5PO4 and the solution adjusted to pH 6.7.
The reaction was then stirred for 50 h exposed to the atmosphere at 40
°C. At the end of the reaction, 10% of the DHS remained unreacted.
Products consisted of gallic acid (14%), protocatechuic acid (12%),
tricarballylic acid (14%), and pyrogallol (3%). Extraction (2×) with
EtOAc to remove pyrogallol was followed by acidification of the
solution to pH 3.6 and extraction (2×) with EtOAc. Drying of the
organic layer and concentration gave a solid (0.26 g). Chromatography
using octadecyl-functionalized silica gel (86 mL) eluted with CH3OH/
H2O (1:9, v/v, adjusted to pH 2.7) afforded pure gallic acid (0.13 g,
13%).
Oxygenation of Dihydrogallic Acid in Phosphate Buffer.
A
5-6% solution of DHS in 0.75 mL of 4 M Na1.5H1.5PO4 was isomerized
to dihydrogallic acid, acidified and extracted (3×) to remove proto-
catechuic acid as described above. Two final extractions with cyclo-
hexane removed EtOAc. The aqueous solution was neutralized with
dilute NaOH to make l M Na1.3H1.7PO4, stoppered under air in a 10
mL flask, and then incubated at 37 °C in a rotary shaker oven. After
12 h, the solution was concentrated under vacuum, transferred to NMR
tubes, diluted with D2O and reference, and sparged with N2. Yields
obtained from integration of these NMR spectra are 3% gallic acid,
6% pyrogallol and 35% tricarballylic acid.
Analysis of Aerobic Reactions of DHS in Aqueous Phosphate
Solutions. Three aqueous solutions of DHS (0.12 g, 0.70 mmol) and
8 mL of l.0 M Nal.5Hl.5PO4 were made in 50 mL Erlenmeyer flasks.
The reaction mixtures were briefly sparged with O2, stoppered, and
then incubated at 37 °C in a rotary shaker oven. Aliquots (0.70 mL)
were taken at t0 and at subsequent timed intervals from each of the
three parallel reactions. After weighing, 10 vol/wt% D2O containing
1 wt% of maleic acid was added to each aliquot followed by thorough
mixing and sparging with N2. 1H NMR with solvent suppression were
acquired for each aliquot and analyzed by integrating the aromatic
signals for protocatechuic acid (3H), gallic acid (2H) and pyrogallol
(3H); the vinyl signals for DHS (lH) and maleic acid (2H); and the
highest field multiplet for tricarballylic acid (2H). In cases where
dihydrogallic acid or other high field products were detected, the
integrations of the overlapping signals of DHS and tricarballylic acid
were subtracted from the integrals. Analysis for each component in
each spectrum was reduced to a mol percentage of the initial DHS.
Averages and standard deviations (σn-l ) were based on aliquots taken
at the same timed intervals from the three parallel reactions.
(15) (a) Kikugawa, K.; Kunugi, A.; Kurechi, T. In Food Antioxidants;
B. J. F. Hudson, Ed.; Elsevier: New York, 1990; p 73. (b) Cuvelier, M.-
E.; Richard, H.; Berset, C. Biosci. Biotech. Biochem. 1992, 56, 324. (c)
Marinova, E. M.; Yanishlieva, N. V. J. Am. Oil. Chem. Soc. 1994, 71, 427.
(16) (a) Dressler, H.; Holter, S. N. In Kirk-Othmer Encylcopedia of
Chemical Technology; Grayson, M., Eckroth, D., Eastman, C. I., Klingsberg,
A., Spiro, L., Wachsman, G., Wainwright, M., Eds.; Wiley: New York,
1982; Vol. 18, p 670. (b) Weetal, H. H. Biotechnol. Bioeng. 1985, 27, 124.
(17) Rubel, T. Vitamin E. Manufacture; Noyes Development: Park Ridge,
NJ, 1969.
Analysis of Anaerobic Reactions of DHS in Aqueous Phosphate
Solutions. A solution of DHS (0.011 g, 0.064 mmol) and D2O (0.96
g containing 1 wt% maleic acid and 0.59 wt% pivalic acid) in 0.75
mL of l M K2HPO4 was deoxygenated directly in an NMR tube by
sparging 1-3 min with N2 gas introduced through a fine capillary. The
initial NMR spectrum gave molar integration ratios of DHS (82.8%),
maleic acid (10.2%) and pivalic acid (7.0%). After standing at room
temperature for two days, the molar composition of this solution was
protocatechuic acid (32.3%), dihydrogallic acid (49.4%), maleic acid
(10.6%) and pivalic acid (7.7%). These data indicate that the yields
(18) Lerner, M. Chem. Mark. Rep. 1995, 14 (August 7).
(19) (a) Chem. Mark. Rep. 1996, 28 (August 26). (b) Lepree, J. Chem.
Mark. Rep. 1995, 16 (August 29).
(20) Smallcombe, S. H. J. Am. Chem. Soc. 1993, 115, 4776.