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Ascorbic acid-2-sulfate of the brine shrimp, Artemia salina
ARCHIVES
OF
BIOCHEMISTRY
Ascorbic
Division,
163,
BIOPHYSICS
Acid-2-Sulfate
A. D. BOND,’ Biology
.4ND
207-214
of the
B. W. McCLELLAND,2 Oak
(1972)
Brine
Shrimp,
J. R,. EINSTEIN,
Artemia AND
F. J. FIKARIORE
Ridge National Laboratory,3 and the University of Tennessee-Oak School of Biomedical Sciences, Oak Ridge, Tennessee 37830 Received
June
salina
Ridge
Graduate
19, 1972
Methods are described for the preparation of ascorbic acid-a-sulfate and for its isolation in very pure form in yields of approximately 50%. Our procedure was similar to those used by others for the synthesis of ascorbic acid-3-sulfate. A comparison of our material with ascorbic acid-3-sulfate from another laboratory demonstrated that the two were indistinguishable. Chemical and physical characteristics of this ascorbate derivative suggested to us that it was a 2-sulfate rather than the 3-sulfate previously reported and X-ray crystallographic studies of the barium salts have now demonstrated that this compound is 2-0-sulfonatoascorbic acid. Comparison of the synthetic compound with that found in brine shrimp showed them to be identical. It can be concluded. therefore, that 2-0-sulfonato-L-ascorbic acid is the nat,llrally occurring derivative.
Clark et al. (1) reported the oxidativc transfer of phosphate from hydroquinone monophosphate esters to various acceptors, including adenosine diphosphate. Others (2, 3) confirmed this work and extended it to ot,her monoacyl esters of hydroquinones. Recognized similarities in the structure of hydroquinones, catechols, and ascorbic acid evoked proposals that enol esters of ascorbic acid should also undergo oxidative group transfer reactions (4, 5). Clark et al. (4) prepared both the 2- and X-phenylphosphoryl ascorbic acid derivatives and reported that the phosphate group was oxidatively transferred to ethanol. Ford and Ruoff (5) prepared a sulfate ester of ascorbic acid which they, and others (6, 7), found t’o be a participant in oxidative sulfations. 1 During the course of this research, 8. D. Bond was a USPHS Fellow, NIH Grant 1 F03 ilM51218-01, and an ORAU Research Participant. 2 Postdoctoral Investigator supported by subcontract No. 3322 from the Biology Division of Oak Ridge National Laboratory to the University of Tennessee. 3 Oak Ridge National Laboratory is operated by the Union Carbide Corporation under contract with the United States Atomic Energy Commission. 207 Copyright All rights
@ 1972:by Academic Press, of reproduction in any form
Inc. reserved.
That ascorbic acid influences the integrity of connective tissue is well-known, but its act’ual function remains a mystery. The possibility of a sulfate derivative of ascorbic acid part’icipating in the biosynt’hesis of sulfat,cd mucopolysaccharides did not escape the attent’ion of those cited above. The discovcrg of ascorbic sulfate in brine shrimp cyst’s by Jlead and Finamorc (8) and isolation of t,he same compound from various animal sourc(‘s by Tolbert et al. (9) demonstrated that ascorbic sulfate was a compound of widcspread biological distribut’ion. It was not, just a laboratory curiosity and perhaps wa,sa new biological sulfating agent. Since the chemical properties of the various isomers of ascorbic sulfate Lvould be expected t.o be very different from c>ach other, the structure of this compound must bc known if one would determine its function in metabolic pathways. Previous reports on ascorbic sulfate clearly established that the sulfate resides on either the 2- or X-position (for numbering, seeFig. 2)) and it was crrotleously assumed by ot#hershhat’ it, was the 3-sulfate. This report includes evidence to support our conclusion that both the synthetic and
20s
BOND
the natural ascorbic sulfates sulfonato-L-ascorbic acid. EXPERIMENTAL
are
2-0-
SECTION
Synthesis of Ascorbic Sulfate Preparation of cyclohexylidene ascorbic acid. n-ascorbic acid (Fisher reagent) was converted to 5,6-O-cyclohexylidene-L-ascorbic acid (CHAA) by a modification of the method described by Von Schuching and Frye (10). Fifty grams of ascorbic acid was finely powdered and added to 250 ml of redistilled cyclohexanone to which had been added 5 ml of concentrated H&S04 with rapid stirring at 4°C. This mixture was magnetically stirred in a glass-stoppered flask in the cold for 48 hr. It was then poured into 500 ml of ice water and quickly extracted with two 500~ml portions of dry, peroxide-free ether. The extracts were combined and washed twice with 250-ml portions of ice water, then dried with anhydrous NapSO, Ether was removed under vacuum to a volume of about 209 ml. Heptane was added slowly to the combined dried ext,ract while stirring with a glass rod to induce crystallization. When crystals began to form, approximately 1 liter of heptane was added and the mixture was placed in the cold room overnight. This material was recrystallized from etherheptane as pure white needles, stable over long periods in a desiccator. The yield was 80%. Formation and isolation of ascorbic sulfate. CHAA was then sulfated by the method of Ford (ll), using pyridine-sulfur trioxide in freshly distilled dimethylformamide. After 24 hr, 4 vol of cold water were added and the solution passed through a Dowex 5O(H+) column to hydrolyze the protective ketal from the 5- and 6-positions. A 3 X 24 cm column was used for 2.5 g of the CHAA preparation. Ascorbic sulfate thus produced was further purified by a modification of the procedure of Mead and Finamore (8). It was applied to a Dowex l-XZ(Cl-) column (3 X 24 cm) previously washed with 0.01 N HCl in 0.1 N NaCl. The column was washed with the same HCl-NaCl solution until no uv-absorbing material was found in the effluent. After further washing of the column with water until the effluent was neutral, ascorbic sulfate was eluted by a 0.1-1.0 M NaCl linear gradient. That material absorbing at 254 nm and eluting at approximately 0.35 M NaCl was acidified to pH 2 with HCl and then applied to an activated charcoal column (2 X 15 cm) as described by Mead and Finamore (8), with the following change. The activated charcoal column was prepared by washing with 1 M HCl followed by water until the effluent was neutral. It was then equilibrated with 0.01 M HCl before applying the sample. Ascorbic sulfate adsorbed to charcoal was washed with 500
ET
AL.
ml of 0.01 N HCl and then with 100 ml water before being eluted with a liter of eluant [ethanol, concentrated NH,OH, water (2:1:2) v/v/v]. Great care was taken to collect the eluate only after its pH rose above neutrality. This eluate was reduced in volume on the flash evaporator, then fractionated on a DEAE-cellulose (HC03-) column exactly as described by Mead and Finamore (8). The same procedure for purification was subsequently used to purify ascorbic sulfate from brine shrimp and other sources.
Crystallization Sulfate
and
Analysis
of Ascorbic
The pure diammonium salt obtained above was converted to free acid, barium salt, or dipotassium salt by passing a dilute solution of it through a Dowex 50 column in the appropriate form. The barium salt was crystallized by reducing the volume of effluent under vacuum and adding a few drops of ethanol. The pure white crystals were subsequently recrystallized several times in pure distilled water. Elemental analyses of the barium salt were performed by Galbraith Laboratories, Knoxville, TN. X-ray analysis. Crystals of the barium salt of synthetic ascorbic sulfate suitable for X-ray experiments were grown from water solution, either by evaporation or by diffusion of methanol through the vapor phase into the solution. Crystals were colorless prisms extended along the a axis. Preliminary diffraction photographs to establish the space group and approximate unit-cell dimensions were made with a Weissenberg camera and Cu Ka radiation. A crystal specimen selected for intensity measurements, roughly 0.1 X 0.2 X 0.4 mm in size, was cemented to a glass fiber, dipped repeatedly into liquid nitrogen, and mounted on an Oak Ridge computer-controlled diffractometer (12). Angle data for 12 reflections in the range 49” < 28 < 56’ (MO Kcv~ radiation) were used to refine the unit-cell parameters. All 1423 independent reflections with niobium-filtered MO Ka radiation and 20 2 56” were measured using s/28 step scans. The data, corrected for Lorentz-polarization and absorption effects, were used as coefficients in a three-dimentional Patterson synthesis (program FORDAPER of A. Zalkin, modified by G. Brunton). The positions of all nonhydrogen atoms were found by distinguishing the correct image from the centrosymmetrically related image through chemical criteria. Full-matrix least squares (program XFLS-3, a modification of program ORFLS; Ref. 13) were used to refine the structure, including subsequently all the hydrogen atoms, which were found in three-dimensional
Artpnlia
salina:
BSCORBIC
difference Fourier syntheses. The drawing was made on a Cal-Comp plotter with the aid of the program ORTEP (14). Spectrophotometric analysis. An infrared spectrum of the dipotassium salt of ascorbic sulfate was obtained on a Perkin-Elmer model 457 spectrophotometer. The KBr pellet was prepared by dissolving the dipotassium salt in water, adding 1 g KBr per mg ascorbic sulfate and reducing the resulting solution to dryness under vacuum. Ultraviolet spectra were obtained for various salts in aqueous solut,ions using a Beckman model DB spectrophotometer. Electrophoretic and chromatographic analysis. Electrophoresis of ascorbic acid sulfate was carried out in 0.1 M acetate buffer (pH 4.65) at 970 V and 7 mA for 25 min at room temperature, using 5 X 20.cm MN300 cellulose thin-layer chromatography (tic) plates obtained from Brinkman Instrument Company. Chromatography of ascorbic sulfat,e was performed with MN300 cellulose tic plates using the following three solvent systems: (1) met,hanol/formic acid/water (80: 15: 5 v/v/v) ; (2) methanol/coned NHaOH/water (60:30:30 v/v/v); (3) sat (NHI)804/0.1 M NHaCzH302/2propanol (79:19:2 v/v/v) (see Table I). The latter system was also used for paper chromatography with What,man No. 1 paper. Ascorbic sulfate was detected on tic plates by uv absorption and FeCls-methanol spray. Bscorbic acid-3.sulfate was graciously provided by B. M. Tolbert for comparison with our preparation. Acid properties of ascorbicsulfate. A Beckman model-G pH meter was used for all pH determinat,ions. The pK, of ascorbic acid sulfate was determined a.s described by Mead and Finamore (8).
Oxidati/le Transfer of Sulfate Group Ascorbic Sulfate
from
Oxidative transfer of the sulfate group was studied by oxidizing 5,6-0-isopropylidene ascorbic acid sulfate with Hz02 . The barium salt of this
ACID-2-SULFATE
209
ascorbic acid derivative was prepared by the method of Ford (11). It was dissolved in anhydrous dimethylsulfoxide at a concentration of 100 mg per 2 ml solvent and then 50 mg p-nitrophenol was dissolved in this solution. One milliliter of 30y0 Hz02 was added; the final solution was stirred and then allowed to stand at room temperature for 24 hr before products were analyzed. A similar experiment was performed in which dimethylsulfoxide was replaced by water. Products were examined by thin-layer chromatography. p-Nitrophenyl sulfate was chromatographed on MN300 cellulose tic plates using n-butanol/ethanol/water (80:24:40 v/v/v). It was detected by uv absorption and by the yellow color formed on exposure to NHaOH vapor after hydrolysis RESULTS
Characterization of Ascorbic Sulfate Spectrophotometric properties. The yield of 20sulfonatoascorbic acid, diammonium salt from this procedure approaches 50 %. Figure 1 represents the ir spectra of its potassium salts isolated from brine shrimp and from chemical synthesis. They are identical, with the exception of minor differences of intensity and a small, sharp absorption at 945 cm-’ which varied in intensity with the preparations. Ultraviolet spectra of ascorbic sulfate from both sourceshad a maximum at 232 nm in 0.01 N HCl that shifted to 254 nm in neutral or alkaline solutions with a concomitant increase in extinction coefficient of 55 %. The molar extinction coefficients previously reported (8) were in error, the new values being 1.1 X lo4 in acid and 1.7 X lo4 in base. The same properties were exhibited by the 3-sulfate supplied by Tolbrrt. Chromatographic and electrophoreticproperties. Results of thin layer chromatography
WAVENUMBER
FIG. 1. Infrared spectra of the dipotassium sources (O-0) and from brine shrimp (-).
salts of ascorbic acid sulfate from synthetic
210
BOND
are compiled in Table I. Ascorbic sulfate from the two sourcesgive the same Rf value in each developing solvent system even though the solvents have widely differing characteristics. A slight trace of contaminant in the natural material was detected by tic but appeared to be destroyed on paper after the longer development times required and was undetectable by other techniques. Electrophoretic mobilities on tic plates were identical. Acidic dissociation constants. Titration of ascorbic sulfate from each source revealed the samepK,%of 2.75. No attempt was made to determine the pK,, of the sulfate group. Elemental analysis. Elemental analyses of the barium salt of ascorbic sulfate are reported in Table II with calculated percentages based on two water molecules of hydration. The two water molecules were also detected by crystallographic studies. TABLE
I
CHROMATOGRAPHY OF ASCORBIC ACID ON TLC CELLULOSE Compound
I
I II
Oxidative Sulfate Transfer from Ascorbic Xulfate
1
Solvent 2
Solvent
3
Oxidation of 5,6-0-isopropylidene ascorbic sulfate by HzOs in aqueous medium quickly TABLE
sulfateO.73(0.79)a
0.SS0.88(0.41)” 0.88(0.44)=
sulfate
0.88
0.88 0.87
TABLE
II
ELEMENTAL AN.LLYSIS OF 2-0.SULFONATO-LASCORBIC ACID, BARIUM SALT, DIHYDR~TE (CsHloOnSBa) Observed percentage 16.80 2.41 41.39 7.60 31.96 lOO.lG
Calculated percentage 16.85 2.36 41.17 7.50 32.12 100.00
Ba S 0 0 0 0 0 0 0 0 0 w w c c
(1)
(2) (3) (4) (5)
(‘3) (7)
03) (9) (1)
(2) (1)
(2)
III
ATOMIC POSITIONS"
ATOM
0.73
e Trace of uv-absorbing substance, disappearing on exposure to air or light for a few hours.
C H 0 S Ba
X-ray crystallographic analysis of ascorbic sulfate. Crystals of the barium salt of synthetic ascorbic sulfate are triclinic, with unitcell parameters a = 50.201(l), b = 6.951 (l), and c = 8.732 (1) A; and cx = 99.54 (l), p = 93.29 (l), and y = 109.12 (1)“. With one molecule per unit cell and two water molecules of hydration, d(calcd) = 2.431 g/cm3, while d(meas) = 2.44 (3) g/cm3 as determined by flotation. The space group is thus PI. Least-squares refinement of the structure (varying the positional parameters of all 29 at’oms, anisotropic thermal parameters for the nonhydrogen atoms and isotropic ones for the hydrogen atoms, and an isot’ropic extinction parameter) has yielded an R factor of 0.009. The atomic coordinates are listed in Table III. The average positional estimated standard deviations are about 0.0025 & for oxygen and carbon atoms, and 0.06 A for hydrogens. The structure is depicted in Fig. 2, which shows many of the important, int’eratomic distances and bond angles. Dct,ails of structure will be presented elsewhere.
RF value Solvent
Ascorbic acid (from brine shrimp) Ascorbic acid (synthetic)
SULFATE
ET AL.
x
Y
0 0 2848 5796 7740 8524 2342 6957 -9853405 5712 6245 3845 1685 4735 764 1813 6859 12713626 5765 6207 5494 1374 9143 9390 5677 7176 3037 6273
z 0 - 1123 2536 551 2018 4018 2657 5904 - 2157 - 1289 - 1024 9282 6654 2747 1870
X’Y
ATOM c C ‘2 c H H H H H H H H H H
(3) (4) (5) (6) (05) (06) (WlA) (WlB) (W2A) (WZB) (C4) (C5) (C6A) (CGB)
z
1370 4725 2511 300147354009 318426104087 515027285490 551 217 263 301 17 594 523 132 826 645 248 974 803 987 628 857 807 618 218 519 490 140 170 426 702 322 522 500 368 632 I
I
(1 Triclinic coordinates X lo4 for nonhydrogen atoms, X 103 for hydrogens. W denotes water oxygen. Hydrogen atoms are labeled according to the atoms to which they are attached.
Artemia
salina: ASCORBIC
211
ACID-2-SULFATE
Gi
O(8) FIG. 2. ORTEP drawing (Johnson, 1970) of the molecular configuration of 2-O-sulfonatoL-ascorbic acid ion. All nonhydrogen atoms are represented by 59% probability ellipsoids of thermal displacement. Bond distances are given in A, bond angles in degrees. Bond angles not indicated include O(4)-C(4)-C(5) = 110.6; O(5)-C(5)-C(6) = 112.5; 0(2)-S-O(7) = 99.9; 0(7)-S-O(9) = 115.3; and 0(8)-S-O(9) = 113.1 degrees.
produced a heavy white precipitate of BaSOl as previously reported (5). However, in dimethylsulfoxide solution, only a trace of precipitate was formed after 24 hr. Chromatography of the reaction mixture yielded only one spot from aqueous solution and three from dimethylsulfoxide solution. When compared with known samples, the Rf = 0.98 spot corresponded to p-nitrophenol and was found in both solutions. An Rf = 0.53 spot corresponded to the 5,6-0-isopropylidene ascorbic sulfate, and the Rf = 0.85 spot corresponded to p-nitrophenyl sulfate. The latt.er two were observed only in the nonaqueous solution (Table IV).
TABLE
IV
CHROMATOGRAPHY OF WV-ABSORBING REACTION PRODUCTS FROM OXIDATION OF ISOPROPYLIDENE ASCORBIC SULFATE AND P-NITROPHENOL Compound Aqueous solution products Nonaqueous solution products p-Nitrophenol Isopropylidene ascorbic sulfate p-Nitrophenylsulfate
RF -
-
0.98
0.53
0.85
0.98
-
-
0.53
-
0.98 -
-
0.85
-
on adsorption and ion-exchange chromatography, provided further evidence that Since ultraviolet and infrared spectra, these compounds were identical and that we Rf in three different solvent systems, elec- were working with the same compound. Previous reports concerning ascorbic sultrophoretic mobilities at acid pH, and acid dissociation constants of natural and syn- fate have clearly demonstrated that the thetic ascorbic sulfate are identical, we sulfate must reside either at the 2- or 3conclude that the natural and synthetic position. Based on indirect evidence, sulfate was assigned to the 3-position by Ford and forms of this substance are also identical. Furthermore, cochromatography of our Ruoff (5) and every subsequent report rematerial with a sample of Tolbert’s ascorbic peated this assignment. However, the ability acid-3sulfate, coupled with identical phys- to form FeCL complexes, an important ical properties and identical behavior of each factor in making the original assignment of DISCUSSION
212
BOND
ET
AL.
structure, is not restricted to the 3-isomer as TABLE V supposed (4, 15). ACID STRENGTH OF ASCORBIC ACID DERIVATIVES Numerous ascorbic acid derivatives have AND SUBSTITUTED ENOLS been synthesized, and a comparison of their Compound ph P& properties with those of ascorbic sulfate from brine shrimp should allow the structure acida 4.1 , 11.8 of the latter to be inferred. Both 2- and Ascorbic 3-Methylascorbic acidb 7.8 3-acyl derivatives have been prepared, but, 2-Methylascorbic acid” 3.4 unfortunately, contradictory structural as2-Phenylphosphoryl <4 <4 signments have been made. Kobayashi (16) ascorbic acid6 prepared several diesters of ascorbic acid, one 3-Phenylphosphoryl <4 7.2 of which was designated as a 2,6-diacyl-nascorbic acidb ascorbic acid. Later, Nomura and Sugimoto Ascorbic sulfate (strong acid) 2.75 (17) synthesized some dibenzoyl esters and Phenol” 9.9 p-Phenolsulfonic acidd concluded that Kobayashi’s 2,6-diester was (strong acid) acidd (strong acid) / YO in fact a 3,6-diester. While Nomura’s in- m-Phenolsulfonic p-Nitrophenola 7.2 terpretation was based on ir and nmr spectra, o-Nitrophenola 7.2 he admitted ambiguity in the data. Tanaka and Yamamoto (18) then prepared a variety 0 Handbook of Chemistry and Physics, 47th of ascorbic acyl esters, compared their ed. (1967). The Chemical Rubber Co., Cleveland, properties, and assigned structures which OH , pp. D-86, 87. supported Kobayashi and contradicted *Personal communication from V. M. Clark. Nomura. Clark et al. (4) synthesized 2- and c Ettlinger et al., 1961 (Ref. 23). 3-phosphoryl ascorbic acids and determined d H. Zollinger (1953) Nature (London) i72; 257. their properties; he subsequently identified them (personal communication). From analogous properties of these isomers, one con- ascorbic acid correspondsroughly to a phenyl cludes that Kobayashi, Tanaka and ester. Consequently, the 2-isomer should be Yamamoto, and Clark et al. agree on struc- more stable thermodynamically. Imai (19) tural assignment and that Nomura and observed a rapid migration of the acyl group from Nomura’s “&benzoyl” ester in dilute Sugimoto’s interpretations are inverted. Clark observed that substitution of a 2- or alkali. Migration should occur in a direction and, therefore, 3-position with a phenylphosphoryl group of increasing stability Nomura’s structural assignment was in error. decreasedthe pK, of the neighboring enol. A Tanaka and Yamamoto (18) observed a number of pK, values for acyl ascorbic acids distinct pattern in the uv spectra of ascorbic and substituted enols are compared in Table acid derivatives which was also seen by V. It is a well-known fact that substitution by an electron-withdrawing group causesthe Clark (personal communication). Table VI pK, of a conjugated enol to decrease. How- lists uv maxima and extinction coefficients ever, even as powerful a substituent as a for some of these, and suggeststhat a simple nitro group causes a decrease of less than rule can be formulated relating structure and three units. Nomura and Sugimoto (17) re- spectral properties. Substitution of the 3-OH ported an inconsistent pattern. That is, they effects little change in uv spectrum from that claimed that benzoylation of the 2-OH of unsubstituted ascorbic acid unlessthe free caused the pK. of the 3-OH to increase from enol becomes dissociated. Dissociation produces a bathochromic shift to about 260-265 4.1 to 6.4, whereas benzoylation of the 3-OH nm with little change in molar extinction caused the pK, of the 2-OH to decrease by coefficient. However, substitution of the nearly nine units. Reversing their assignment 2-OH causesa hypsochromic shift of about would yield a consistent decrease in each 10 nm for both the undissociated molecule case and eliminate disagreement with the and the anion. Dissociation of the 3-OH has other investigators. Furthermore, the 3- a hyperchromic effect resulting in an increase benzoyl ascorbic acid is a vinylog of a mixed of extinction coefficient of about 50 70. acid anhydride, whereas the 2-benzoyl From its spectral characteristics in acid
i g..
Artemia
salina:
ASCORBIC TABLE
UV
SPECTRAL
CHARACTERISTICS
Compound
3-Phenylphosphoryl 2-Phenylphosphoryl Ascorbic sulfate (L Personal
ascorbic ascorbic
communication
VI OF SUBSTITUTED
ASCORBIC
Acid
Ascorbic acida 3-Methylascorbic acidu 2,3-Dimethyl ascorbic acida acida acid”
from
V. M.
ACIDS
Neutral
max
e x 18
max
243 244 235
10 9 g
265 244 -
237 233 232
8 9.6 11
238 257 254
Base
Ex
103
max
6 x lo3
16.5 9 -
276 -
-
8 14.5 17
263 258 254
8 15 17
9 -
Clark.
and neutral solution, and the acidity of ascorbic sulfate, it would appear that this molecule is a %-isomer rather than the 3isomer. We have subjected the pure crystalline barium salt to X-ray crystallographic analyses and have shown conclusively that it is, in fact, 2-0-sulfonato-L-ascorbic acid, barium salt. Widespread natural occurrence of this compound (9, 20) implies that ascorbic sulfate may have a function in sulfate metabolism, and ascorbate is known to be required for proper sulfation of mucopolysaccharides. To explain this function, previous reports have claimed oxidative transfer of sulfate from ascorbic sulfate and suggested that a high-transfer-potential sulfate was formed by the oxidation (5-7, 21). However, the acceptors used did not establish formation of such a compound. Detection of p-nitrophenyl sulfate as an oxidat’ion product of isopropylidene ascorbic sulfate and p-nitrophenol in nonaqueous solution determined that a high-transfer-potential sulfate had been formed (22). Robbins est’imated the AG of hydrolysis for p-nitrophenylsulfate equal to -15 kcal/mole, which is comparable to that for 3’-phosphoadenosine-5’phosphosulfate. We must conclude, therefore, that 2-0-sulfonato-L-ascorbic acid is a widely distributed natural product which has the capacity to form a high-transferpotential sulfate during an in vitro oxidation process. It thus could participate in in viva sulfation processes as suggested by Mumma and Verlangieri (al). ACKNOWLEDGMENT We are grateful to C. II. Wei with the X-ray crystallographic
213
ACID-2-SULFATE
for his assistance analysis.
REFERENCES V. M., HUTCHINSON, D. W., KIRBY, G. W., AND TODD, SIR ALEXANDER (1961) J. Chem. Sot. 715. BOND, A., AND MASON, H. S. (1962) Biochem. Biophys. Res. Commun. 9, 574. WF,IDMAN, S. W., MAYERS, D. F., ZABORSKY, 0. R., AND KAYSI.;R, E. T. (1967) J. Amer. Chem. Sot. 89, 4555. CLARK, V. M., HERSHEY, J. W. B., AND HUTCHINSON, D. W. (1966) Esperientia 22, 425. FORD, E. A., AND RUOFF, P. M. (1965) Chem. Commun. 630. CHU, T. M., AND SLAUNWHITE, W. R., JR. (1968) Steroids 12, 309. MUMMA, R. 0. (1968) Biochim. Biophys. Acta 166, 571. MEAD, C. G., AND FINAMORE, F. J. (1969) Biochemistry 8, 2652. TOLBERT, B. M., ISHERWOOD, D. J., ATCWELY, R. W., AND BAKER, E. M. (1971) Fed. Proc. 30, 529. VON SCHUCHING, S. L., AND FRYI<, G. H. (1966) Biochem. S. 93, 652. FORD, E. A. (1968) Doctoral dissertation, Syracuse University, Syracuse, NY, 1967; Diss. AbstT. B 28, 4059. BUSING, W. R., ELLISON, K. D., LI:VY, H. A., KING, S. P., AND ROSEBERRY, R. T. (1968) The Oak Ridge Computer Controlled X-ray Diffractometer. Report ORNL-4143, Oak Ridge National Laboratory, Oak Ridge, TN. BUSING, W. R., MARTIN, K. O., AND LEVY, H. A. (1962) ORFLS, A Fortran Cryst,allographic Least-Squares Program. ORNLTM-305, Oak Ridge National Laboratory, Oak Ridge, TN. JOHNSON, C. K. (1970) ORTEP, A Fortran Thermal-Ellipsoid Plot Program for Crystal-structure Illustration. ORNL-3794, 2nd revision, Oak Ridge Nat onal Laborat,ory, Oak Ridge, TN.
1. CLARK,
2. 3.
4.
5. 6. 7. 8.
9.
10. 11.
12.
13.
14.
214
BOND H., AND MORIMOTO, S. (1971) Chem. Pharm. Bull. (Tokyo) 19, 335 KOBAYASHI, K. (1965) Japan Patent Application 9134. NOMURA, H., AND SUGIMOTO, K. (1966) Chem. Pharm. Bull. (Tokyo) 14, 1039. TANAKA, H., AND YAMAMOTO, R. (1966) Yakugaku Zasshi 86, 376. IMAI, Y. (1966) Chem. Pharm. Bull. (Tokyo) 14, 1045. BBKER, E. M. III, HAMMER, D. C., MARCH,
15. NOMURA,
16. 17. 18. 19. 20.
ET AL. S. C., TOLBERT, B. M., AND CANHAM, J. E. (1971) Science 173, 826. 21. MUMMA, R. O., AND VERLANGIERI, A. J. (1971) Fed. Proc. 30, 370. 22. ROBBINS, P. (1962) in The Enzymes (Boyer, P. D., Lardy, H. A., and MyrbSick, K., eds.), 2nd ed., Vol. 6, p. 363. Academic Press, New York. 23. ETTLINGER, M. G., DATEO, G., HARRISON, B., MALOY, T., AND THOMPSON, C. (1961) Proc. Nat. Acad. Sci. USA 47, 1875.
OF
BIOCHEMISTRY
Ascorbic
Division,
163,
BIOPHYSICS
Acid-2-Sulfate
A. D. BOND,’ Biology
.4ND
207-214
of the
B. W. McCLELLAND,2 Oak
(1972)
Brine
Shrimp,
J. R,. EINSTEIN,
Artemia AND
F. J. FIKARIORE
Ridge National Laboratory,3 and the University of Tennessee-Oak School of Biomedical Sciences, Oak Ridge, Tennessee 37830 Received
June
salina
Ridge
Graduate
19, 1972
Methods are described for the preparation of ascorbic acid-a-sulfate and for its isolation in very pure form in yields of approximately 50%. Our procedure was similar to those used by others for the synthesis of ascorbic acid-3-sulfate. A comparison of our material with ascorbic acid-3-sulfate from another laboratory demonstrated that the two were indistinguishable. Chemical and physical characteristics of this ascorbate derivative suggested to us that it was a 2-sulfate rather than the 3-sulfate previously reported and X-ray crystallographic studies of the barium salts have now demonstrated that this compound is 2-0-sulfonatoascorbic acid. Comparison of the synthetic compound with that found in brine shrimp showed them to be identical. It can be concluded. therefore, that 2-0-sulfonato-L-ascorbic acid is the nat,llrally occurring derivative.
Clark et al. (1) reported the oxidativc transfer of phosphate from hydroquinone monophosphate esters to various acceptors, including adenosine diphosphate. Others (2, 3) confirmed this work and extended it to ot,her monoacyl esters of hydroquinones. Recognized similarities in the structure of hydroquinones, catechols, and ascorbic acid evoked proposals that enol esters of ascorbic acid should also undergo oxidative group transfer reactions (4, 5). Clark et al. (4) prepared both the 2- and X-phenylphosphoryl ascorbic acid derivatives and reported that the phosphate group was oxidatively transferred to ethanol. Ford and Ruoff (5) prepared a sulfate ester of ascorbic acid which they, and others (6, 7), found t’o be a participant in oxidative sulfations. 1 During the course of this research, 8. D. Bond was a USPHS Fellow, NIH Grant 1 F03 ilM51218-01, and an ORAU Research Participant. 2 Postdoctoral Investigator supported by subcontract No. 3322 from the Biology Division of Oak Ridge National Laboratory to the University of Tennessee. 3 Oak Ridge National Laboratory is operated by the Union Carbide Corporation under contract with the United States Atomic Energy Commission. 207 Copyright All rights
@ 1972:by Academic Press, of reproduction in any form
Inc. reserved.
That ascorbic acid influences the integrity of connective tissue is well-known, but its act’ual function remains a mystery. The possibility of a sulfate derivative of ascorbic acid part’icipating in the biosynt’hesis of sulfat,cd mucopolysaccharides did not escape the attent’ion of those cited above. The discovcrg of ascorbic sulfate in brine shrimp cyst’s by Jlead and Finamorc (8) and isolation of t,he same compound from various animal sourc(‘s by Tolbert et al. (9) demonstrated that ascorbic sulfate was a compound of widcspread biological distribut’ion. It was not, just a laboratory curiosity and perhaps wa,sa new biological sulfating agent. Since the chemical properties of the various isomers of ascorbic sulfate Lvould be expected t.o be very different from c>ach other, the structure of this compound must bc known if one would determine its function in metabolic pathways. Previous reports on ascorbic sulfate clearly established that the sulfate resides on either the 2- or X-position (for numbering, seeFig. 2)) and it was crrotleously assumed by ot#hershhat’ it, was the 3-sulfate. This report includes evidence to support our conclusion that both the synthetic and
20s
BOND
the natural ascorbic sulfates sulfonato-L-ascorbic acid. EXPERIMENTAL
are
2-0-
SECTION
Synthesis of Ascorbic Sulfate Preparation of cyclohexylidene ascorbic acid. n-ascorbic acid (Fisher reagent) was converted to 5,6-O-cyclohexylidene-L-ascorbic acid (CHAA) by a modification of the method described by Von Schuching and Frye (10). Fifty grams of ascorbic acid was finely powdered and added to 250 ml of redistilled cyclohexanone to which had been added 5 ml of concentrated H&S04 with rapid stirring at 4°C. This mixture was magnetically stirred in a glass-stoppered flask in the cold for 48 hr. It was then poured into 500 ml of ice water and quickly extracted with two 500~ml portions of dry, peroxide-free ether. The extracts were combined and washed twice with 250-ml portions of ice water, then dried with anhydrous NapSO, Ether was removed under vacuum to a volume of about 209 ml. Heptane was added slowly to the combined dried ext,ract while stirring with a glass rod to induce crystallization. When crystals began to form, approximately 1 liter of heptane was added and the mixture was placed in the cold room overnight. This material was recrystallized from etherheptane as pure white needles, stable over long periods in a desiccator. The yield was 80%. Formation and isolation of ascorbic sulfate. CHAA was then sulfated by the method of Ford (ll), using pyridine-sulfur trioxide in freshly distilled dimethylformamide. After 24 hr, 4 vol of cold water were added and the solution passed through a Dowex 5O(H+) column to hydrolyze the protective ketal from the 5- and 6-positions. A 3 X 24 cm column was used for 2.5 g of the CHAA preparation. Ascorbic sulfate thus produced was further purified by a modification of the procedure of Mead and Finamore (8). It was applied to a Dowex l-XZ(Cl-) column (3 X 24 cm) previously washed with 0.01 N HCl in 0.1 N NaCl. The column was washed with the same HCl-NaCl solution until no uv-absorbing material was found in the effluent. After further washing of the column with water until the effluent was neutral, ascorbic sulfate was eluted by a 0.1-1.0 M NaCl linear gradient. That material absorbing at 254 nm and eluting at approximately 0.35 M NaCl was acidified to pH 2 with HCl and then applied to an activated charcoal column (2 X 15 cm) as described by Mead and Finamore (8), with the following change. The activated charcoal column was prepared by washing with 1 M HCl followed by water until the effluent was neutral. It was then equilibrated with 0.01 M HCl before applying the sample. Ascorbic sulfate adsorbed to charcoal was washed with 500
ET
AL.
ml of 0.01 N HCl and then with 100 ml water before being eluted with a liter of eluant [ethanol, concentrated NH,OH, water (2:1:2) v/v/v]. Great care was taken to collect the eluate only after its pH rose above neutrality. This eluate was reduced in volume on the flash evaporator, then fractionated on a DEAE-cellulose (HC03-) column exactly as described by Mead and Finamore (8). The same procedure for purification was subsequently used to purify ascorbic sulfate from brine shrimp and other sources.
Crystallization Sulfate
and
Analysis
of Ascorbic
The pure diammonium salt obtained above was converted to free acid, barium salt, or dipotassium salt by passing a dilute solution of it through a Dowex 50 column in the appropriate form. The barium salt was crystallized by reducing the volume of effluent under vacuum and adding a few drops of ethanol. The pure white crystals were subsequently recrystallized several times in pure distilled water. Elemental analyses of the barium salt were performed by Galbraith Laboratories, Knoxville, TN. X-ray analysis. Crystals of the barium salt of synthetic ascorbic sulfate suitable for X-ray experiments were grown from water solution, either by evaporation or by diffusion of methanol through the vapor phase into the solution. Crystals were colorless prisms extended along the a axis. Preliminary diffraction photographs to establish the space group and approximate unit-cell dimensions were made with a Weissenberg camera and Cu Ka radiation. A crystal specimen selected for intensity measurements, roughly 0.1 X 0.2 X 0.4 mm in size, was cemented to a glass fiber, dipped repeatedly into liquid nitrogen, and mounted on an Oak Ridge computer-controlled diffractometer (12). Angle data for 12 reflections in the range 49” < 28 < 56’ (MO Kcv~ radiation) were used to refine the unit-cell parameters. All 1423 independent reflections with niobium-filtered MO Ka radiation and 20 2 56” were measured using s/28 step scans. The data, corrected for Lorentz-polarization and absorption effects, were used as coefficients in a three-dimentional Patterson synthesis (program FORDAPER of A. Zalkin, modified by G. Brunton). The positions of all nonhydrogen atoms were found by distinguishing the correct image from the centrosymmetrically related image through chemical criteria. Full-matrix least squares (program XFLS-3, a modification of program ORFLS; Ref. 13) were used to refine the structure, including subsequently all the hydrogen atoms, which were found in three-dimensional
Artpnlia
salina:
BSCORBIC
difference Fourier syntheses. The drawing was made on a Cal-Comp plotter with the aid of the program ORTEP (14). Spectrophotometric analysis. An infrared spectrum of the dipotassium salt of ascorbic sulfate was obtained on a Perkin-Elmer model 457 spectrophotometer. The KBr pellet was prepared by dissolving the dipotassium salt in water, adding 1 g KBr per mg ascorbic sulfate and reducing the resulting solution to dryness under vacuum. Ultraviolet spectra were obtained for various salts in aqueous solut,ions using a Beckman model DB spectrophotometer. Electrophoretic and chromatographic analysis. Electrophoresis of ascorbic acid sulfate was carried out in 0.1 M acetate buffer (pH 4.65) at 970 V and 7 mA for 25 min at room temperature, using 5 X 20.cm MN300 cellulose thin-layer chromatography (tic) plates obtained from Brinkman Instrument Company. Chromatography of ascorbic sulfat,e was performed with MN300 cellulose tic plates using the following three solvent systems: (1) met,hanol/formic acid/water (80: 15: 5 v/v/v) ; (2) methanol/coned NHaOH/water (60:30:30 v/v/v); (3) sat (NHI)804/0.1 M NHaCzH302/2propanol (79:19:2 v/v/v) (see Table I). The latter system was also used for paper chromatography with What,man No. 1 paper. Ascorbic sulfate was detected on tic plates by uv absorption and FeCls-methanol spray. Bscorbic acid-3.sulfate was graciously provided by B. M. Tolbert for comparison with our preparation. Acid properties of ascorbicsulfate. A Beckman model-G pH meter was used for all pH determinat,ions. The pK, of ascorbic acid sulfate was determined a.s described by Mead and Finamore (8).
Oxidati/le Transfer of Sulfate Group Ascorbic Sulfate
from
Oxidative transfer of the sulfate group was studied by oxidizing 5,6-0-isopropylidene ascorbic acid sulfate with Hz02 . The barium salt of this
ACID-2-SULFATE
209
ascorbic acid derivative was prepared by the method of Ford (11). It was dissolved in anhydrous dimethylsulfoxide at a concentration of 100 mg per 2 ml solvent and then 50 mg p-nitrophenol was dissolved in this solution. One milliliter of 30y0 Hz02 was added; the final solution was stirred and then allowed to stand at room temperature for 24 hr before products were analyzed. A similar experiment was performed in which dimethylsulfoxide was replaced by water. Products were examined by thin-layer chromatography. p-Nitrophenyl sulfate was chromatographed on MN300 cellulose tic plates using n-butanol/ethanol/water (80:24:40 v/v/v). It was detected by uv absorption and by the yellow color formed on exposure to NHaOH vapor after hydrolysis RESULTS
Characterization of Ascorbic Sulfate Spectrophotometric properties. The yield of 20sulfonatoascorbic acid, diammonium salt from this procedure approaches 50 %. Figure 1 represents the ir spectra of its potassium salts isolated from brine shrimp and from chemical synthesis. They are identical, with the exception of minor differences of intensity and a small, sharp absorption at 945 cm-’ which varied in intensity with the preparations. Ultraviolet spectra of ascorbic sulfate from both sourceshad a maximum at 232 nm in 0.01 N HCl that shifted to 254 nm in neutral or alkaline solutions with a concomitant increase in extinction coefficient of 55 %. The molar extinction coefficients previously reported (8) were in error, the new values being 1.1 X lo4 in acid and 1.7 X lo4 in base. The same properties were exhibited by the 3-sulfate supplied by Tolbrrt. Chromatographic and electrophoreticproperties. Results of thin layer chromatography
WAVENUMBER
FIG. 1. Infrared spectra of the dipotassium sources (O-0) and from brine shrimp (-).
salts of ascorbic acid sulfate from synthetic
210
BOND
are compiled in Table I. Ascorbic sulfate from the two sourcesgive the same Rf value in each developing solvent system even though the solvents have widely differing characteristics. A slight trace of contaminant in the natural material was detected by tic but appeared to be destroyed on paper after the longer development times required and was undetectable by other techniques. Electrophoretic mobilities on tic plates were identical. Acidic dissociation constants. Titration of ascorbic sulfate from each source revealed the samepK,%of 2.75. No attempt was made to determine the pK,, of the sulfate group. Elemental analysis. Elemental analyses of the barium salt of ascorbic sulfate are reported in Table II with calculated percentages based on two water molecules of hydration. The two water molecules were also detected by crystallographic studies. TABLE
I
CHROMATOGRAPHY OF ASCORBIC ACID ON TLC CELLULOSE Compound
I
I II
Oxidative Sulfate Transfer from Ascorbic Xulfate
1
Solvent 2
Solvent
3
Oxidation of 5,6-0-isopropylidene ascorbic sulfate by HzOs in aqueous medium quickly TABLE
sulfateO.73(0.79)a
0.SS0.88(0.41)” 0.88(0.44)=
sulfate
0.88
0.88 0.87
TABLE
II
ELEMENTAL AN.LLYSIS OF 2-0.SULFONATO-LASCORBIC ACID, BARIUM SALT, DIHYDR~TE (CsHloOnSBa) Observed percentage 16.80 2.41 41.39 7.60 31.96 lOO.lG
Calculated percentage 16.85 2.36 41.17 7.50 32.12 100.00
Ba S 0 0 0 0 0 0 0 0 0 w w c c
(1)
(2) (3) (4) (5)
(‘3) (7)
03) (9) (1)
(2) (1)
(2)
III
ATOMIC POSITIONS"
ATOM
0.73
e Trace of uv-absorbing substance, disappearing on exposure to air or light for a few hours.
C H 0 S Ba
X-ray crystallographic analysis of ascorbic sulfate. Crystals of the barium salt of synthetic ascorbic sulfate are triclinic, with unitcell parameters a = 50.201(l), b = 6.951 (l), and c = 8.732 (1) A; and cx = 99.54 (l), p = 93.29 (l), and y = 109.12 (1)“. With one molecule per unit cell and two water molecules of hydration, d(calcd) = 2.431 g/cm3, while d(meas) = 2.44 (3) g/cm3 as determined by flotation. The space group is thus PI. Least-squares refinement of the structure (varying the positional parameters of all 29 at’oms, anisotropic thermal parameters for the nonhydrogen atoms and isotropic ones for the hydrogen atoms, and an isot’ropic extinction parameter) has yielded an R factor of 0.009. The atomic coordinates are listed in Table III. The average positional estimated standard deviations are about 0.0025 & for oxygen and carbon atoms, and 0.06 A for hydrogens. The structure is depicted in Fig. 2, which shows many of the important, int’eratomic distances and bond angles. Dct,ails of structure will be presented elsewhere.
RF value Solvent
Ascorbic acid (from brine shrimp) Ascorbic acid (synthetic)
SULFATE
ET AL.
x
Y
0 0 2848 5796 7740 8524 2342 6957 -9853405 5712 6245 3845 1685 4735 764 1813 6859 12713626 5765 6207 5494 1374 9143 9390 5677 7176 3037 6273
z 0 - 1123 2536 551 2018 4018 2657 5904 - 2157 - 1289 - 1024 9282 6654 2747 1870
X’Y
ATOM c C ‘2 c H H H H H H H H H H
(3) (4) (5) (6) (05) (06) (WlA) (WlB) (W2A) (WZB) (C4) (C5) (C6A) (CGB)
z
1370 4725 2511 300147354009 318426104087 515027285490 551 217 263 301 17 594 523 132 826 645 248 974 803 987 628 857 807 618 218 519 490 140 170 426 702 322 522 500 368 632 I
I
(1 Triclinic coordinates X lo4 for nonhydrogen atoms, X 103 for hydrogens. W denotes water oxygen. Hydrogen atoms are labeled according to the atoms to which they are attached.
Artemia
salina: ASCORBIC
211
ACID-2-SULFATE
Gi
O(8) FIG. 2. ORTEP drawing (Johnson, 1970) of the molecular configuration of 2-O-sulfonatoL-ascorbic acid ion. All nonhydrogen atoms are represented by 59% probability ellipsoids of thermal displacement. Bond distances are given in A, bond angles in degrees. Bond angles not indicated include O(4)-C(4)-C(5) = 110.6; O(5)-C(5)-C(6) = 112.5; 0(2)-S-O(7) = 99.9; 0(7)-S-O(9) = 115.3; and 0(8)-S-O(9) = 113.1 degrees.
produced a heavy white precipitate of BaSOl as previously reported (5). However, in dimethylsulfoxide solution, only a trace of precipitate was formed after 24 hr. Chromatography of the reaction mixture yielded only one spot from aqueous solution and three from dimethylsulfoxide solution. When compared with known samples, the Rf = 0.98 spot corresponded to p-nitrophenol and was found in both solutions. An Rf = 0.53 spot corresponded to the 5,6-0-isopropylidene ascorbic sulfate, and the Rf = 0.85 spot corresponded to p-nitrophenyl sulfate. The latt.er two were observed only in the nonaqueous solution (Table IV).
TABLE
IV
CHROMATOGRAPHY OF WV-ABSORBING REACTION PRODUCTS FROM OXIDATION OF ISOPROPYLIDENE ASCORBIC SULFATE AND P-NITROPHENOL Compound Aqueous solution products Nonaqueous solution products p-Nitrophenol Isopropylidene ascorbic sulfate p-Nitrophenylsulfate
RF -
-
0.98
0.53
0.85
0.98
-
-
0.53
-
0.98 -
-
0.85
-
on adsorption and ion-exchange chromatography, provided further evidence that Since ultraviolet and infrared spectra, these compounds were identical and that we Rf in three different solvent systems, elec- were working with the same compound. Previous reports concerning ascorbic sultrophoretic mobilities at acid pH, and acid dissociation constants of natural and syn- fate have clearly demonstrated that the thetic ascorbic sulfate are identical, we sulfate must reside either at the 2- or 3conclude that the natural and synthetic position. Based on indirect evidence, sulfate was assigned to the 3-position by Ford and forms of this substance are also identical. Furthermore, cochromatography of our Ruoff (5) and every subsequent report rematerial with a sample of Tolbert’s ascorbic peated this assignment. However, the ability acid-3sulfate, coupled with identical phys- to form FeCL complexes, an important ical properties and identical behavior of each factor in making the original assignment of DISCUSSION
212
BOND
ET
AL.
structure, is not restricted to the 3-isomer as TABLE V supposed (4, 15). ACID STRENGTH OF ASCORBIC ACID DERIVATIVES Numerous ascorbic acid derivatives have AND SUBSTITUTED ENOLS been synthesized, and a comparison of their Compound ph P& properties with those of ascorbic sulfate from brine shrimp should allow the structure acida 4.1 , 11.8 of the latter to be inferred. Both 2- and Ascorbic 3-Methylascorbic acidb 7.8 3-acyl derivatives have been prepared, but, 2-Methylascorbic acid” 3.4 unfortunately, contradictory structural as2-Phenylphosphoryl <4 <4 signments have been made. Kobayashi (16) ascorbic acid6 prepared several diesters of ascorbic acid, one 3-Phenylphosphoryl <4 7.2 of which was designated as a 2,6-diacyl-nascorbic acidb ascorbic acid. Later, Nomura and Sugimoto Ascorbic sulfate (strong acid) 2.75 (17) synthesized some dibenzoyl esters and Phenol” 9.9 p-Phenolsulfonic acidd concluded that Kobayashi’s 2,6-diester was (strong acid) acidd (strong acid) / YO in fact a 3,6-diester. While Nomura’s in- m-Phenolsulfonic p-Nitrophenola 7.2 terpretation was based on ir and nmr spectra, o-Nitrophenola 7.2 he admitted ambiguity in the data. Tanaka and Yamamoto (18) then prepared a variety 0 Handbook of Chemistry and Physics, 47th of ascorbic acyl esters, compared their ed. (1967). The Chemical Rubber Co., Cleveland, properties, and assigned structures which OH , pp. D-86, 87. supported Kobayashi and contradicted *Personal communication from V. M. Clark. Nomura. Clark et al. (4) synthesized 2- and c Ettlinger et al., 1961 (Ref. 23). 3-phosphoryl ascorbic acids and determined d H. Zollinger (1953) Nature (London) i72; 257. their properties; he subsequently identified them (personal communication). From analogous properties of these isomers, one con- ascorbic acid correspondsroughly to a phenyl cludes that Kobayashi, Tanaka and ester. Consequently, the 2-isomer should be Yamamoto, and Clark et al. agree on struc- more stable thermodynamically. Imai (19) tural assignment and that Nomura and observed a rapid migration of the acyl group from Nomura’s “&benzoyl” ester in dilute Sugimoto’s interpretations are inverted. Clark observed that substitution of a 2- or alkali. Migration should occur in a direction and, therefore, 3-position with a phenylphosphoryl group of increasing stability Nomura’s structural assignment was in error. decreasedthe pK, of the neighboring enol. A Tanaka and Yamamoto (18) observed a number of pK, values for acyl ascorbic acids distinct pattern in the uv spectra of ascorbic and substituted enols are compared in Table acid derivatives which was also seen by V. It is a well-known fact that substitution by an electron-withdrawing group causesthe Clark (personal communication). Table VI pK, of a conjugated enol to decrease. How- lists uv maxima and extinction coefficients ever, even as powerful a substituent as a for some of these, and suggeststhat a simple nitro group causes a decrease of less than rule can be formulated relating structure and three units. Nomura and Sugimoto (17) re- spectral properties. Substitution of the 3-OH ported an inconsistent pattern. That is, they effects little change in uv spectrum from that claimed that benzoylation of the 2-OH of unsubstituted ascorbic acid unlessthe free caused the pK. of the 3-OH to increase from enol becomes dissociated. Dissociation produces a bathochromic shift to about 260-265 4.1 to 6.4, whereas benzoylation of the 3-OH nm with little change in molar extinction caused the pK, of the 2-OH to decrease by coefficient. However, substitution of the nearly nine units. Reversing their assignment 2-OH causesa hypsochromic shift of about would yield a consistent decrease in each 10 nm for both the undissociated molecule case and eliminate disagreement with the and the anion. Dissociation of the 3-OH has other investigators. Furthermore, the 3- a hyperchromic effect resulting in an increase benzoyl ascorbic acid is a vinylog of a mixed of extinction coefficient of about 50 70. acid anhydride, whereas the 2-benzoyl From its spectral characteristics in acid
i g..
Artemia
salina:
ASCORBIC TABLE
UV
SPECTRAL
CHARACTERISTICS
Compound
3-Phenylphosphoryl 2-Phenylphosphoryl Ascorbic sulfate (L Personal
ascorbic ascorbic
communication
VI OF SUBSTITUTED
ASCORBIC
Acid
Ascorbic acida 3-Methylascorbic acidu 2,3-Dimethyl ascorbic acida acida acid”
from
V. M.
ACIDS
Neutral
max
e x 18
max
243 244 235
10 9 g
265 244 -
237 233 232
8 9.6 11
238 257 254
Base
Ex
103
max
6 x lo3
16.5 9 -
276 -
-
8 14.5 17
263 258 254
8 15 17
9 -
Clark.
and neutral solution, and the acidity of ascorbic sulfate, it would appear that this molecule is a %-isomer rather than the 3isomer. We have subjected the pure crystalline barium salt to X-ray crystallographic analyses and have shown conclusively that it is, in fact, 2-0-sulfonato-L-ascorbic acid, barium salt. Widespread natural occurrence of this compound (9, 20) implies that ascorbic sulfate may have a function in sulfate metabolism, and ascorbate is known to be required for proper sulfation of mucopolysaccharides. To explain this function, previous reports have claimed oxidative transfer of sulfate from ascorbic sulfate and suggested that a high-transfer-potential sulfate was formed by the oxidation (5-7, 21). However, the acceptors used did not establish formation of such a compound. Detection of p-nitrophenyl sulfate as an oxidat’ion product of isopropylidene ascorbic sulfate and p-nitrophenol in nonaqueous solution determined that a high-transfer-potential sulfate had been formed (22). Robbins est’imated the AG of hydrolysis for p-nitrophenylsulfate equal to -15 kcal/mole, which is comparable to that for 3’-phosphoadenosine-5’phosphosulfate. We must conclude, therefore, that 2-0-sulfonato-L-ascorbic acid is a widely distributed natural product which has the capacity to form a high-transferpotential sulfate during an in vitro oxidation process. It thus could participate in in viva sulfation processes as suggested by Mumma and Verlangieri (al). ACKNOWLEDGMENT We are grateful to C. II. Wei with the X-ray crystallographic
213
ACID-2-SULFATE
for his assistance analysis.
REFERENCES V. M., HUTCHINSON, D. W., KIRBY, G. W., AND TODD, SIR ALEXANDER (1961) J. Chem. Sot. 715. BOND, A., AND MASON, H. S. (1962) Biochem. Biophys. Res. Commun. 9, 574. WF,IDMAN, S. W., MAYERS, D. F., ZABORSKY, 0. R., AND KAYSI.;R, E. T. (1967) J. Amer. Chem. Sot. 89, 4555. CLARK, V. M., HERSHEY, J. W. B., AND HUTCHINSON, D. W. (1966) Esperientia 22, 425. FORD, E. A., AND RUOFF, P. M. (1965) Chem. Commun. 630. CHU, T. M., AND SLAUNWHITE, W. R., JR. (1968) Steroids 12, 309. MUMMA, R. 0. (1968) Biochim. Biophys. Acta 166, 571. MEAD, C. G., AND FINAMORE, F. J. (1969) Biochemistry 8, 2652. TOLBERT, B. M., ISHERWOOD, D. J., ATCWELY, R. W., AND BAKER, E. M. (1971) Fed. Proc. 30, 529. VON SCHUCHING, S. L., AND FRYI<, G. H. (1966) Biochem. S. 93, 652. FORD, E. A. (1968) Doctoral dissertation, Syracuse University, Syracuse, NY, 1967; Diss. AbstT. B 28, 4059. BUSING, W. R., ELLISON, K. D., LI:VY, H. A., KING, S. P., AND ROSEBERRY, R. T. (1968) The Oak Ridge Computer Controlled X-ray Diffractometer. Report ORNL-4143, Oak Ridge National Laboratory, Oak Ridge, TN. BUSING, W. R., MARTIN, K. O., AND LEVY, H. A. (1962) ORFLS, A Fortran Cryst,allographic Least-Squares Program. ORNLTM-305, Oak Ridge National Laboratory, Oak Ridge, TN. JOHNSON, C. K. (1970) ORTEP, A Fortran Thermal-Ellipsoid Plot Program for Crystal-structure Illustration. ORNL-3794, 2nd revision, Oak Ridge Nat onal Laborat,ory, Oak Ridge, TN.
1. CLARK,
2. 3.
4.
5. 6. 7. 8.
9.
10. 11.
12.
13.
14.
214
BOND H., AND MORIMOTO, S. (1971) Chem. Pharm. Bull. (Tokyo) 19, 335 KOBAYASHI, K. (1965) Japan Patent Application 9134. NOMURA, H., AND SUGIMOTO, K. (1966) Chem. Pharm. Bull. (Tokyo) 14, 1039. TANAKA, H., AND YAMAMOTO, R. (1966) Yakugaku Zasshi 86, 376. IMAI, Y. (1966) Chem. Pharm. Bull. (Tokyo) 14, 1045. BBKER, E. M. III, HAMMER, D. C., MARCH,
15. NOMURA,
16. 17. 18. 19. 20.
ET AL. S. C., TOLBERT, B. M., AND CANHAM, J. E. (1971) Science 173, 826. 21. MUMMA, R. O., AND VERLANGIERI, A. J. (1971) Fed. Proc. 30, 370. 22. ROBBINS, P. (1962) in The Enzymes (Boyer, P. D., Lardy, H. A., and MyrbSick, K., eds.), 2nd ed., Vol. 6, p. 363. Academic Press, New York. 23. ETTLINGER, M. G., DATEO, G., HARRISON, B., MALOY, T., AND THOMPSON, C. (1961) Proc. Nat. Acad. Sci. USA 47, 1875.