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Selective hydroxyl sulfation by a dicyclohexylcarbodiimide-mediated reaction
BIOCHIMICA ET BIOPHYSICA ACTA
149
S I-I O RT C O M M U N I C A T I O N S
BK~ 23478
Selective hydroxyl sulfation by a dicyclohexylcarbodiimide-mediated reaction A great number of naturally occurring alkyl and aryl sulfates have been found in both plant and animal tissues1, ~. In the present investigation, mild reaction conditions were sought under which biologically labile compounds could be sulfated easily. A dicyclohexylcarbodiimide-mediated sulfation reaction ~ was examined using radiochemical techniques to determine whether sulfate esters could be synthesized in good yields, and whether reaction conditions could be found which would permit the selective sulfation of hydroxyl groups. Two sets of reaction conditions, here designated as (I) "dilute" and (2) "concentrate", were examined. Under the "concentrate" conditions, both alcoholic and phenolic groups were sulfated in high yield, there being no selectivity. However, under the "dilute" conditions, selective hydroxyl sulfation did occur, alcohols being sulfated in good yields whereas phenols were not sulfated. Hence, the selective monosulfation of polyhydroxy molecules such as ~-estradiol is now possible. Previous methods required the addition and removal of selective blocking groups 4. Radiochemical techniques were employed in all the reactions to verify the formation of ~sS-labeled products. Under the "dilute" conditions the following procedure was used: I mmole of labeled conc. H,SO 4 dissolved in 12 ml of dimethylformamide was added to a solution containing 5 mmoles of dicyclohexylcarbodiimide and I mmole of a selected hydroxy compound dissolved in 28 ml of dimethylformamide at 0 °. The "concentrate" conditions contained the same number of mmoles of reactants as the "dilute" conditions, except that only one-tenth the amount of the solvent (dimethylformamide) was used. These reaction conditions were chosen in order to obtain standard conditions for comparison; they are not necessarily optimal for the synthesis of the sulfate esters. After 15 rain, aliquots were removed from the reaction mixtures and separated by thin-layer chromatography (chloroform-methanol-water; 65:25:4, v / v / v ) a n d paper chromatography (phenol-water; Ioo:4o , w/w). Monosulfate products were visualized on the chromatograms by selective sprays and radioautographic techniques. The yield for each ~S-labeled monosulfate was determined, from the thin-layer and/or paper chromatograms, as the relative percentage of ~*~;S~monosulfate (counts/rain) to the total amount of ~S (counts/rain) present. The chromatograms were sectioned, and counted in a Tri-Carb (Packard) liquid scintillation spectrometer. The purity of the reagents used was checked by gas-liquid chromatography, thin-layer chromatography, paper chromatography, and b y melting and boiling points. The structure of the alkyl monosulfate, synthesized by the dicyclohexylcarbodiimidemediated reaction, was verified by elemental, infrared, and melting point analyses ~. Biochim. Biophys. Acta, 177 (1969) 149-I51
150
SHORT COMMUNICATIONS
Further confirmation of the monosulfate structure was obtained by thin-layer co-, chromatography of the dicyclohexylcarbodiimide-synthesized lnOnosulfates wit~ pyridine-SOa-synthesized monosulfates ~ in five different so!vent systems. Also, >h (~ M HC1) hydrolysis of the products yielded inorganic sulfate and the respective alcohol (confirmed by gas-liquid chromatographic data). This is characteristic of sulfate esters a. Several selected monosulfate structures were confirmed by mass spectral analysis utilizing laser beam ionizing techniques v. A typical thinqayer chromatogram is shown in Figs. IA and ~E.
~ntI
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. . . .
.
.
F
J
. _ ('~).
~,
.
"g ~ ® ] ~
~
]
~
1
origin I a
~
~
b
~
~"
~
d
e
a
b
c
d
e
Fig. -~A. R a d i o a u t o g r a m d r a w i n g of t h e t e t r a d e c y l s u l f a t e e x p e r i m e n t a nd i t s c ont ro!s : a. T e t r a d e c a n o l a n d d i m e t h y l f o r m a m i d e , b. D i c y c l o h e x y l c a r b o d i i m i d e , Hza~SOa a n d d i m e t h y l f o r m a m i d e . c. T e t r a d e c a n o l , H=a~SO, a n d d i m e t h y l f o r m a m i d e , d. T e t r a d e c a n o l , d i c y c l o h e x y l c a r b o d i i m i d e , H2asSO, a n d d i m e t h y l f o r m a m i d e ( " d i l u t e " r e a c t i o n c o n d i t i o n s / , e. Pyridine-a~SOa, t e t r a d e c a n o l and dimethylformamide. Fig. I B . T h e s a m e t h i n - l a y e r c h r o m a t o g r a p h i c p l a t e as in Fig. I A b u t v i s u a l i z e d w i t h iodine (the t h i n - l a y e r c h r o m a t o g r a p h i c p l a t e w a s d e v e l o p e d w i t h c h l o r o f o r m - m e t h a n o l -wa~er, 05 : 25 : ¢.
v!~lv). TABLE YIELD
I
DATA
FOR
THE
H y d e o x y compou~cd
DICYCLOHEXYLCARBODIIMIDE-MEDIATED
Reaction conditiO~
SULFATION
OF
H~DROXY
COMPOUNDS
Total ~zumbex q[ ass-gabded p~oduc~,s observed a~d the combined } ield Thice-layer chromatography
Paper chromatography
No.
No.
Yield (%)
Y i e l d (%
I --OctanoI
Dilute Concentrate
~ z
78 Ioo
~ z
77 99
CyclohexanoI
Dilute Concentrate Dilute Concentrate Dilute Concentrate DiInte Concentrate Dilute D i l a t e + 0. 5 m m o l e H2SO a
i 2 ~ z ~ 2 o 2 • i
45 95 80 93 < I 95
~ 2
42 96
o g o 2
o 93 o 99
Dilute
•
45
I-Tetradecanol Pheno! ~-Naphthol ~-Estradio! Cholesterol
* E x p e r i m e n t not conducted.
Biochim: Biophys. Act•, •77 (I969) 1 4 9 - I 5 I
28 65
SHORT COMMUNICATIONS
I5I
The monosulfate esters of a number of primary and secondary alcohols were formed in good yields in both the "dilute" and "concentrate" conditions. A selective number of these alcohols are presented in Table I. Phenolic hydroxyl groups, for example in phenol, ~-naphthol and fi-estradiol, were not sulfated under the "dilute" conditions but were sulfated in good yields under the "concentrate" conditions. In addition to the monosulfates, one other ~S-labeled product was obtained under the "concentrate" conditions. This additional product appears to be a sulfate ester anhydride, analogous to the sulfonic anhydrides reported by t{HORANA s, and is currently being investigated. A practical application of the selective sulfation was demonstrated by the direct synthesis of estradiol-IT/3-sulfate. Elemental, ultraviolet, infrared, and melting point analyses confirmed the structure~, showing that the IT-hydroxy group was selectively 0 OS--O~0
Estradio[-~ 7B- sulfate
sulfated and not the phenolic group. Details of the preparation of gram quantities of steroid sulfates and structural proof will be presented at a later date. The selectivity of this dicyclohexylcarbodiimide-mediated sulfation will have important biological applications, particularly in the synthesis of aaS-labeled steroid sulfates. This work was supported in part by the U.S. Public Health Service Grant AM o8481 and the Pennsylvania Agricultural Experiment Station.
Department of Biochemistry, Department of Entomology, The Pennsylvania State University, Pesticide Research Laboratory and Graduate Study Center, University Park, Pa. 168o2 (U.S.A.)
CHARLES P . ~{0IBERG RALPH O. MUMMA
I J. D. GRXGORY AND P. \¥. ROBIn,S, Ann. Rev. Biochem., 29 (I96o) 347. 2 E. ]~AULIEU, C. CORPECHOT, ~'. ~DRAY, ]~. E~ILIOZZI, iVf. LEBEAU, P. MAUVAIS-JARVIS ANI~ P. ROBEL, in G. PINCUS, Recent Progress in Hormone Research, Academic Press, New York, 1965, p. 411. 3 IR. O. MUMMA, Lipids, I (1966) 221. 4 R. "Y. KIRDANI, Steroids, 6 (1965) 845. 5 E. ]~. GILBERT, Sulfonation and Related Reactions, Interscience, New York, 1965, p. 3516 13. D. BATTS, f . Chem. Soc. (B), (1966) 551. 7 J" ~'ASTOLA,A, J. PIRO.NE AND R. O. MUMV~A,I6th Annual Conference on Mass Spectrometry and Allied Topics, Pittsburgh, Pa., ~968. 8 14£. I~D.KttORANA, Can. J. Chem., 31 (I953) 585.
Received September zoth, 1968 Biochim. Biophys. Acta, 177 (1969) I49-15I
149
S I-I O RT C O M M U N I C A T I O N S
BK~ 23478
Selective hydroxyl sulfation by a dicyclohexylcarbodiimide-mediated reaction A great number of naturally occurring alkyl and aryl sulfates have been found in both plant and animal tissues1, ~. In the present investigation, mild reaction conditions were sought under which biologically labile compounds could be sulfated easily. A dicyclohexylcarbodiimide-mediated sulfation reaction ~ was examined using radiochemical techniques to determine whether sulfate esters could be synthesized in good yields, and whether reaction conditions could be found which would permit the selective sulfation of hydroxyl groups. Two sets of reaction conditions, here designated as (I) "dilute" and (2) "concentrate", were examined. Under the "concentrate" conditions, both alcoholic and phenolic groups were sulfated in high yield, there being no selectivity. However, under the "dilute" conditions, selective hydroxyl sulfation did occur, alcohols being sulfated in good yields whereas phenols were not sulfated. Hence, the selective monosulfation of polyhydroxy molecules such as ~-estradiol is now possible. Previous methods required the addition and removal of selective blocking groups 4. Radiochemical techniques were employed in all the reactions to verify the formation of ~sS-labeled products. Under the "dilute" conditions the following procedure was used: I mmole of labeled conc. H,SO 4 dissolved in 12 ml of dimethylformamide was added to a solution containing 5 mmoles of dicyclohexylcarbodiimide and I mmole of a selected hydroxy compound dissolved in 28 ml of dimethylformamide at 0 °. The "concentrate" conditions contained the same number of mmoles of reactants as the "dilute" conditions, except that only one-tenth the amount of the solvent (dimethylformamide) was used. These reaction conditions were chosen in order to obtain standard conditions for comparison; they are not necessarily optimal for the synthesis of the sulfate esters. After 15 rain, aliquots were removed from the reaction mixtures and separated by thin-layer chromatography (chloroform-methanol-water; 65:25:4, v / v / v ) a n d paper chromatography (phenol-water; Ioo:4o , w/w). Monosulfate products were visualized on the chromatograms by selective sprays and radioautographic techniques. The yield for each ~S-labeled monosulfate was determined, from the thin-layer and/or paper chromatograms, as the relative percentage of ~*~;S~monosulfate (counts/rain) to the total amount of ~S (counts/rain) present. The chromatograms were sectioned, and counted in a Tri-Carb (Packard) liquid scintillation spectrometer. The purity of the reagents used was checked by gas-liquid chromatography, thin-layer chromatography, paper chromatography, and b y melting and boiling points. The structure of the alkyl monosulfate, synthesized by the dicyclohexylcarbodiimidemediated reaction, was verified by elemental, infrared, and melting point analyses ~. Biochim. Biophys. Acta, 177 (1969) 149-I51
150
SHORT COMMUNICATIONS
Further confirmation of the monosulfate structure was obtained by thin-layer co-, chromatography of the dicyclohexylcarbodiimide-synthesized lnOnosulfates wit~ pyridine-SOa-synthesized monosulfates ~ in five different so!vent systems. Also, >h (~ M HC1) hydrolysis of the products yielded inorganic sulfate and the respective alcohol (confirmed by gas-liquid chromatographic data). This is characteristic of sulfate esters a. Several selected monosulfate structures were confirmed by mass spectral analysis utilizing laser beam ionizing techniques v. A typical thinqayer chromatogram is shown in Figs. IA and ~E.
~ntI
(Y~
. . . .
.
.
F
J
. _ ('~).
~,
.
"g ~ ® ] ~
~
]
~
1
origin I a
~
~
b
~
~"
~
d
e
a
b
c
d
e
Fig. -~A. R a d i o a u t o g r a m d r a w i n g of t h e t e t r a d e c y l s u l f a t e e x p e r i m e n t a nd i t s c ont ro!s : a. T e t r a d e c a n o l a n d d i m e t h y l f o r m a m i d e , b. D i c y c l o h e x y l c a r b o d i i m i d e , Hza~SOa a n d d i m e t h y l f o r m a m i d e . c. T e t r a d e c a n o l , H=a~SO, a n d d i m e t h y l f o r m a m i d e , d. T e t r a d e c a n o l , d i c y c l o h e x y l c a r b o d i i m i d e , H2asSO, a n d d i m e t h y l f o r m a m i d e ( " d i l u t e " r e a c t i o n c o n d i t i o n s / , e. Pyridine-a~SOa, t e t r a d e c a n o l and dimethylformamide. Fig. I B . T h e s a m e t h i n - l a y e r c h r o m a t o g r a p h i c p l a t e as in Fig. I A b u t v i s u a l i z e d w i t h iodine (the t h i n - l a y e r c h r o m a t o g r a p h i c p l a t e w a s d e v e l o p e d w i t h c h l o r o f o r m - m e t h a n o l -wa~er, 05 : 25 : ¢.
v!~lv). TABLE YIELD
I
DATA
FOR
THE
H y d e o x y compou~cd
DICYCLOHEXYLCARBODIIMIDE-MEDIATED
Reaction conditiO~
SULFATION
OF
H~DROXY
COMPOUNDS
Total ~zumbex q[ ass-gabded p~oduc~,s observed a~d the combined } ield Thice-layer chromatography
Paper chromatography
No.
No.
Yield (%)
Y i e l d (%
I --OctanoI
Dilute Concentrate
~ z
78 Ioo
~ z
77 99
CyclohexanoI
Dilute Concentrate Dilute Concentrate Dilute Concentrate DiInte Concentrate Dilute D i l a t e + 0. 5 m m o l e H2SO a
i 2 ~ z ~ 2 o 2 • i
45 95 80 93 < I 95
~ 2
42 96
o g o 2
o 93 o 99
Dilute
•
45
I-Tetradecanol Pheno! ~-Naphthol ~-Estradio! Cholesterol
* E x p e r i m e n t not conducted.
Biochim: Biophys. Act•, •77 (I969) 1 4 9 - I 5 I
28 65
SHORT COMMUNICATIONS
I5I
The monosulfate esters of a number of primary and secondary alcohols were formed in good yields in both the "dilute" and "concentrate" conditions. A selective number of these alcohols are presented in Table I. Phenolic hydroxyl groups, for example in phenol, ~-naphthol and fi-estradiol, were not sulfated under the "dilute" conditions but were sulfated in good yields under the "concentrate" conditions. In addition to the monosulfates, one other ~S-labeled product was obtained under the "concentrate" conditions. This additional product appears to be a sulfate ester anhydride, analogous to the sulfonic anhydrides reported by t{HORANA s, and is currently being investigated. A practical application of the selective sulfation was demonstrated by the direct synthesis of estradiol-IT/3-sulfate. Elemental, ultraviolet, infrared, and melting point analyses confirmed the structure~, showing that the IT-hydroxy group was selectively 0 OS--O~0
Estradio[-~ 7B- sulfate
sulfated and not the phenolic group. Details of the preparation of gram quantities of steroid sulfates and structural proof will be presented at a later date. The selectivity of this dicyclohexylcarbodiimide-mediated sulfation will have important biological applications, particularly in the synthesis of aaS-labeled steroid sulfates. This work was supported in part by the U.S. Public Health Service Grant AM o8481 and the Pennsylvania Agricultural Experiment Station.
Department of Biochemistry, Department of Entomology, The Pennsylvania State University, Pesticide Research Laboratory and Graduate Study Center, University Park, Pa. 168o2 (U.S.A.)
CHARLES P . ~{0IBERG RALPH O. MUMMA
I J. D. GRXGORY AND P. \¥. ROBIn,S, Ann. Rev. Biochem., 29 (I96o) 347. 2 E. ]~AULIEU, C. CORPECHOT, ~'. ~DRAY, ]~. E~ILIOZZI, iVf. LEBEAU, P. MAUVAIS-JARVIS ANI~ P. ROBEL, in G. PINCUS, Recent Progress in Hormone Research, Academic Press, New York, 1965, p. 411. 3 IR. O. MUMMA, Lipids, I (1966) 221. 4 R. "Y. KIRDANI, Steroids, 6 (1965) 845. 5 E. ]~. GILBERT, Sulfonation and Related Reactions, Interscience, New York, 1965, p. 3516 13. D. BATTS, f . Chem. Soc. (B), (1966) 551. 7 J" ~'ASTOLA,A, J. PIRO.NE AND R. O. MUMV~A,I6th Annual Conference on Mass Spectrometry and Allied Topics, Pittsburgh, Pa., ~968. 8 14£. I~D.KttORANA, Can. J. Chem., 31 (I953) 585.
Received September zoth, 1968 Biochim. Biophys. Acta, 177 (1969) I49-15I