44
A.F. Hartog, R. Wever / Journal of Molecular Catalysis B: Enzymatic 129 (2016) 43–46
wise and the solution was stirred for 24 h. 10 mL of water was
added drop wise and the dioxane removed by evaporation. The
10 mL water phase was extracted twice with 10 mL ethyl acetate
and water was removed by evaporated to almost complete dry-
ness. 20 mL of methanol was added resulting in a precipitate that
was removed and discarded. After standing white crystals were
formed in the solution, these were collected on a glass filter and
washed with methanol. HPLC, IR, and 1H and 13C NMR identified
this as NHS sulfate (sodium salt, 0.93 g).
Fig. 1. Scheme of the synthesis of N-hydroxysuccinimide-sulfate (1).
observed [24]. Recently it was demonstrated [28] that by substrate
engineering a number of carbohydrate derivatives could be sul-
fated by this bacterial enzyme. However, a broad applicability of the
cation of the sulfated product to remove toxic p-nitrophenol and
the donor p-NPS limiting the application of the enzymatic system
in the synthesis of products with a pharmaceutical application.
In our search for compounds that were sulfated by the
bacterial arylsulfotransferase we observed [24] that when N-
hydroxysuccinimide (1) was added to a solution of p-nitrophenol
sulfate in the presence of the enzyme that the concentration of p-
nitrophenol sulfate decreased. We concluded that sulfate transfer
might have occurred and decided to synthesize the sulfate ester of
NHS as a reference compound and to test its application as a sulfate
donor for the enzyme.
2.4. Enzymatic synthesis of 3-sulfo-17-ˇ-estradiol (3) using
NHS-sulfate as donor
17--Estradiol (2) (0.024 g, 0.088 mmol) was dissolved in
600 L acetone and 5 mL Tris-glycine (100 mM, pH 9) was added. To
the milky solution 500 L of 12 U/ml AST was added. The resulting
solution (1 unit/ml, 1.6 M AST, 15 mM 17--estradiol) was stirred
and NHS-sulfate Na salt (0.021 g, 0.1 mmol) was added (Fig. 2). The
addition was repeated 3 times every hour. After 4 h acetic acid was
added to pH 6 and the reaction mixture evaporated to almost dry-
ness. The residue was solubilized in 20 mL methanol and 5 g silica
gel was added. After evaporation to almost dryness the silica gel
was poured onto a silica column and the silica gel column was
eluted with acetonitrile (0.5% acetic acid/5 to 10% methanol). NHS
eluted first followed by (3). Due to complete hydrolyses of NHS-
sulfate at pH > 7 only NHS was found in the first fractions. TLC and
HPLC were used to check the fraction and these were pooled and
evaporated to complete dryness. Pure 3 (0.028 g, 68% isolated yield)
was obtained.
2. Experimental
2.1. HPLC analysis and NMR
All chemicals used were purchased from Sigma-Aldrich. Reac-
tion progress was monitored by HPLC. Samples from the reaction
mixture were diluted 100 times with acetonitrile/water (1:1), and
25 L was injected onto a Nucleosil 100-5C-18HD column (plus
guard column), equilibrated with NH4H2PO4 buffer (pH 6.3, 25 mM)
containing TBAP (ion-pairing reagent, tetrabutylammonium phos-
phate, 5 mM) and 5% acetonitrile (buffer A). The HPLC dual pump
system was an Agilent 1100 system with a 0–80% gradient (37 min
run, 0.4 mL min−1 flow rate) of acetonitrile (90% aq.), containing
TBAP (5 mM) (buffer B). The UV absorbance of the compounds was
monitored at 210 and 300 nm. Peak areas of the detected substrates
and products were used to calculate the degree of sulfate transfer.
1H and 13C NMR spectroscopy was carried out with a Bruker NMR
instrument at 400 or 500 MHz. Samples for NMR measurements
were dissolved in [D6]DMSO.
2.5. Enzymatic synthesis of bisphenol A 4,4ꢀ-bisulfate (5) using
NHS-sulfate (1) as a donor
Bisphenol A (4) (0.046 g, 0.2 mmol) was added to 14 mL Tris-
glycine (100 mM, pH 9). To the milky solution 700 L of 47 U/ml
AST was added. The resulting solution (2.2 unit AST/ml) was stirred
and (1) (0.035 g, 0.16 mmol) was added. The addition of (1) was
repeated 5 times every hour. After 2 h the colorless solution clar-
ified. Each additions of (1) was followed by the addition of 0.05 g
solid Tris to keep the pH between pH 8.5 and 9. After 6 h the reac-
tion was stopped by the addition of acetic acid and (5) was purified
by silica gel chromatography as described for (3). Pure 5 (0.055 g,
70% isolated yield) was obtained.
3. Results and discussion
2.2. Heterologous overproduction and purification of AST
(4-hydroxyphenyl) propane) (4) were tested as acceptors in the
enzymatic sulfation with the NHS-sulfate as a donor. The first two
compounds have previously been sulfated by the arylsulfotrans-
ferase using p-nitrophenol sulfate. Bisphenol A is a well-known
endocrine-disrupting chemical [33]. After exposure the latter com-
pound is secreted in its sulfated form by the human body. As
shown before the sulfation of E-resveratrol is complex since it has
three OH groups that may become sulfated resulting in the for-
mation of mono, di- and trisulfated resveratrol [25]. On a small
scale (1 mL) enzymatic sulfation of resveratrol was tested by the
daily addition of NHS-sulfate. After 3 days resveratrol was con-
verted completely into its sulfated forms. The HPLC traces in the
Supporting information shows this conversion in detail. Similarly
on a small scale (1 mL) 17--estradiol (2) and bisphenol A (2,2-bis
(4-hydroxyphenyl) propane) (4) were completely sulfated enzy-
matically (not shown) in 4–6 h by adding each hour one equivalent
of solid NHS-sulfate (Na salt) keeping the pH at 9 by addition of
solid Tris.
The AST was expressed and purified using a method developed
earlier with minor modifications [24,25].
Esters of NHS are widely used in peptide synthesis, in the
amino groups in peptides [29–31]. However, there is hardly
any literature on the synthesis of the NHS-sulfate ester (1-
(sulfooxy)-2,5-pyrrolidinedione, CAS 127007-81-2). Only a patent
[32] describes its synthesis (outlined below in Fig. 1) and its appli-
cation as a cross linker of cellulose fibers.
0.5 g of a mixture of sodium hydride (NaH) in mineral oil (60%)
was washed 2 times with petroleum ether. The solid was dis-
persed in 8 mL dry dioxane under nitrogen and 1.15 g (10 mmol)
of N-hydroxysuccinimide was added wise at 20 ◦C. After 1 h 16 mL
dry dioxane with 2 g (11 mmol) triethylamine-SO3 was added drop