Rapid and efficient microwave-assisted synthesis of highly sulfated organic scaffolds

Article abstract of DOI:10.1016/j.tetlet.2007.07.100

Sulfation of multiple hydroxylated small organic molecules is fraught with problems of poor yield, multitude of products, and long reaction times. We have developed a rapid microwave-based method for synthesis of highly sulfated small organic molecules, w

Full text of DOI:10.1016/j.tetlet.2007.07.100

Tetrahedron Letters 48 (2007) 6754–6758
Rapid and efficient microwave-assisted synthesis
of highly sulfated organic scaffolds
Arjun Raghuraman,a Muhammad Riaz,a Michael Hindleb and Umesh R. Desaia,*
aDepartment of Medicinal Chemistry and Institute for Structural Biology and Drug Discovery,
Virginia Commonwealth University, Richmond, VA, USA
bDepartment of Pharmaceutics, Virginia Commonwealth University, Richmond, VA, USA
Received 14 June 2007; revised 11 July 2007; accepted 14 July 2007
Available online 24 July 2007
Abstract—Sulfation of multiple hydroxylated small organic molecules is fraught with problems of poor yield, multitude of products,
and long reaction times. We have developed a rapid microwave-based method for synthesis of highly sulfated small organic mole-
cules, which affords the per-sulfated product in moderate to excellent yields and high purity. The method is expected to be of value in
the discovery of per-sulfated organic molecules as mimics of glycosaminoglycans, which are being increasingly recognized as mod-
ulators of key physiological functions.
Ó 2007 Elsevier Ltd. All rights reserved.
Recent work in our laboratory shows that designed
highly sulfated, aromatic, small organic molecules pos-
sess interesting physico-chemical and biological proper-
ties.1–5 Biochemically, these molecules form multiple
ionic as well as non-ionic interactions, which form the
backbone of most protein-recognition elements. Struc-
turally, these represent mimics of glycosaminoglycans
(GAGs), which are increasingly being recognized as
modulators of key physiological functions,6,7 while
toxicologically, the sulfated structure represents a
highly water-soluble, already-metabolized form that is
expected to possess minimal toxicity. Despite these
novel features, highly sulfated organic molecules remain
largely unexplored.
Theoretically, this method could be extended for the
synthesis of highly sulfated drug-like molecules, yet
practically it is a synthetic nightmare because these
molecules possess significantly higher negative charge
density.2 The major challenge is driving the reaction to
completion in order to sulfate all available hydroxyl
groups (alcoholic or phenolic) on the substrate. As the
number of –OH groups increase on a small scaffold, sul-
fation becomes progressively difficult because of anion
crowding, resulting in numerous partially sulfated side-
products.
A further challenge is the isolation of the chemically
pure per-sulfated product, which requires aqueous isol-
ation techniques. Yields in the range of 11% and 100%
have been reported, yet the presence of inorganic salts
arising from the use of buffers and salts lead to signifi-
cant inconsistencies and inaccuracies. Additionally,
instability of highly anionic products introduce limita-
tions on reaction times and temperature.13 This is likely
to be especially true for highly sulfated, aromatic, small
organic molecules, which are expected to be less stable
than the saccharide scaffolds.
A major limitation in exploring these novel structures is
their challenging synthesis. Nearly all small organic sul-
fates reported in the literature are mono- or di-sulfated
molecules,8–12 typically prepared using sulfur trioxide
complexes with amines in a highly polar solvent
(DMF or DMA). Sulfation of such organic scaffolds
may require as many as 13 h and temperatures as high
as 95 °C in the presence of a large excess of the sulfating
complexes,8–12 while sugars, which contain multiple
–OH groups, require reaction times in the range of
12 h to several days.13–19
To avoid these problems with one-step sulfation, we
recently synthesized some small aromatic per-sulfated
structures using a two-step approach involving the
2,2,2-trichloroethyl protecting group.20 The two-step
protection–deprotection protocol resolved some of the
*
Corresponding author. Tel.: +1 804 828 7328; fax: +1 804 827
3664; e-mail: urdesai@vcu.edu
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.07.100

A. Raghuraman et al. / Tetrahedron Letters 48 (2007) 6754–6758
6755
problems of the direct sulfation approach, yet required
careful real-time monitoring of the reaction by RP-
HPLC to prevent product degradation and was not par-
ticularly applicable to substrates that were acid and/or
metal sensitive. These limitations led us to seek an alter-
native sulfation approach, which can be rapid, efficient,
and widely applicable to a number of poly-hydroxy
scaffolds.
studies, 6 and 10 M equiv of the sulfating complex and
base, respectively, were chosen.
To assess the effect of solvent, we chose to evaluate
nitromethane and DMF, both of which are solvents
with high dielectric constant and known to be micro-
wave-friendly. While only 23.2% and 17.2% of per-
sulfated product 1s was formed from 1 in 10 min at
100 °C in CH3NO2 and DMF, respectively, 47.4% of
the product was formed in CH3CN (Table 1, entries
10 and 11). Thus, our initial choice of CH3CN proved
to be optimal. To assess the effect of temperature
and reaction time, sulfation was performed for 10–
30 min at 40 to 120 °C. While 30 min were required to
yield 91.2% of 1s at 100 °C, only 10 min were needed
for 90.8% conversion at 120 °C. In striking contrast,
no product was detected at 40 °C within 10 min. Finally,
SO3Æpy/py was found to give nearly twice as much
per-sulfated product as SO3ÆMe3N/Et3N (entries 7
and 12) in 10 min at 100 °C. Since pyridine is ꢀ10,000-
fold weaker base in comparison to Et3N, this result sug-
gests general base catalysis as the predominant mecha-
nism of sulfation rather than a process involving
deprotonation of the substrate followed by nucleophilic
attack.
We hypothesized that significant rate enhancements
are likely to be achieved using microwaves, especially
because the ionic sulfated product may couple to micro-
waves through ionic conduction, for example, in
CH3CN.21 CH3CN was chosen as the solvent over the
commonly used DMF because (a) it can be evaporated
at lower temperature (thus aiding isolation) and (b) it
was likely to solubilize the per-sulfated product with
an amine counter-ion. We also hypothesized that intro-
ducing free base in the reaction mixture should promote
the difficult per-sulfation reaction.
Sulfation of 122 with SO3ÆMe3N complex (6 equiv per
–OH group) at 100 °C in the absence of free base gave
only 4.7% of per-sulfated product 1s in 20 min (Table
1, entry 1).23 Inclusion of 1 equiv of free Et3N per
–OH group resulted in 13.5% conversion (entry 2), while
79.8% of 1s was formed with 5 equiv of Et3N (entry 3).
Further increase to 10 equiv Et3N per –OH group had a
negligible increase in the yield of 1s (entry 4). Increasing
the proportion of SO3ÆMe3N per –OH group from 1 to
9 M equiv (Table 1, entries 5–8) gradually increased
the yield of the per-sulfated product from 14.5% to
79.5%, while further increase to 12 equiv was found to
be not particularly advantageous (entry 9). For further
Appropriate control reactions in the absence of micro-
waves using two different substrates—1 and 3 (entries
1 and 3 in Table 2)—at 60 °C in DMF with no free base
showed poor product yields. For example, it took 24 h
in the absence of microwaves to yield 1s in 60% yield,
while 3s was not detected even after 24 h (Table 2, entry
3). These results highlight the importance of microwaves
in achieving rapid per-sulfation.
Table 1. Optimization of microwave-assisted sulfation of tetrahydroisoquinoline derivative 1
OH
OS
HO
HO
SO
SO
MW; CH3CN
N
1
N
40 — 100 °C, 5—30 min
OS
OH
6 Equiv Me3N•SO3 per OH grp
O
O
10 Equiv Et3N per OH grp
1s (S=SO3Na)
Time (min)
Temperature (°C)
Modifications to conditions
HPLC yield (%)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
20
20
20
20
10
10
10
10
10
10
10
10
10
10
10
30
100
100
100
100
100
100
100
100
100
100
100
100
40
No base
4.7
13.5
79.8
80.0
14.5
46.1
47.4
79.5
80.7
17.2
23.2
82.3
0
1 equiv Et3N per OH grp
5 equiv Et3N per OH grp
a
—
1 equiv SO3ÆMe3N per OH grp
3 equiv SO3ÆMe3N per OH grp
a
—
9 equiv SO3ÆMe3N per OH grp
12 equiv SO3ÆMe3N per OH grp
DMF as solvent
CH3NO2 as solvent
With 6 equiv SO3Æpy/OH grp and 10 equiv py/OH grp as base
a
—
a
70
120
100
—
18.2
90.8
91.2
a
—
a
—
a Reaction conditions here are as listed above with no additional modifications.

6756
A. Raghuraman et al. / Tetrahedron Letters 48 (2007) 6754–6758
Table 2. Microwave-assisted sulfation of poly-hydroxyl substrates
Microwaves, 100 °C, 20-30 min
Substrate
Product
Et3N (10 equiv/OH), SO3•Me3N
(6 equiv/OH), CH3CN
Substrate
Producta
Isolated yield (%)
OH
OS
HO
HO
SO
SO
1
85
N
N
OH
OS
OS
O
O
O
O
1s
1
OS
OH
OH
HO
HO
SO
SO
2
3
87
N
N
O
2
2s
OS
OH
OH
SO
SO
OS
OS
HO
HO
54b
N
N
OH
O
3
3s
OH
OS
COOEt
COOEt
HO
HO
OH
SO
SO
OS
4
5
74
N
O
N
O
4s
4
CH2OH
O
OH
CH2OS
CH2OH
CH2OS
O
O
O
84c
OS
OH
OH
OS
OS
5s
5
OS
H3C
OH
H3C
6
94c
SO
HO
6s
6
SO
HO
H
H
SOH2C
SO
HOH2C
HO
O
H
O
H
7
72d
O
O
SO
OS
HO
OH
SO
HO
7s
7
CHO
CHO
8
97c,e
HO
SO
8s
8
a S: SO3Na.
b With 9 equiv of SO3ÆMe3N/OH group.
c With SO3Æpy (6–9 equiv/OH group) and pyridine as base.
d Reaction conditions: 120 °C, 10 min, SO3Æpy (12 equiv/OH group) and pyridine as base.
e S = SO3ÆpyH+.

A. Raghuraman et al. / Tetrahedron Letters 48 (2007) 6754–6758
6757
Having optimized the reaction conditions, we assessed
References and notes
whether the method works for a variety of different sub-
strates. Per-sulfation of 2 proceeded smoothly in a man-
ner identical to 1 (Table 2).24 More importantly, per-
sulfation of 3, containing the crowded 3,4,5-trihydroxy
moiety, was achieved under microwave conditions in
an isolated yield of 54%, while the conventional proce-
dure completely failed to give 3s. Finally, microwave-
assisted per-sulfation also works extremely well for
substrates 5 through 8 containing one to six –OH
groups. Interestingly, 5 and 6 gave a mixture of products
with SO3ÆMe3N, but yielded the per-sulfated products
with SO3Æpy.
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Several points make the microwave-assisted synthetic
protocol particularly attractive. (A) The method appears
to tolerate a range of functional groups including amide
(Table 2, entries 1–4), ester (entry 4), aldehyde (entry 8)
and double bond (entry 7). The relatively high isolated
yields (ꢀ70–95%) in each case make the reaction espe-
cially suitable for library construction. (B) The methods
work equally well for substrates containing one –OH
group to those that contain six –OH groups. This is
important because the small size of these molecules
introduces considerable anion–anion repulsion as the
number of sulfate groups increases. (C) The method ap-
plies equally well to alcoholic and phenolic –OH groups,
especially with SO3Æpy complex. (D) The method pro-
vides high purity per-sulfated product that is readily iso-
lated using an aqueous G10 filtration column. Typically,
the purity of these highly water soluble, per-sulfated,
small, organic molecules was found to be more than
95% using reverse polarity capillary electrophoresis
(see Supplementary data). (E) The method is particu-
larly suitable for quantitative isolation of small amounts
(<10 mg) of the per-sulfated products, however could be
linearly scaled up at least 20-fold without affecting the
yields to a significant extent.
8. Santos, G. A.; Murray, A. P.; Pujol, C. A.; Damonte, E.
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In summary, we have developed a rapid and high yield-
ing microwave-based synthesis of variably functional-
ized, per-sulfated organic molecules. The protocol is
expected to greatly facilitate the construction of a
library of per-sulfated, small organic molecules for
screening as glycosaminoglycan mimetics.
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¨
22. See Supplementary data for synthesis of starting materials.
23. RP-HPLC profile showed peaks from 4.3 to 6.0 min, in
addition to one at 9.0 min. The peak at 4.3 was
subsequently isolated after optimization of conditions
and determined to be per-sulfated (1s). The peak at
9.0 min was identified as 1 by comparison with synthet-
ically pure sample. Conversions (%) were determined by
area normalization.
24. Representative procedure for per-sulfation: To a stirred
solution of the poly-alcohol (20 mg, 0.066 mmol) in
MeCN (1 mL) at rt, Et3N (0.4 mL, 2.9 mmol) and
Me3NÆSO3 (220 mg, 1.6 mmol) were added. The reaction
vessel was sealed and micro-waved (CEM-discover micro-
wave synthesizer) for 20 min at 100 °C. The reaction was
repeated for four times and the reaction mixture was
pooled for isolation of the product. The MeCN layer was
decanted and pooled, while the residue was washed with
MeCN (5 mL) and centrifuged. The combined MeCN
layers were concentrated in vacuo. Water (5 mL) was
added to the residue and stirred for 10 min. The water
Acknowledgments
This work was made possible by the financial support
from the National Heart, Lung and Blood Institute
(RO1 HL069975 and R41 HL081972) and the American
Heart Association—National Center (EIA 0640053).
Supplementary data
Preparation and characterization (NMR, mass, and cap-
illary electrophoretic data) of the molecules prepared.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.2007.
07.100.

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A. Raghuraman et al. / Tetrahedron Letters 48 (2007) 6754–6758
layer was concentrated to approximately 2 mL, loaded
(m, 3H, isomers I–III); ESI (Àve) m/z Calcd for
C19H15NNa4O19S4 [(MÀNa)À] 757.88; found, 757.5.
Compound 5s: 1H NMR (DMSO, 400 MHz) d: 7.34 (d,
1H, J = 8.0 Hz), 7.16 (t, 1H, J = 8.0 Hz), 6.95 (t, 1H,
J = 8.0 Hz), 6.89 (d, 1H, J = 8.0 Hz), 5.24 (d, 1H,
J = 8.0 Hz), 4.86 (s,2H), 4.62 (d, 2H, J = 4.0 Hz), 4.35
(m, 1H), 4.21–4.27 (m, 1H), 3.93–3.99 (m, 1H), 3.75 (m,
1H); ESI (Àve) m/z Calcd for C16H10NNa5O21S5
[(MÀNa)À] 772.81; found, 772.7. Compound 6s: 1H
NMR (DMSO, 400 MHz) d: 7.1 (d, J = 8.4 Hz, 1H),
6.81–6.83 (m, 2H), 4.02 (t, J = 8.0 Hz, 1H), 2.71–2.74 (m,
2H), 2.21–2.25 (m, 1H), 2.08–2.12 (m, 1H), 1.87–2.00 (m,
2H), 1.75–1.78 (m, 1H), 1.48–1.60 (m, 2H), 1.09–1.34 (m,
6H), 0.66 (s, 3H); ESI (Àve) m/z Calcd for C18H22Na2O8S2
[(MÀNa)À] 453.07; found, 453.1. Compound 7s: 1H NMR
(DMSO, 400 MHz) d: 7.56 (s, 1H), 7.54 (s, 1H), 6.99–7.10
(m, 7H), 6.61 (s, 1H), 5.36 (s, 1H), 4.63 (s, 1H), 4.59 (s,
1H), 4.39 (s, 1H), 4.14–4.18 (m, 1H), 4.08 (d, J = 8.8 Hz,
1H) 3.81 (t, J = 10 Hz, 1H); ESI (Àve) m/z Calcd for
C20H16Na6O26S6 [(MÀNa)À] 978.77; found, 979.0. Com-
onto a Sephadex G10 column (ꢀ160 cm) and chromato-
graphed using water as eluent. Fractions were combined
based on RP-HPLC profiles, concentrated and re-loaded
onto a SP Sephadex C25 column for sodium exchange.
Appropriate fractions were pooled, concentrated in vacuo,
and lyophilized to obtain a white powder. Spectral
characteristics of the final sulfated compounds are as
follows: 1s: 1H NMR (DMSO, 400 MHz) d: 7.29–7.30 (m,
2H), 6.94–6.97 (m, 3H), 4.58 (s, 2H, isomer I), 4.48 (s, 2H,
isomer II), 3.58 (s, 2H, isomer II), 3.50 (s, 2H, isomer I),
2.66 (br, 2H, isomer I and II); ESI (Àve) m/z Calcd for
C16H11NNa4O17S4 [(MÀNa)À] 685.86; found, 686.1.
Compound 2s: 1H NMR (DMSO, 400 MHz) d: 7.65 (d,
J = 2.4 Hz, 1H), 7.57 (d, J = 8.4 Hz, 1H), 7.29 (s, 2H),
6.99 (dd, J = 8.4, 1.6 Hz, 1H), 4.54 (s, 2H), 3.70 (br, 2H),
2.69 (t, J = 4.8 Hz, 2H); ESI (Àve) m/z Calcd for
C16H11NNa4O17S4 [(MÀNa)À] 685.86; found, 686.0.
Compound 3s: 1H NMR (DMSO, 400 MHz) d: 7.37 (s,
2H), 7.29 (s, 2H), 4.54 (s, 2H), 3.53 (s, 2H), 2.68 (s, 2H);
ESI (Àve) m/z Calcd for C16H10NNa5O21S5 [(MÀNa)À]
803.79; found, 804.1. Compound 4s: 1H NMR (DMSO,
400 MHz) d: 6.96–7.70 (m, 5H), 4.83–5.16 (m, 1H isomers
I–III), 4.18–4.44 (m, 2H, isomers I–III), 3.55–3.61 (m, 2H,
isomers I–III), 2.99–3.13 (m, 2H, isomers I–III), 1.08–1.16
1
pound 8s: H NMR (DMSO, 400 MHz) d: 9.76 (s, 1H),
8.94–8.96 (m, 2H), 8.62–8.68 (m, 1H), 8.09–8.14 (m, 2H),
7.72–7.75 (m, 2H), 6.92–6.95 (m, 2H); ESI (Àve) m/z
Calcd for C12H11NO5S [(MÀpyH+)À] 200.99; found,
200.8.