Postprint of: Tetrahedron Letters Volume 52, Issue 3, 19 January 2011, Pages 441–443
Microwave-assisted sulfonation of heparin oligosaccharides
Susana Maza, Jose L. de Paz, Pedro M. Nieto
Instituto de Investigaciones Químicas, Centro de Investigaciones Científicas Isla de La Cartuja,
CSIC and US, Américo Vespucio, 49, 41092 Sevilla, Spain
Abstract
The use of microwaves for the efficient and fast O-sulfonation of heparin oligosaccharide
intermediates has been reported for the first time. Experimental problems typically associated
with this chemical reaction, such as poor isolated yields and long reaction times, have been
avoided with the present method. The efficiency of this protocol was demonstrated by the
high-yielding sulfonation of a series of oligosaccharides using SO3·Me3N complex. Microwaveassisted sulfonation is expected to greatly facilitate the preparation of heparin
oligosaccharides, a crucial step for understanding the role of these complex carbohydrates in
biological processes.
Keywords
Microwave-assisted synthesis; Sulfation; Heparin; Oligosaccharides; Sulfonation
Heparin is a highly sulfated and heterogeneous polymer that belongs to the family of
glycosaminoglycans (GAGs) and participates in a plethora of biological processes.1 Heparin is
formed by disaccharide repeating units of d-glucosamine 1→4 linked to either l-iduronic or dglucuronic acid. This basic disaccharide may contain O-sulfo groups at positions 3 and 6 of the
glucosamine unit and position 2 of the uronic acid unit. In addition, the glucosamine nitrogen
may be sulfonated, acetylated, or may remain unsubstituted. This structural variability,
particularly the sulfation pattern, is responsible for the specific interaction of heparin with a
wide variety of proteins. 2, 3 and 4
Chemical synthesis has been extensively employed to access structurally defined heparin
oligosaccharides in order to determine structure–activity relationships for specific heparin
sequences.5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15 However, synthesis of heparin
oligosaccharides still remains a considerable challenge. The traditional approach involves, after
assembly of the protected oligosaccharide, deprotection of hydroxyl groups to be sulfonated,
and subsequent sulfonation prior global deprotection. Importantly, deprotection/sulfonation
steps may require more time that the assembly of the fully protected oligosaccharide.
Moreover, these final steps on valuable and elaborated compounds often result in low to
moderate isolated overall yields of the desired heparin oligosaccharides. In particular, good
1
isolated yields of the sulfonation reactions can be difficult to attain due to problems associated
with the purification and manipulation of highly polar sulfated products.
O-Sulfonation16 and 17 of hydroxyl-containing heparin oligosaccharide precursors is typically
carried out by using a sulfur trioxide complex such as SO3·Et3N or SO3·Me3N at 50–60 °C in
DMF. Alternatively, SO3·Py complex in pyridine as solvent has been also employed.
Oligosaccharides containing multiple hydroxyl groups usually require a large excess of the
sulfonating complex and prolonged reaction times, up to several days. In some cases, even
under harsh conditions, sulfonation yields are only moderate because the reaction is not
completed and numerous partially sulfated side-products are detected.18 Standard strategy
often involves running several reaction cycles with fresh sulfonating complex, after purification
of partially sulfated crude mixtures. Differences in reported yields and reaction times could be
attributed to different protecting group distributions and/or sulfonating reagent source. It is
noteworthy to mention that instability of highly sulfated heparin oligosaccharides introduces
certain limitations on the use of high temperatures during prolonged reaction times.
Therefore, more rapid and efficient methods for the preparation of highly sulfated heparin
oligosaccharides are needed.19, 20 and 21 In the past few years, microwave irradiation has
been extensively used for chemical synthesis.22 One of the main advantages of microwaveassisted synthesis is the dramatic reduction in reaction times, from days and hours to minutes
and seconds.22 Microwave-induced rate enhancements are likely to be obtained in reactions
involving ionic charged molecules and polar solvents. Therefore, sulfonation reaction is
especially appropriate for the application of microwave irradiation. However, microwaveassisted sulfonations have surprisingly received very little attention in the literature.23 and 24
To the best of our knowledge, microwaves have not been previously employed to synthesize
sulfated GAGs, including heparin oligosaccharides.
Here, we report a rapid and high-yielding method for O-sulfonation of heparin-like
oligosaccharide intermediates using microwaves. This protocol is expected to greatly
accelerate the preparation of these highly-sulfated carbohydrates, avoiding experimental
problems associated with the use of sulfur trioxide complexes under standard conditions.
We started this study with attempts to synthesize sulfated disaccharide 2 from 1 (Table 1)
using typical treatment with SO3·Me3N at 60 °C in DMF (conventional heating in an oil bath,
entry 1). Close monitoring by TLC revealed very low reaction rate. A large excess of the
sulfonating agent (20 equiv per OH group) and long reaction time (seven days) were required
to drive the reaction to completion. Similar problems were previously found in the Osulfonation of certain partially protected heparin oligosaccharides.18 Microwave heating
greatly reduced the reaction time under the same conditions with 5 equiv of SO3·Me3N
complex per OH group (entry 2). Pure disaccharide 2 was obtained in excellent yield after
purification by Sephadex LH-20 column chromatography. This result highlights the effect of
microwaves on O-sulfonation reaction. To evaluate the effect of temperature, sulfonation was
performed at 100 °C (entries 3 and 4). It took only 15 min to yield 2 in 91% yield, while 1 h
microwave irradiation slightly increased the yield to 98%.25 Interestingly, N-sulfonation of the
carbamate moiety was not detected under these conditions.26 Similar results were obtained
by alternative use of SO3·Py complex at 60 °C in pyridine (entry 5).
2
After having optimized the microwave-assisted O-sulfonation of model disaccharide 1, we
applied this protocol to more complex heparin-like oligosaccharides. O-Sulfonation of
tetrasaccharide 3 was found to be particularly problematic with conventional heating
conditions (Scheme 1). Extensive treatment with SO3·Me3N gave a mixture of partially
sulfated products. No reaction progress was observed in the presence of a large excess of the
sulfonating complex after long reaction time. After Sephadex LH-20 gel filtration, the crude
material was further treated with SO3·Py complex. Reverse-phase C-18 chromatography27
was required to obtain pure tetrasaccharide 4 in a moderate 43% yield. However, the same
reaction proceeded smoothly with microwave heating to afford 428 in excellent yield after
simple gel filtration (Scheme 1).
Next goal was the preparation of hexasaccharide 6 containing 7 sulfate groups (Table 2).
Microwave-assisted sulfonation was performed with SO3·Me3N complex at 100 °C for 5–30
min to optimize the required reaction time (entries 1–3). 30 min were required to produce 629
in excellent 90% yield. Interestingly, treatment with SO3·Py complex produced a mixture of
partially sulfated derivatives as indicated by TLC monitoring (entry 4). Identical crude mixtures,
with no reaction progress, were obtained after both increasing the temperature up to 100 °C
and elongation of the reaction time. Similar results were previously reported in the sulfonation
of certain heparin-like oligosaccharides using SO3·Py complex.26
In conclusion, a microwave-assisted protocol for the per-O-sulfonation of heparin-like
oligosaccharide precursors has been reported. Application of microwave irradiation
dramatically reduced reaction times, allowing for the rapid and high-yielding sulfonation of
products ranging from di- to hexasaccharides. Interestingly, short reaction times of microwaveassisted reactions (15–30 min) allowed for the use of higher temperatures than for
conventional sulfonations. This method is expected to greatly accelerate the preparation of
these molecules, overcoming one of the most important bottle-necks of GAG synthesis.
Acknowledgments
We thank the CSIC (Grant 200880I041), the Spanish Ministry of Science and Innovation (Grant
CTQ2009-07168), Junta de Andalucía (Grant P07-FQM-02969, and ‘Incentivo a Proyecto
Internacional’), and the European Union (FEDER support and Marie Curie Reintegration Grant)
for financial support.
3
References and notes
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4
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25. General procedure for the microwave-assisted per-O-sulfonation: Microwave-based
sulfonation reactions were performed using a Biotage Initiator synthesizer in sealed reaction
vessels. Poly-hydroxylated sugar (7–14 μmol, 1.0 equiv), sulfur trioxide–trimethylamine
complex (5 equiv per OH; Sigma–Aldrich; this complex had been previously washed with H2O,
MeOH, and CH2Cl2 and dried under high vacuum) and a magnetic stirrer bar were placed in a 5
mL microwave reaction vial and fitted with a septum, which was then pierced with a needle.
5
The closed vial was then evacuated under high vacuum and left to dry for 12 h. Ar was let in,
dry DMF (1.0 mL) was added and the reaction mixture was subjected to microwave radiation
for 15–30 min at 100 °C (80 W average power). The reaction was cooled to room temperature
and quenched with Et3N (150 μL). MeOH (1 mL) and CH2Cl2 (1 mL) were added, and the
solution was layered on the top of a Sephadex LH-20 chromatography column which was
eluted with CH2Cl2/MeOH (1:1) to obtain the corresponding triethylammonium salt (90–99%
yield). The sodium salt was obtained by elution from a column of Dowex 50WX4-Na+ with
MeOH/H2O 3:1. Characterization of compound 2 (triethylammonium salt; in the NMR data
unit A refers to the reducing end monosaccharide): TLC (EtOAc–pyridine–H2O–AcOH 10:5:3:1)
Rf 0.51; 1H NMR (500 MHz, MeOD): δ 7.43–7.25 (m, 20H, Ar), 5.18 (br s, 1H, H-1A), 5.06 (m,
3H, H-1B, CH2(NHCO2Bn)), 4.90–4.65 (m, 7H, H-5A (4.74), CH2(OBn)), 4.49 (br s, 1H, H-2A),
4.33 (dd, 1H, J5,6a = 2.8 Hz, J6a,6b = 10.7 Hz, H-6aB), 4.25 (br t, 1H, H-3A), 4.20 (dd, 1H, J5,6b =
1.9 Hz, H-6bB), 4.14 (br t, 1H, H-4A), 3.97–3.93 (m, 2H, H-5B, H-3B), 3.80 (m, 1H, CH2–O), 3.66
(t, 1H, J3,4 = J4,5 = 9.4 Hz, H-4B), 3.59 (m, 1H, CH2–O), 3.32 (m, 3H, H-2B, CH2–N), 3.19 (q,
Et3NH+), 1.32 (t, Et3NH+); 13C NMR (125 MHz, MeOD) (Significant data from HSQC
experiment): δ 99.4 (C-1A), 96.5 (C-1B), 79.7 (C-3B), 77.6 (C-4B), 72.8 (C-3A), 71.9 (C-4A), 71.7
(C-2A), 70.2 (C-5B), 67.1 (C-5A), 66.8 (CH2–O), 65.5 (C-6B), 63.4 (C-2B); ESI MS: m/z: calcd for
C43H47N4O19S2: 987.2; found: 986.7 [M+2H]−; calcd for C61H94N7O19S2: 1292.6; found:
1292.1 [M+3Et3NH+H]+.
26. C. Noti, J.L. de Paz, L. Polito, P.H. Seeberger
Chem. Eur. J., 12 (2006), pp. 8664–8686
27. A. Dilhas, R. Lucas, L. Loureiro-Morais, Y. Hersant, D. Bonnaffe
J. Comb. Chem., 10 (2008), pp. 166–169
28. Characterization of compound 4 (sodium salt): TLC (EtOAc–pyridine–H2O–AcOH 10:5:3:1)
Rf 0.44; 1H NMR (500 MHz, MeOD): δ 7.46–7.17 (m, 30H, Ar), 5.49 (br s, 1H, H-1C), 5.22 (br s,
1H, H-1A), 5.10 (d, 1H, J1,2 = 3.7 Hz, H-1B), 5.06 (br s, 2H, CH2(NHCO2Bn)), 5.01 (d, 1H, J1,2 =
3.7 Hz, H-1D), 5.00 (d, 1H, J4,5 = 1.5 Hz, H-5C), 4.90–4.64 (m, 10H, CH2(OBn), H-5A (4.83)), 4.60
(br s, 1H, H-2C), 4.52 (br s, 1H, H-2A), 4.42 (d, 1H, CH2(OBn)), 4.35–4.31 (m, 2H, H-6aB, H-6aD),
4.27–4.22 (m, 3H, H-3A, H-3C, H-6bB), 4.16 (br s, 1H, H-4A), 4.12 (br d, 1H, H-6bD), 4.05 (t, 1H,
J3,4 = J4,5 = 9.6 Hz, H-4B), 3.97 (m, 2H, H-4C, H-5B), 3.85 (m, 2H, H-3D, CH2–O), 3.74 (m, 2H, H3B, H-5D), 3.67–3.60 (m, 2H, H-4D, CH2–O), 3.47 (dd, 1H, J2,3 = 10.3 Hz, H-2D), 3.37 (m, 2H,
CH2–N), 3.29 (dd, 1H, J2,3 = 10.3 Hz, H-2B); 13C NMR (125 MHz, MeOD) (Significant data from
HSQC experiment): δ 100.6 (C-1A), 98.2 (C-1C), 96.6 (C-1D), 96.3 (C-1B), 81.5 (C-3D), 79.6 (C3B), 76.2, 76.1, 75.6, 73.1, 73.1 (CH2(OBn)), 72.4 (C-3A), 72.1 (C-2A, C-4B), 72.0 (C-4C), 71.7 (C3C, C-4A), 71.3 (C-5B), 71.2 (C-5D), 70.7 (C-2C, C-4D), 68.2 (CH2–O), 67.8 (C-5A), 67.3 (C-5C),
67.2 (CH2(NHCO2Bn)), 67.1 (C-6B), 66.6 (C-6D), 65.3 (C-2B), 65.0 (C-2D), 41.4 (CH2–N); ESI MS:
m/z: calcd for C69H73N7O35S4K2: 882.6; found: 882.4 [M+2H+2 K]2−; calcd for
C75H90N8O35S4: 895.2; found: 894.9 [M+3H+Et3NH]2−; calcd for C69H71N7O35S4K2Na2:
904.6; found: 904.9 [M+2K+2Na]2−.
29. Characterization of compound 6 (sodium salt): TLC (EtOAc–pyridine–H2O–AcOH 8:5:3:1) Rf
0.17; 1H NMR (500 MHz, MeOD): δ 7.52–7.18 (m, 35H, Ar), 5.53 (br s, 1H, H-1E), 5.47 (br s, 1H,
6
H-1C), 5.22 (br s, 1H, H-1A), 5.21 (d, 1H, CH2(OBn)), 5.12 (d, 1H, J1,2 = 3.7 Hz, H-1B or H-1D),
5.09 (d, 1H, J1,2 = 3.8 Hz, H-1B or H-1D), 5.07 (br s, 3H, H-1F, CH2(NHCO2Bn)), 5.04 (br s, 1H,
H-5C), 5.03 (br s, 1H, H-5E), 4.90–4.79 (m, 3H, CH2(OBn), H-5A (4.82)), 4.77–4.59 (m, 7H,
CH2(OBn), H-2E (4.62)), 4.53 (br s, 2H, H-2C, H-2A), 4.50 (m, 2H, CH2(OBn), H-6aF), 4.49–4.42
(m, 2H, CH2(OBn)), 4.36–4.15 (m, 11H, H-4F, H-6aB, H-6aD, H-3E, H-3C, H-3A, H-6bB, H-6bD, H6bF, H-4A, H-4C), 4.07 (m, 2H, H-4B, H-4D), 4.03 (m, 1H, H-4E), 4.00 (m, 1H, H-5B, or H-5D),
3.94 (m, 1H, H-5F), 3.90–3.73 (m, 4H, H-3F, CH2–O, H-5B or H-5D, H-3B or H-3D), 3.71–3.60 (m,
2H, H-3B, or H-3D, CH2–O), 3.45–3.39 (m, 2H, H-2F, H-2B, or H-2D), 3.37 (m, 2H, CH2–N), 3.31
(m, 1H, H-2B, or H-2D); 13C NMR (125 MHz, MeOD) (Significant data from HSQC experiment):
δ 100.1 (C-1A), 98.6 (C-1E), 98.5 (C-1C), 96.8, 96.1 (C-1B, C-1D), 95.8 (C-1F), 80.2 (C-3F), 80.1,
79.7 (C-3B, C-3D), 78.0 (C-4F), 76.7, 76.5, 76.1, 73.5, 73.3 (CH2(OBn)), 72.9 (C-3A or C-3C), 72.7
(C-2A), 72.3 (C-4B, C-4D), 72.1 (C-4A), 71.5 (C-4E, C-2C), 71.4 (C-3E, C-3A, or C-3C), 71.3, 71.2
(C-5B, C-5D), 71.1 (C-4C, C-5F), 70.9 (C-2E), 68.3 (CH2–O), 68.1 (C-5A), 67.8 (C-6F), 67.5 (C-5C,
C-5E), 67.4 (CH2(NHCO2Bn), C-6B, C-6D), 65.9, 65.3 (C-2B, C-2D), 64.5 (C-2F), 41.5 (CH2–N); ESI
MS: m/z: calcd for C142H237N19O54S7: 1648.2; found: 1648.3 [M+9Et3NH+3H]2+.
7
Table 1
Table 1. Microwave-assisted sulfonation of disaccharide 1
Entry Sulfonating agent Solvent
SO3·Me3N
1
(20 equiv per OH DMF
group)
SO3·Me3N (5 equiv
2
DMF
per OH group)
SO3·Me3N (5 equiv
3
DMF
per OH group)
SO3·Me3N (5 equiv
4
DMF
per OH group)
SO3·Py
(5 equiv
5
pyridine
per OH group)
a Conventional heating (oil bath).
b Estimated by TLC analysis.
Temperature
(°C)
Reaction
time
Isolated yield
(%)
60a
7d
Quantitativeb
60
7h
94
100
1h
98
100
15 min
91
60
15 min
Quantitative
8
Table 2
Table 2. Microwave-assisted sulfonation of hexasaccharide 5
Entry Sulfonating agent
Solvent
SO3·Me3N (5 equiv
DMF
per OH group)
SO3·Me3N (5 equiv
2
DMF
per OH group)
SO3·Me3N (5 equiv
3
DMF
per OH group)
SO3·Py (5 equiv per
4
pyridine
OH group)
a Estimated by TLC analysis.
1
Temperature
(°C)
Reaction
time
Isolated
yield (%)
100
5 min
20a
100
20 min
80a
100
30 min
90
60→100
5 min→2 h 25a
9
Scheme 1
Scheme 1. Sulfonation of tetrasaccharide 3. Reagents and conditions: (a) SO3·Me3N (>10
equiv per OH group), DMF, 60 °C, ⩾3 d; then SO3·Py (10 equiv. per OH group), pyridine, room
temperature, 48 h, 43%; (b) SO3·Me3N (5 equiv per OH group), DMF, 100 °C, microwaves, 30
min, 95%.
10