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HOOC
OH
O
O
O
HOOC
O
OH
HO
O
O
HO
O
Heparosan
O
OH
HO
NHAc
HO
O
OH
NaOH
NHAc
N
-sulf otransf erase
HOOC
OH
O
O
O
HOOC
O
OH
HO
O
O
O
HO
O
OH
HO
NH
SO
3
-
HO
O
OH
NH
SO
3
-
6OST
HOOC
O
SO
3
-
O
O
O
HOOC
O
SO
3
-
O
HO
O
O
O
HO
O
OH
HO
NHSO
3
-
HO
O
OH
NSO
3
-
3OST1
HOOC
OSO
3
-
O
O
O
HOOC
O
OSO
3
-
HO
O
-
O
3
S
O
O
O
O
HO
HO
HO
NHSO
3
-
O
OH
NHSO
3
-
Recomparin
Fig. 7
Enzymatic synthesis of anticoagulant polysaccharide Recomparin. The synthesis was
begun with heparosan. The acetyl group was removed by sodium hydroxide to yield the GlcNH
2
unit. The resultant GlcNH
2
unit was then
N
-sulfated by NST. After
N
-sulfated HS polysaccharide
was generated, it was further modified by 6-OST and 3-OST1 to generate Recomparin
entry step [42]. It has been shown that 3-OST-3 modified HS is necessary for gD
binding and that the octasaccharide is the minimum length for sufficient binding
[26]. The octasaccharide exhibited the activity in inhibiting HSV-1 via saturating
gD, a key envelope protein for promoting the entry of viral particles into the cells.
HS that is modified by 3-OST isoforms, with the exception of 3-OST-1, binds to
gD and serves as an entry receptor for HSV-1 [29, 53]. In this experiment, 3-OST
isoform 3 was used. 3-OST isoform 3 transfers the sulfo group to the GlcN residue
that is linked to an IdoUA2S unit at the nonreducing end [20].
The synthesis of 3-
O
-sulfated octasaccharide was completed by incubating
purified 3-OST-3 enzyme and an octasaccharide substrate (Fig. 8). The octasac-
charide substrate was purified from partially depolymerized heparin with hep-
arin lyases. This 3-
O
-sulfated octasaccharide possesses a binding constant (K
d
)
of 19
M as determined by affinity co-electrophoresis [29], which is compa-
rable to the gD-binding 3-
O
-sulfated octasaccharide previously isolated from
HS [26]. Further cell based assay [56, 57] demonstrated that this 3-
O
-sulfated
μ
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