13-01-2022 дата публикации
Номер: US20220010344A1
Принадлежит:
The present invention includes methods for preparing anticoagulant polysaccharides using several non-naturally occurring, engineered sulfotransferase enzymes that are designed to react with aryl sulfate compounds instead of the natural substrate, PAPS, to facilitate sulfo group transfer to polysaccharide sulfo group acceptors. Suitable aryl sulfate compounds include, but are not limited to, p-nitrophenyl sulfate or 4-nitrocatechol sulfate. Anticoagulant polysaccharides produced by methods of the present invention comprise N-, 3-O-, 6-O-sulfated glucosamine residues and 2-O sulfated hexuronic acid residues, have comparable anticoagulant activity compared to commercially-available anticoagulant polysaccharides, and can be utilized to form truncated anticoagulant polysaccharides having a reduced molecular weight. 1. A method of enzymatically synthesizing an N- , 2-O , 3-O , 6-O sulfated , heparan sulfate (N ,2 ,3 ,6-HS) product in the absence of 3′-phosphoadenosine 5′-phosphosulfate (PAPS) , the method comprising the following steps:a. providing a starting polysaccharide mixture comprising N-sulfated heparosan;b. combining the starting polysaccharide mixture with a first sulfotransferase reaction mixture comprising a hexuronyl 2-O sulfotransferase enzyme (2OST) and a sulfo group donor, the sulfo group donor consisting of an aryl sulfate compound, to form a first product mixture, the first product mixture comprising an N-, 2-O sulfated heparan sulfate (N,2-HS) product;c. combining the first product mixture with a second sulfotransferase reaction mixture comprising a glucosaminyl 6-O sulfotransferase enzyme (6OST) and a sulfo group donor, the sulfo group donor consisting of an aryl sulfate compound, to form a second product mixture, the second product mixture comprising an N-, 2-O, 6-O sulfated heparan sulfate (N,2,6-HS) product;d. combining the second product mixture with a third sulfotransferase reaction mixture comprising a glucosaminyl 3-O sulfotransferase enzyme ( ...
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