Membranes with bound oligonucleotides and peptides
First Claim
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1. A modified membrane comprising a polymeric membrane having a protected nucleoside or amino acid linked thereto.
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Abstract
A method is provided for synthesizing oligonucleotides and peptides directly onto a membrane. The method provides a means for generating membrane affinity supports. A modified membrane for the method of direct synthesis is also provided.
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Citations
42 Claims
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1. A modified membrane comprising a polymeric membrane having a protected nucleoside or amino acid linked thereto.
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2. A modified membrane represented by the formula:
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space="preserve" listing-type="equation">P--X--Y--N--Z--S.sup.Wwherein P is a polymeric membrane; X is a functional group on the membrane; Y--N--Z is a linker wherein N is a spacer molecule and Y and Z are the same or different functional groups, the linker being bound to the membrane through its functional group Y to the functional group X; and SW is a protected nucleoside or amino acid, SW being bound to the linker through the functional group Z of the linker.
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3. A modified membrane of claim 2, wherein the membrane P comprises a flat, permeable polymeric material of porous structure which contains functional groups within its constituent monomeric units for attachment of the spacer group.
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4. A modified membrane of claim 3, wherein the monomers are acrylic or methacrylic acid esters having a free alcohol or ester function for attachment of the spacer group.
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5. A modified membrane of claim 3, wherein the polymeric material is a cross-linked polymer selected from the group consisting of polydialkylsilandiols, polydialkylsiloxanes, polyvinyl alcohols, polyoxymethylenes and polyoxyethylenes.
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6. A modified membrane of claim 2, wherein the membrane is a flat, permeable, polymeric material having a porous structure wherein a functional group for attachment of the spacer has been introduced.
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7. A modified membrane of claim 6, wherein the polymeric material comprises polystyrenes, polysulfones containing aromatic residues, polyesters, polyamides, polycarbonates, polyvinylidene difluoride and polyvinyl acetate.
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8. A modified membrane of claim 2, wherein the membrane is a flat, permeable, polymeric material having a porous structure onto which are grafted moieties containing functional groups.
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9. A modified membrane of claim 8, wherein the polymeric material comprises polysulfones, polytetrafluoroethylene, polyethylene, polypropylene or polyvinyllidene difluoride.
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10. A modified membrane of claim 2, wherein the membrane is a flat, permeable, polymeric material having a porous structure onto which is coated a second polymeric material containing free functional groups for attachment of the spacer.
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11. A modified membrane of claim 10, wherein the polymeric material comprises polysulfones, polytetrafluoroethylene, polyethylene, polypropylene or polyvinylidene difluoride.
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12. A modified membrane of claim 10, wherein the second polymeric material comprises acrylic or methacrylic acid esters having a free alcohol or ester function for attachment of the spacer group.
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13. A modified membrane of claim 2 wherein N is --(CH2)n --, where n is 1-20.
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14. A modified membrane of claim 2 wherein N is --NH--(CH2)m --NHCO--(CH2)m --CO--, where m is 1-6.
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15. A modified membrane of claim 2 wherein N is oligoglycine.
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16. A modified membrane of claim 2 wherein Y and Z are individually selected from the group consisting of ##STR5##
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17. A modified membrane of claim 2 wherein SW represents a nucleoside of the formula:
- ##STR6## wherein BW'"'"' is a nucleoside base having a protecting group for protection of exocyclic amino groups; and
W" is a H or a protecting group for the 2'"'"' hydroxy group.
- ##STR6## wherein BW'"'"' is a nucleoside base having a protecting group for protection of exocyclic amino groups; and
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18. A modified membrane of claim 17 wherein W" is a trityl group, an acyl group or a silyl group.
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19. A modified membrane of claim 2 wherein SW represents an amino acid of the formula:
- ##STR7## wherein U represents an amino acid side chain;
W'"'"' represents protecting groups for the side chain of the amino acid;
W" represents a protecting group for the amino group of the amino acid;
W"'"'"' represents a protecting group for the carboxy group of the amino acid.
- ##STR7## wherein U represents an amino acid side chain;
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20. A modified membrane of claim 19 wherein SW is norleucine attached to the linker through its carboxyl group.
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21. A modified membrane of claim 20, wherein the primary amino group of the nucleocine is protected with fluoroenyl-methyloxycarbonyl.
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22. A method of synthesizing an oligonucleotide comprising sequentially coupling nucleotide to a modified membrane of claim 17.
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23. A method of synthesizing a peptide comprising sequentially coupling amino acids to a modified membrane of claim 19.
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24. A method of synthesizing an oligonucleotide comprising the steps of:
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a. providing a modified membrane represented by the formula;
space="preserve" listing-type="equation">P--X--Y--N--Z--S.sup.Wwherein P is a polymeric membrane; X is a functional group on the membrane; Y--N--Z is a linker wherein N is a spacer molecule and Y and Z are the same or different functional groups, the linker being bound to the membrane through the functional group X; and SW is a protected nucleoside, SW being bound to the linker through the functional group Z of the linker; b. coupling a nucleoside phosphoramidite to the nucleoside SW to produce a membrane-bound nucleoside-nucleotide having a phosphite triester linkage; c. oxidizing the phosphite triester to form a phosphate triester linkage; and d. sequentially coupling additional nucleoside phosphoramidites to the membrane-bound nucleoside-nucleotide, and after each coupling step, oxidizing the resulting phosphite triester linkage to a phosphate triester to produce a membrane-bound polynucleotide.
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25. A method of claim 24, wherein the nucleside phosphoramidite is a nucleoside beta-cyanoethyl phosphoramidite.
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26. A method of claim 24, further comprising removing the protecting groups from the membrane bound polynucleotide.
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27. A method of claim 26, wherein the synthesized polynucleotide is cleaved from the membrane.
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28. A membrane-bound polynucleotide produced by the method of claim 24.
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29. A method of synthesizing a peptide, comprising the steps of:
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a. providing a modified membrane of the formula
space="preserve" listing-type="equation">P--X--Y--N--Z--S.sup.Wwherein P is a polymeric membrane; X is a functional group on the membrane; Y--N--Z is a linker wherein N is a spacer molecule and Y and Z are the same or differential functional groups, the linker being bound to the membrane through the functional group X; and SW is a amino acid, SW being bound to the linker through the functional group Z of the linker; and b. sequentially coupling amino acids to SW to produce a membrane-bound peptide.
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30. A method of claim 29, further comprising the step of removing the protecting groups from the membrane-bound peptide.
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31. A method of claim 29, wherein the synthesized peptide is cleaved from the membrane.
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32. A membrane-bound peptide produced by the method of claim 29.
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33. A method of claim 29, wherein SW represents a norleucine.
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34. A method of claim 29, wherein a second linker group is attached to SW, the linker group providing an functional group for coupling of subsequent protected amino acids.
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35. A method of claim 34, wherein the linker is p-hydroxymethylphenoxyacetic acid.
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36. A membrane having an oligonucleotide attached through a terminus of the oligonucleotide.
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37. A membrane having an peptide attached through a terminus of the peptide.
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38. A membrane having an attached oligonucleotide or peptide represented by the formula:
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space="preserve" listing-type="equation">P--X--Y--N--Z--(--S.sup.W).sub.nwherein P is a polymeric membrane; X is a functional group on the membrane; Y--N--Z is a linker wherein N is a spacer molecule and Y and Z are the same or different functional groups, the linker being bound to the membrane through the functional group X; and (SW)n represents an oligonucleotide or peptide comprised of protected or partly protected nucleotides or amino acids, wherein n is the number of nucleotide or amino acid units of the oligonucleotide, (SW)n being bound to the linker through the functional group Z of the linker.
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39. A membrane having an attached oligonucleotide or peptide represented by the formula:
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space="preserve" listing-type="equation">P--X--Y--N--Z--(--S).sub.nwherein P is a polymeric membrane; X is a functional group on the membrane; Y--N--Z is a linker wherein N is a spacer molecule and Y and Z are the same or different functional groups, the linker being bound to the membrane through the functional group X; and (S)n represents an oligonucleotide or peptide, wherein n is the number of nucleotide or amino acid units of the oligonucleotide or peptide, (S)n being bound to the linker through the functional group Z of the linker.
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40. A modified membrane of the formula:
- ##STR8## wherein P is a polyvinylidene difluoride;
m is 1 through 6; BW is a nucleoside base having a protecting group for protection of exocylic amino groups; and DMT is the 4,4-dimethoxytrityl protecting group.
- ##STR8## wherein P is a polyvinylidene difluoride;
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41. A modified membrane of the formula:
- ##STR9## wherein P is a membrane comprising polypropylene coated with hydroxyporpylacrylate;
n=1-20; Nle is norleucine; UW is an amino acid side chain; and Fmoc is a fluorenylmethoxycarbonyl protecting group.
- ##STR9## wherein P is a membrane comprising polypropylene coated with hydroxyporpylacrylate;
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42. A modified membrane of claim 33, wherein an amino acid is linked to norleucine by ester linkage with a benzyl alcoholic linker.
Specification