Thermolabile phosphorus protecting groups, associated intermediates and methods of use
IPC분류정보
국가/구분
United States(US) Patent
등록
국제특허분류(IPC7판)
C07H-021/00
C07H-021/02
C07H-021/04
출원번호
US-0792799
(2001-02-23)
발명자
/ 주소
Beaucage, Serge L.
Wilk, Andrzej
Grajkowski, Andrzej
출원인 / 주소
The United States of America as represented by the Department of Health and Human Services
대리인 / 주소
Leydig, Voit & Mayer, Ltd.
인용정보
피인용 횟수 :
9인용 특허 :
65
초록▼
The invention provides a method of thermally deprotecting the internucleosidic phosphorus linkage of an oligonucleotide, which method comprises heating a protected oligonucleotide in a fluid medium at a substantially neutral pH, so as to deprotect the oligonucleotide.The present invention further pr
The invention provides a method of thermally deprotecting the internucleosidic phosphorus linkage of an oligonucleotide, which method comprises heating a protected oligonucleotide in a fluid medium at a substantially neutral pH, so as to deprotect the oligonucleotide.The present invention further provides a method of synthesizing an oligonucleotide using the thermal deprotection method described above, and novel oligonucleotides and intermediates that incorporate the thermolabile protecting group used in accordance with the present invention.
대표청구항▼
1. A method of deprotecting an oligonucleotide, which method comprises heating an oligonucleotide of the formula:in a fluid medium, at a substantially neutral pH, at a temperature up to about 100° C. to produce an oligonucleotide of the formula:wherein:R is a thermolabile protecting group of th
1. A method of deprotecting an oligonucleotide, which method comprises heating an oligonucleotide of the formula:in a fluid medium, at a substantially neutral pH, at a temperature up to about 100° C. to produce an oligonucleotide of the formula:wherein:R is a thermolabile protecting group of the formula:R 1 is H, R 1a , OR 1a , SR 1a or NR 1a R 1a′ , wherein R 1a and R 1a′ are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl; or, when R 1 is NR 1a R 1a′ , R 1a and R 1a′ , together with the nitrogen atom to which they are bonded, comprise a heterocycle containing from 3 to about 7 atoms in the ring skeleton thereof;X 1 is O, S or Se;X is O or S;Z is O, S, NR 2a , CR 2a R 2a′ or CR 2a R 2a CR 2b R 2b′ , wherein R 2a , R 2a′ , R 2b and R 2b′ are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl; orR 1a or R 1a′ , in combination with any of R 2a , R 2a′ , R 2b or R 2b′ , together with C═X of the protecting group to which they are bonded, comprise a ring containing from 3 to about 7 atoms in the skeleton thereof;provided that R 1 is not R 1a when Z is S, Z is not CR 2a R 2a′ or CR 2a R 2a′ CR 2b R 2b′ when R 1 is SR 1a , and Z is not O or S when R 1 is H;R 2 , R 2′ , R 3 and are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl, orR 2 or R 2′ , in combination wit R 3 or R 3′ , together with the carbon atoms to which they are bonded, comprise a cyclic substituent of the formula:wherein p is an integer from 0-6 and a-d are the same or different and each is selected from the group consisting of H, an alkyl, a nitro, a dialkylamino, an alkoxy, an alkylthio, a cyano and a halogen, provided that the aromatic ring, which bears substituents a-d, is one carbon removed from the phosphate oxygen of formula (IIIa),wherein R 1 , R 2a , R 2a′ , R 2b , R 2b′ , R 2 , R 2′ , R 3 or R 3′ is unsubstituted or substituted with one or more substituents, which are the same or different, selected from the group consisting of OR 8 , CN, NO 2 , N 3 , and a halogen, wherein R 8 is H or an alkyl;R 4 and R 15 are the same or different and each is H, a hydroxyl protecting group, or a solid support;Q 1 is a nucleoside, an oligonucleotide or an oligomer comprising an oligonucleotide;n is an integer from 1 to about 300; andQ is a nucleoside, an oligonucleotide or an oligomer comprising an oligonucleotide and, when n is greater than 1, each Q is independently selected, provided that the deprotection is not by an enzyme. 2. The method of claim 1, wherein Q or Q 1 comprises a nucleoside of the formula:wherein:B is a labeling group, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, a heteroaryl, a heterocycloalkyl, an aralkyl, an amino, an alkylamino, a dialkylamino, a purine, a pyrimidine, adenine, guanine, cytosine, uracil, or thymine, wherein B is unsubstituted or substituted with one or more substituants, which are the same or different, selected from the group consisting of a nucleobase protecting group, R 11 , OR 11 , NHR 11 , NR 11 R 12 , N═C—NR 11′ R 12′ , CN, NO 2 , N 3 , and a halogen, wherein R 11 and R 12 are the same or different and each is H, an alkyl or an acyl, and R 11 and R 12′ are the same or different and each is an alkyl or R 11′ and R 12′ , together with the nitrogen atom to which they are bonded, form a heterocycle containing 3 to about 7 atoms in the ring skeleton thereof; andE is H, a halogen, OR 13 , NHR 13 , or NR 13 R 14 , wherein R 13 and R 14 are the same or different and each is H, a protecting group, an alkyl, or an acyl. 3. The method of claim 1, wherein R 1 is H, an alkyl or NR 1a R 1a′ , wherein R 1a and R 1a′ , together with the nitrogen atom to which they are bonded, comprise a heterocycle containing from 3 to about 7 atoms in the ring skeleton thereof. 4. The method of claim 1, wherein X 1 is S. 5. The method of claim 1, wherein Z is CR 2a R 2a′ or CR 2a R 2a′ CR 2b R 2b′ and R 2a , R 2a′ , R 2b and R 2b′ are the same or different and each is H or an alkyl. 6. The method of claim 1, wherein R 2 or R 2′ is H or an alkyl. 7. The method of claim 1, wherein R 3 or R 3′ is H, an alkyl or an 8. The method of claim 1, wherein R is a protecting group of the formula: 9. The method of claim 1, wherein the temperature is from about 50° C. to about 90° C. 10. The method of claim 1, wherein the deprotection is carried out at about pH 7. 11. The method of claim 1, wherein the fluid medium contains water. 12. A method of producing an oligonucleotide, which method comprises(a) reacting a nucleophile of the formula: 4 —O—Q 1 —OHwith an electrophile of the formula:wherein W is a dialkylamino group that is displaced by the nucleophile, under conditions to displace W and produce an adduct comprising a tricoordinated phosphorus atom;(b) reacting the product obtained in step (a) with a reagent selected from the group consisting of oxidizing agents, sulfurizing agents, and selenizing agents to produce a protected oligonucleotide of the formula:(c) cleaving R 15 from the protected oligonucleotide from step (b) to produce a nucleophile;(d) optionally repeating steps (a)-(c) until an oligomer of a specified length is obtained; and(e) heating the product from step (c) or (d) in a fluid medium, at a substantially neutral pH, at a temperature up to about 100° C. to produce a deprotected oligonucleotide of the formula:wherein R is a thermolabile protecting group of the formula:R 1 is H, R 1a , OR 1a , SR 1a or NR 1a R 1a′ , wherein R 1a and R 1a′ are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl; or, when R 1 is NR 1a R 1a′ , R 1a and R 1a′ , together with the nitrogen atom to which they are bonded, comprise a heterocycle containing from 3 to about 7 atoms in the ring skeleton thereof;X 1 is O, S or Se;X is O or S;Z is O, NR 2a , CR 2a B 2a′ or CR 2a R 2a′ CR 2b R 2b′ , wherein R 2a , R 2a′ , R 2b and R 2b′ are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl; orR 1a or R 1a′ , in combination with any of R 2a , R 2a′ , R 2b or R 2b′ , together with C═X of the protecting group to which they are bonded, comprise a ring containing from 3 to about 7 atoms in the skeleton thereof;provided that R 1 is not R 1a when Z is S, Z is not CR 2a R 2a′ or CR 2a R 2a′ CR 2b CR 2b′ when R 1 is SR 1a , and Z is not O or S when R 1 is H;R 2 , R 2′ , R 3 and R 3′ are the same or different and each is H, an alkyl, an alkenyl, alkynyl, a cycloalkyl, an aryl, or an aralkyl, orR 2 or R 2′ , in combination R 3 or R 3′ , together with the carbon atoms to which they are bonded, comprise a cyclic substituent of the formula:wherein p is an integer from 0-6 and a-d are the same or different and each is selected from the group consisting of H, an alkyl, a nitro, an amino, a hydroxyl, a thio, a cyano and a halogen, provided that the aromatic ring, which bears the substituents a-d, is one carbon removed from the phosphate oxygen of formula (IIIa),wherein R 1 , R 2a , R 2a′ , R 2b , R 2b′ R 2 , R 2′ , R 3 or R 3′ is unsubstituted or substituted with one or more substituents, which are the same or different, selected from the group consisting of OR 8 , CN, NO 2 , N 3 , and a halogen, wherein R 8 is H or an alkyl;R 4 is H, a hydroxyl protecting group, or a solid support;R 15 is a hydroxyl protecting group or a solid support;Q 1 is a nucleoside, an oligonucleotide or an oligomer comprising an oligonucleotide;n is an integer from 1 to about 300; andQ is a nucleoside, an oligonueleotide or an oligomer comprising an oligonucleotide and, when a is greater than 1, each Q is independently selected, provided that the deprotection is not by an enzyme.
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이 특허에 인용된 특허 (65)
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Urdea Michael S. (Alamo CA) Horn Thomas (Berkeley CA), Hydroxyl-protecting groups attached to cytidine nucleotide compounds which are orthogonally removable.
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Srivastava Saresh C. (Waltham MA) Raza Syed K. (Waltham MA), N-protected-2′-O-methyl-and N-protected-3′-O-methyl-ribonucleosides and their phosphoramidite derivatives.
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Engels Joachim (Kronberg/Taunus VA DEX) Jger Alfred (Charlottesville VA), Process for the preparation of nucleoside alkyl-aralkyl- and aryl-phosphonites and -phosphonates.
Reddy M. Parameswara (Brea CA) Farooqui Firdous (La Habra CA), Processes for synthesizing nucleotides and modified nucleotides using N4protected deoxycytidines.
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Reddy M. Parameswara (Brea CA) Farooqui Firdous (LaHabra CA) Hanna Naeem B. (Fullerton CA), Synthesis of DNA using substituted phenylacetyl-protected nucleotides.
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