CA2024182A1 - Probes - Google Patents
ProbesInfo
- Publication number
- CA2024182A1 CA2024182A1 CA002024182A CA2024182A CA2024182A1 CA 2024182 A1 CA2024182 A1 CA 2024182A1 CA 002024182 A CA002024182 A CA 002024182A CA 2024182 A CA2024182 A CA 2024182A CA 2024182 A1 CA2024182 A1 CA 2024182A1
- Authority
- CA
- Canada
- Prior art keywords
- hydrogen
- probe
- beta
- diphenylcarbamoyl
- pyridone
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6832—Enhancement of hybridisation reaction
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
- C12Q1/701—Specific hybridization probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10S435/00—Chemistry: molecular biology and microbiology
- Y10S435/805—Test papers
Abstract
07-21(281)A
IMPROVED PROBES
ABSTRACT
The probes of the invention are able to withstand higher temperatures, thereby allowing unmatched probes and mismatched probes to be washed off at higher hybridization stringency, thereby elimi-nating background readings and improving ease and accuracy of probe use.
IMPROVED PROBES
ABSTRACT
The probes of the invention are able to withstand higher temperatures, thereby allowing unmatched probes and mismatched probes to be washed off at higher hybridization stringency, thereby elimi-nating background readings and improving ease and accuracy of probe use.
Description
~2~32 07-21 ( 281 )A
IMPROVED PROBES
BACKGROUND OF T~E INVENTION
This invention is directed to nov~l nucleic acid probes that can be used wi~h stringent hybridi-zation conditions thereby decreasing background readings and improving the ease and accuracy of probe use.
In recombinant DNA technology and related fields such as diagnostics it is necessary to possess means for detecting specific nucleic acid sequences.
The basic building blocks from which nucleic acids are constructed are nucleotides. The major nucleotides found in living cells are adenosine, guanosine, cytidine, thymidine and uridine. Nucleic acids contain the information for the transfer of genetic information from one generation to the ne~t, as well , as for the expression of this information through protein syn~hesis. Therefore, genetic materials can be evaluated and manipulated on ~he basis o~ nucleo-tide sequences. Based on ~his understanding of genetic makeup, probes can be designed to perform a variety of functions in the field of recombinant DNA
technology and related areas.
Probes are oligom~ric or poly~eric nucleic acid molecules with complementary sequences to a nucleic acid sequence of interest. Through the use of probes, the ability to identify, localize, and detect nucleic acid sequences can be readily accomplished.
Probes are not only useful in laboratory techniques and diagnostics, but are also useful in the field of therapeutics.
~ 2 L~g 2 -2- 07-21(281)A
Typically, probes are used in determining the presence of a specific nucleic acid sequence.
This lnvolves digesting DNA or RNA with restrlctlon endonucleases to produce smaller fragments. The S fragments are separated by molecular weight and transferred and bound to a filter. The filter is then incubated with 1abelled probes consisting of single strands of DNA or RNA with nucleotide sequences complementary to a specific region in the DNA or RNA
that is being detected. If the labelled probe binds to one of the nucleic acid bands on the filter, the sequence of interest resides in that band. Thus, probes may be used to determine the presence of specific genes, pathogens, human and non-hu~an DNA or RNA, natural or foreign DNA or RNA and a variety of other detectable compositions.
Whether a hybrid nucleic acid molecule forms accurate~y between a probe and target sequence depends on two important factors. The probe should bind only to the desired target sequence, and the hybrid nucleic acid molecule thus formed should be correctly and sensitively identified. Therefore, it is of great advantage to have a probe that is both capable of recognizing and hybridizing to only its complement and is also capable of being accurately and sensitively identified.
The use of probes in determining the pre-sence of specific genomic regions has been possible for some time, see Souther~, J. Mol. Biol.,98:503, 1975. ~lbarella et al., U.S. Patent No. 4,S63,417, describe hybridization which occurs b2tween sample ` nucleic acid and the probe in nucleic acid hybridiza-tion assays detected by an antibody tha~ binds to intercalation complexes formed in association with hybridized probe. The use of probes containing psor-2~
IMPROVED PROBES
BACKGROUND OF T~E INVENTION
This invention is directed to nov~l nucleic acid probes that can be used wi~h stringent hybridi-zation conditions thereby decreasing background readings and improving the ease and accuracy of probe use.
In recombinant DNA technology and related fields such as diagnostics it is necessary to possess means for detecting specific nucleic acid sequences.
The basic building blocks from which nucleic acids are constructed are nucleotides. The major nucleotides found in living cells are adenosine, guanosine, cytidine, thymidine and uridine. Nucleic acids contain the information for the transfer of genetic information from one generation to the ne~t, as well , as for the expression of this information through protein syn~hesis. Therefore, genetic materials can be evaluated and manipulated on ~he basis o~ nucleo-tide sequences. Based on ~his understanding of genetic makeup, probes can be designed to perform a variety of functions in the field of recombinant DNA
technology and related areas.
Probes are oligom~ric or poly~eric nucleic acid molecules with complementary sequences to a nucleic acid sequence of interest. Through the use of probes, the ability to identify, localize, and detect nucleic acid sequences can be readily accomplished.
Probes are not only useful in laboratory techniques and diagnostics, but are also useful in the field of therapeutics.
~ 2 L~g 2 -2- 07-21(281)A
Typically, probes are used in determining the presence of a specific nucleic acid sequence.
This lnvolves digesting DNA or RNA with restrlctlon endonucleases to produce smaller fragments. The S fragments are separated by molecular weight and transferred and bound to a filter. The filter is then incubated with 1abelled probes consisting of single strands of DNA or RNA with nucleotide sequences complementary to a specific region in the DNA or RNA
that is being detected. If the labelled probe binds to one of the nucleic acid bands on the filter, the sequence of interest resides in that band. Thus, probes may be used to determine the presence of specific genes, pathogens, human and non-hu~an DNA or RNA, natural or foreign DNA or RNA and a variety of other detectable compositions.
Whether a hybrid nucleic acid molecule forms accurate~y between a probe and target sequence depends on two important factors. The probe should bind only to the desired target sequence, and the hybrid nucleic acid molecule thus formed should be correctly and sensitively identified. Therefore, it is of great advantage to have a probe that is both capable of recognizing and hybridizing to only its complement and is also capable of being accurately and sensitively identified.
The use of probes in determining the pre-sence of specific genomic regions has been possible for some time, see Souther~, J. Mol. Biol.,98:503, 1975. ~lbarella et al., U.S. Patent No. 4,S63,417, describe hybridization which occurs b2tween sample ` nucleic acid and the probe in nucleic acid hybridiza-tion assays detected by an antibody tha~ binds to intercalation complexes formed in association with hybridized probe. The use of probes containing psor-2~
-3- 07-21(281)A
alen derivatives that form covalent bonds with the target DNA when photochemically activated has been described in Yabusaki et al., US Patent No. 4,599,303.
Specific DNA probes in diagnostic microbiology have been disclosed in Falkow et al., U.S. Patent No.
alen derivatives that form covalent bonds with the target DNA when photochemically activated has been described in Yabusaki et al., US Patent No. 4,599,303.
Specific DNA probes in diagnostic microbiology have been disclosed in Falkow et al., U.S. Patent No.
4,358,535. There are a variety of probes available for detection of a variety of nuclelc acid sequences.
Despite ~he variety of probe strateyies available, it would greatly improve probe use to have a probe capable of withstanding stringent hybridization conditions to eliminate background readings and to improve the ease, accuracy and sensitivity of probe u~e.
SUMMARY OF THE INVENTION
The invention relates to single-stranded nucleic acid probes which comprise a nucleoside res-idue of the formula:
0~1 N
o\ R3 in which Rl and R2 i~dependently are Cl-Cs alkyl, C2-Cs alkenyl, halo or hydrogen, and R3 is hydrogen, hydroxy, C6-Cl 4 aryloxy or C1-Cs alkoxy.
In preferred probes of the invention only one R1 or R2 is substituted. Preferred Rl and R2 . .
~2~2 -4- 07-21(281)A
substituents are iodo or vinyl. In more preferred probes of the inventlon Rl, R~ and R3 are hydrogen.
The nucleoside resldue is drawn to indlcate bondlng between the 3' and 5' positions of the ribose or deoxyribose. The nucleoside residue of the lnven-tion is situated in the probe to be complementary to a guanosine r~sidue (where a cytidine would normally reside) of a target sequence. Typically, probes of the invention are molecules of about 15 to 50 nucleotides in which a nucleoside residue of the invention ls at least five (5) nucleotides from both ends of the molecule. Preferred probes of the invention are molecules of 15 to 25 nucleotides. Theoretically, there is no maximum length for a probe of the lnven-tion, however, in order to obtain reasonable hybridi-zation times, probes of 100 nucleotldes or less are recommended.
As used herein, "hybridization" refers to the process in which a strand of nucleotide sequences binds with complementa~y sequences through base pairing. "~ybrid molecule" refers to the complex resulting from hybridization. Further, as used herein, "complementary sequences" refers to sequences of nucleotides that are capable of base pairing.
Furthermore, "base pairing" refers ~o the in~eraction, throuyh hydrogen bonding, that occurs between opposite purine and pyrimidine bases in nucleic acids. Nucleo-tides that form stable base pairs are adenosine and thymidine (uridine in RNA), and guano~ine and cyti-30 dine. Base pairing ~ay occur between two co~pl~men-tary strands of DNA (D~A/DNA), between two RNA strands (RNA/RNA), or between one strand of each (DNA/RN~).
"Target sequence" or "target nucleic acid seguence", as used herein, is defined as the region to which a probe is complementary. DNA, and RNA, as used herein, refer to nucleic acids with a naturally occurring 2 ~ 2 ~
Despite ~he variety of probe strateyies available, it would greatly improve probe use to have a probe capable of withstanding stringent hybridization conditions to eliminate background readings and to improve the ease, accuracy and sensitivity of probe u~e.
SUMMARY OF THE INVENTION
The invention relates to single-stranded nucleic acid probes which comprise a nucleoside res-idue of the formula:
0~1 N
o\ R3 in which Rl and R2 i~dependently are Cl-Cs alkyl, C2-Cs alkenyl, halo or hydrogen, and R3 is hydrogen, hydroxy, C6-Cl 4 aryloxy or C1-Cs alkoxy.
In preferred probes of the invention only one R1 or R2 is substituted. Preferred Rl and R2 . .
~2~2 -4- 07-21(281)A
substituents are iodo or vinyl. In more preferred probes of the inventlon Rl, R~ and R3 are hydrogen.
The nucleoside resldue is drawn to indlcate bondlng between the 3' and 5' positions of the ribose or deoxyribose. The nucleoside residue of the lnven-tion is situated in the probe to be complementary to a guanosine r~sidue (where a cytidine would normally reside) of a target sequence. Typically, probes of the invention are molecules of about 15 to 50 nucleotides in which a nucleoside residue of the invention ls at least five (5) nucleotides from both ends of the molecule. Preferred probes of the invention are molecules of 15 to 25 nucleotides. Theoretically, there is no maximum length for a probe of the lnven-tion, however, in order to obtain reasonable hybridi-zation times, probes of 100 nucleotldes or less are recommended.
As used herein, "hybridization" refers to the process in which a strand of nucleotide sequences binds with complementa~y sequences through base pairing. "~ybrid molecule" refers to the complex resulting from hybridization. Further, as used herein, "complementary sequences" refers to sequences of nucleotides that are capable of base pairing.
Furthermore, "base pairing" refers ~o the in~eraction, throuyh hydrogen bonding, that occurs between opposite purine and pyrimidine bases in nucleic acids. Nucleo-tides that form stable base pairs are adenosine and thymidine (uridine in RNA), and guano~ine and cyti-30 dine. Base pairing ~ay occur between two co~pl~men-tary strands of DNA (D~A/DNA), between two RNA strands (RNA/RNA), or between one strand of each (DNA/RN~).
"Target sequence" or "target nucleic acid seguence", as used herein, is defined as the region to which a probe is complementary. DNA, and RNA, as used herein, refer to nucleic acids with a naturally occurring 2 ~ 2 ~
-5- 07-21~281)A
sugar-phosphate backbone and also modifled backbones including phosphorothloates, d1thlonates, alkyl phosphonates, phosphonates, phosphoramidates, hydrogen phosphonates and a-nucleotides. Compounds of the lnvention herein are named, then followed by a number in parenthesis corresponding to its structure as set forth in Tables I and II.
A probe may contaln more than one nucleoside residue of the inventlon or combinations thereof.
Preferably, there are intervening nucleotldes separa-ting each nucleoside residue of the invention in a probe. For example, a 30 mer probe could have a nucleoside residue of the invention at the 8th and 21st nucleotide. In addition, the invention may be lS used in either deoxyribonucleic acid probes or ribo-nucleic ac1d probes.
To make a probe of the inventlon, a probe is constructed to be complementary to a target sequence of interest. At a position ln the probe that is complementary to a guanosine residue of the target sequence (therefore, at a position in the probe where a cytidine would normally be found), a nucleoside residue of the invention is substituted therefore. The resulting probe contains a nucleoside residue of the invention complementary to a guanidine in the target sequance. The nucleoside residue of the invention is generally placed at least five (5) nucleotides from both ends of the probe. While not wishing to be bound or limited by theory, it is believed that when such a substitution is made, the nucleoside residue of the invention is capable of foImi~g a covalent bond with the target sequence to which it hybridizes. The hybrid molecules made by using probes of this inven-tion and immobilized on filters can be washed at high temperatures where probes not containing a nucleoside residue of the invention wash off of the target sequence at the same temperature. Consequently, ~2~2 -6- 07-21(281)A
probes of the invention offer better specifici~y and ~he separation and removal of unhybridized material lS
greatly enhanced. The efficient removal of incorrectly hybridized probes eliminates background readings and enhances the detectability of correctly hybridi2ed target sequences. Thus, a probe of the lnvention can be used with both improved ease and accuracy over probes without a nucleoside of the lnventlon. It 1S
anticipated that various substitutions to enhance bonding and stability between the nucleosides of the invention and the complementary strand may be made.
The invention further relates to compounds which are useful as intermediates in the preparation of probes of the invention. These compounds have the formula:
R~
o R~
~0-~
O R
R~
in which R1 and R2 independently are C,-Cs alkyl, C2-Cs alkenyl, halo or hydrogen, R3 is hydro~en, halo, Cl-Cs alkoxy or tri(C~-Cs alkyl)substituted silyloxy, R4 is triphenylmethyl, or mono-, di- or 2~2~
sugar-phosphate backbone and also modifled backbones including phosphorothloates, d1thlonates, alkyl phosphonates, phosphonates, phosphoramidates, hydrogen phosphonates and a-nucleotides. Compounds of the lnvention herein are named, then followed by a number in parenthesis corresponding to its structure as set forth in Tables I and II.
A probe may contaln more than one nucleoside residue of the inventlon or combinations thereof.
Preferably, there are intervening nucleotldes separa-ting each nucleoside residue of the invention in a probe. For example, a 30 mer probe could have a nucleoside residue of the invention at the 8th and 21st nucleotide. In addition, the invention may be lS used in either deoxyribonucleic acid probes or ribo-nucleic ac1d probes.
To make a probe of the inventlon, a probe is constructed to be complementary to a target sequence of interest. At a position ln the probe that is complementary to a guanosine residue of the target sequence (therefore, at a position in the probe where a cytidine would normally be found), a nucleoside residue of the invention is substituted therefore. The resulting probe contains a nucleoside residue of the invention complementary to a guanidine in the target sequance. The nucleoside residue of the invention is generally placed at least five (5) nucleotides from both ends of the probe. While not wishing to be bound or limited by theory, it is believed that when such a substitution is made, the nucleoside residue of the invention is capable of foImi~g a covalent bond with the target sequence to which it hybridizes. The hybrid molecules made by using probes of this inven-tion and immobilized on filters can be washed at high temperatures where probes not containing a nucleoside residue of the invention wash off of the target sequence at the same temperature. Consequently, ~2~2 -6- 07-21(281)A
probes of the invention offer better specifici~y and ~he separation and removal of unhybridized material lS
greatly enhanced. The efficient removal of incorrectly hybridized probes eliminates background readings and enhances the detectability of correctly hybridi2ed target sequences. Thus, a probe of the lnvention can be used with both improved ease and accuracy over probes without a nucleoside of the lnventlon. It 1S
anticipated that various substitutions to enhance bonding and stability between the nucleosides of the invention and the complementary strand may be made.
The invention further relates to compounds which are useful as intermediates in the preparation of probes of the invention. These compounds have the formula:
R~
o R~
~0-~
O R
R~
in which R1 and R2 independently are C,-Cs alkyl, C2-Cs alkenyl, halo or hydrogen, R3 is hydro~en, halo, Cl-Cs alkoxy or tri(C~-Cs alkyl)substituted silyloxy, R4 is triphenylmethyl, or mono-, di- or 2~2~
-7- 07-21(2~1)A
trl(CI-Cs alkoxy)substituted tr1phenylmethyl, R5 is hydrogen phosphonate or R'-P-R", in which R' ls ~-cyanoethoxy, Cl-C5 alkoxy or Cl-Cs alkyl and R'' lS
morphollno, or mono- or di(C~-Cs alkyl)substltuted amino, R6 i5 tri(C~-Cs alkyl or Cl-Cs alkoxy) substi-tuted silyl, benzoyl, methylcarbonyl, carbamoyl, or carbamoyl substituted with CI-C5 alkyl, phenyl, acetyl or isobutyryl.
Examples of suitable alkyl radlcals include methyl, ethyl, propyl, isopropyl, butyl, sec butyl, isobutyl, t-butyl, pentyl, isopentyl, and l-methyl-l-butyl. Examples of suitable alkenyl radicals include vinyl, 1-propenyl, 2-propenyl, isopropenyl, l-butenyl, 2-butenyl, isobutenyl, 2-methyl-2-butenyl, l-pentenyl, 2-pentenyl, 3-pentenyl, 4 pentenyl, and isopentenyl.
Examples of suitable alkoxyl radicals include methoxy, ethoxy, propoxy, butoxy and pentoxy.
Examples of suitable tri-substituted silyl radicals include triisopropylsilyl, trimethylsilyl, and t-butyldimethylsilyl. Examples of suitable mono-, di-and tri-alko~y substituted triphenylmethyl includes p-methoxytriphenylmethyl, p~dimethoxytriphenylmethyl, and p-trimethoxytriphenylmethyl. Examples of suitable di-alkyl substituted amino groups include N,N~diisopro-pylamino, N,N-dimethyl~mino, and N,N-diethylamino.
Examples of suitable C6-C~4 aryloxy includes pheno~y, 2-methylphenoxy, 4-methylphenoxy, 2,4-dimethylphenoxy, and naphthoxy. Examples of suitable substituted carbamoyl radicals are diphenylcarbamoyl, diisopropyl-carbamoyl and dimethylcarbamoyl.
Illustrative compounds of ~he invention are - listed below in Table I.
~ 0 2 ~
trl(CI-Cs alkoxy)substituted tr1phenylmethyl, R5 is hydrogen phosphonate or R'-P-R", in which R' ls ~-cyanoethoxy, Cl-C5 alkoxy or Cl-Cs alkyl and R'' lS
morphollno, or mono- or di(C~-Cs alkyl)substltuted amino, R6 i5 tri(C~-Cs alkyl or Cl-Cs alkoxy) substi-tuted silyl, benzoyl, methylcarbonyl, carbamoyl, or carbamoyl substituted with CI-C5 alkyl, phenyl, acetyl or isobutyryl.
Examples of suitable alkyl radlcals include methyl, ethyl, propyl, isopropyl, butyl, sec butyl, isobutyl, t-butyl, pentyl, isopentyl, and l-methyl-l-butyl. Examples of suitable alkenyl radicals include vinyl, 1-propenyl, 2-propenyl, isopropenyl, l-butenyl, 2-butenyl, isobutenyl, 2-methyl-2-butenyl, l-pentenyl, 2-pentenyl, 3-pentenyl, 4 pentenyl, and isopentenyl.
Examples of suitable alkoxyl radicals include methoxy, ethoxy, propoxy, butoxy and pentoxy.
Examples of suitable tri-substituted silyl radicals include triisopropylsilyl, trimethylsilyl, and t-butyldimethylsilyl. Examples of suitable mono-, di-and tri-alko~y substituted triphenylmethyl includes p-methoxytriphenylmethyl, p~dimethoxytriphenylmethyl, and p-trimethoxytriphenylmethyl. Examples of suitable di-alkyl substituted amino groups include N,N~diisopro-pylamino, N,N-dimethyl~mino, and N,N-diethylamino.
Examples of suitable C6-C~4 aryloxy includes pheno~y, 2-methylphenoxy, 4-methylphenoxy, 2,4-dimethylphenoxy, and naphthoxy. Examples of suitable substituted carbamoyl radicals are diphenylcarbamoyl, diisopropyl-carbamoyl and dimethylcarbamoyl.
Illustrative compounds of ~he invention are - listed below in Table I.
~ 0 2 ~
-8- 07-21(281)A
TABLE 1.
OC~O)N(Ph)2 H,CO ~ O
(I Pr)2N ~OCI12CH2CN
4-O-diphenylcarbamoyl-l-~ f s-o- [bls(4-methoxyphenyl) phenylmethyl]-3-O-[bis(l-methylethyl)amlno~-(2-cyanoethoxy)phosphino]-2-deoxy-beta-D-erythro-pento-S furanosyl]-2(1~)-pyridone (9) OC~O)N(Phk H~CO 0~ o ~
~I Pr)2N ~ OCH3 '` !9 4-O-diphenylcarbamoyl-1-15-O-[bis(4-methoxyphenyl) phenylmethyl]-3-O-[bis(l-methylethyl~amino]-methoxy-phosphino]-2-deoxy-beta-~-erythro-pentofuranosyl]-2(1H)-pyridone (10~
, - , ` , .
2~2~2 _9- 07-21~281)A
OC(o)N(ph)2 '~3C0 ~ _yyf ~3 O OSI~I-Pr)3 ~I Pr)2N OCH2CH2CN
Ll 4-O-diphenylcarbamoyl-1-[[[5-O-[(4-methoxyphenyl) diphenylmethyl]-3-O-[bis(l-methylethyl)amino~
(2-cyanoethyl)phosphino]-2-O-tris(l-methylethyl)silyl]
-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone (21) OC(O)N(PI~)2 II~c2~0 ~
/
oO - Sl~ u) IU~
~P
t~Pr)2N OCH2CH2CN
, 4-O-diphenylcarbamoyl-1-[[[5-0-[(4-methoxyphenyl) diphenylmethyl]-3-O-[bis(1-methylethyl)amino~(2-cyanoethyl)phosphino)-2-O-(l,l methylethyl) dimethylsilyl]-beta-D-erythro-pentofuranosyl]-2(1H)~
pyridone (22) ~2~2 -10- 07-21~281)A
OC(O)N(PI~)2 H,CO {~ O ~
O OSI(I Pr)3 (I P~)2N OCH,CH2CN
2~
4-O-diphenylcarbamoyl-3-iodo-1-[[[5-0-[(4-methoxyphenyl) diphenylmethyl]-3-0-[bis(l-methylethyl)amino](2-cyanoethyl) phosphino]-2-O-trls(l-methylethyl)sllyl]-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone ~23) o~(o)N~ph)2 ~3 0~
H,CO~ ~
O OSI(I-Pr)3 ~P~)2N OCH2CH2CN
.- ~
4-O-diphenylcarbamoyl-5-iodo-1-[[[S-O-[(~-methoxyphenyl) diphenylmethyl]-3-O-[bis(l-methylethyl3amino]t2-cyanoethyl) phosphino]-2-O-tris(l-methylethyl)silyl]-beta-D-eryt~ro-pentofuranosyl]-2(1~) pyridone (24) :
.
2~2~
~ 07-21~281)A
oC~O)N~Ph)2 H3CO ~ O
H--P=O
O HN(Et)3-4-O-diphenylcarbamoyl-l-[ E 5 -O- [bis(4-methoxyphenyl) phenylmethyl]-3-0- ~ hydroxyphosphinyl ) -2-deoxy]-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone trl-ethylammonium salt (25).
Oc(o)N(ph)a H~CO ~ O ~
o OS~ Pr)3 H--P =O
~ HN(E~)3~
4-O-diphenylcarbamoyl-1-[[5-0-[(4-methox~phenyl) diphenylmethyl]-3-O-(hydroxyphosphinyl)-2-0-tris-(l-methylethyl)silyl]-bet a-D-erythro -pentofuranosyl]-2(lH~-pyridone triethylammonium salt (26~
.
.
æ~2~-8~
-12- 07-21(281)~
OC~O)N~Ph)2 ~3CO ~ o ~
O - Sl ~ U) \ Me H--P = o O HN~Et)~-~
4-O-diphenylcarbamoyl-1-[[5-0-[(4-methoxyphenyl) diphenylmethyl]-3-O-(hydroxyphosphinyl)-2-O-(l,l-dlmethylethyl)dlmethylsllyl]-beta-D-erythro-pento-furanosyi]-2(lH)-pyrldone trlethylammonium salt (27 OC(O)N(Ph)2 _ N
~:0~ ~
(I Pr)2N OCHaCH2t:~J
s g-O-diphenylcarbamoyl-3-iodo-1-[S-O-tbis (4-methoxyphenyl)phenylmethyl]-[3~0-[bis(1-methylethyl)amino](2-cyanoethoxy)phosphino]-2-deoxy-~eta-D-erythro-pentofuranosyl]-2(lH)-pyridone ~34) 2~2~
-13- 07-21(281)A
oC~o)p~(ph~2 H3CO f ~ I
~,CO~O~
(I Pr)2N ocH2~l~2cN
4-O-diphenylcarbamoyl-5-iodo-1-[S-O-[bis(4-methoxyphenyl)phenylmethyl]-[3-O-[bis(l-methylethyl)amlno](2-cyanoethoxy)phosphino]-2-deoxy-beta-D-erythro~pentofuranosyl]-2tlH)-S pyridone ~35) OC(O)N~PI1)2 Ha~
H~CO~ o~/
(I Pr)2N C)CH2CH2CN
~2 ~
4-O-diphenylcarbamoyl-3-ethenyl-1-[5-O-[bis(4-methoxyphenyl)phenylme~hyl]-[3-0-[bis(1-methylethyl) amino](2-cyanoethoxy)phosphino]-2-deoxy-beta~D-erythro-pentofuranosyl]-2(1~)-pyridone (39) 2~)2~
-14- 07-21(281)A
oC(O)N(Ph~2 D,~3 N
HO ~
-0~1 ~7 4-O-diphenylcarbamoyl-3-ethenyl-1(2-deoxy-beta-D-erythro-pentofuranosyl)-2(lH)-pyrldone (37) OC(O)N(Ph)2 H3CO ~3 H3CO ~ ~
OCH, )2N OCH20H2CN
. 4~
4-O-diphenylcarbamoyl-l-[S-O-[bis(4-methoxyphenyl) phenylmethyl]-3-O-[bis(l-methylethyl)amino(2~
S cyanoethoxy)phosphino~-2-O-methyl-be~a-D-erythro-pentofuranosyl]-2(1~)-pyridone (43) , 2 -15- 07-21(281)A
Typically, the ratio of the extent of hybrldization of perfect and mlsmatched duplexes ls critically dependent upon the temperatures at which hybridization and washing are carried out. Such critical dependence on temperature leads to compromise between the higher temperatures requlred to achieve dissociation of mismatched duplexes and the lower temperatures needed to give the greatest degree of binding and, therefore, the highest sensitivity.
However, when hybridization takes place between a probe of the invention and a complementary nucleic acid sequence, the resulting hybrid molecule exhibits enhanced stabilization resulting in higher melting temperatures, sometimes 25C higher, or more, than a corresponding hybrid molecule formed without a probe of the invention. Therefore, after hybridization, uncomplexed probe and mismatched complexes will be washed off at higher stringency conditions, thereby virtually eliminating all background readings and improving the ease and accuracy of probe use.
In order to incorporate a nucleoside residue of the invention into a probe, in place of one or more cytidine, the nucleoside is converted to a phosphor-amidite derivative that is appropriately protected for DNA synthesis. See S. P. Adams et al., J. Am. Chem.
Soc., 105:661 (1983), L. J. McBride and M. H. Caruthers;
Tetrahedron Lett., 24:245 (19~3), and N. D. Sinha et al., Nucleic Acids Res., 12:4539 (1984). It i~
anticipated that all protecting groups will serve the same function of appropriately protecti~g the various groups of the nucleoside. Examples of appropriate protecting groups include triisopropylsilyloxy, t-butyl-2 ~ 2 ~
-16~ 07-21(281)A
dlmethylsilyloxy, methoxyl, dimethoxytrlphenylmethyl, triphenylmethyl (trltyl), monomethoxytriphenylmethyl, trimethoxytriphenylmethyl, plxyl, N,N-dllsopropylamlne, morpholino, N,N-diethylamlno, N,N-dimethylamine, methyl, ~-cyanoethyl, hydrogen phosphonate, dl-o-anisyl-l-napthylmethyl, and p-anisyl-l-napthylphenylbutyl.
An example of such a phosphoramidite deriva-tive is the 2'-deoxy-3-deazaurldine derivatlve, 4-O-di-phenylcarbamoyl-l-[[5-O-[bis(4-methoxyphenyl)phenylmeth-yl]-3-O-[bis(l-methylethyl)amino]-(2-cyanoethoxy)phos-phino]-2-deoxy-beta-D-erythro-pentofuranosyl] -2(lH)-pyridone (9). The nucleic acids that may be used for purposes of practicing the invention include both the naturally occurring sugar-phosphate backbones as well as modified backbones as generally illustrated by Miller and T'so, Ann. Reports Med. Chem., 23:295 (19~8) and Moran et al., Nuc. Acids Res., 14:5019 (1987). Likewise, the ribo~e forms of the nucleoside of the invention are also converted to the phosphorami-dite form with appropriate protection of the 2'hydroxyl group before being placed into a nucleic acid probe. Methods for incorporating the phosphoramidite into a nucleic acid strand include the use of solid phase, solution phase triesters and ~-phosphonate intermediates as generally illustrated by Froehler et al., Nuc. Acids Res., 14:5399 (lg86), Narang et al., Methods En~., 68:90 (1979) and Ogilvie, K. K. et al., Proc. Natl Acad. Sci. USA, 85:5764 (1988).
After making a probe of the invention, the probe may be used to detect a target sequence. The particular hybridization technique for locating a ~' target sequence is not essential to the inYention.
Typically, hybridization with a normal probe proceeds at about 5C to 10C below the melting temperature (Tm) of a normal phosphodiester probe. Usually, hybridi-2 ~
-17- 07-21(281)A
zation ls carried out in a buffered solution between about pH 6 to 8. Optimal hybrldlzatlon conditions may be determined empirically. Hybrldization for a probe of the invention is carried out in a similar manner.
An lmportant advantage of the probes of this invention is that the resulting hybrid molecules have significantly higher melting temperatures than corres-ponding hybrid molecules which contain no probes of the invention. This higher meltlng temperature permits higher wash temperatures than can be employed for probes without a nucleoside residue of the inven-tion, for example, 50-100C, preferably between about 65-85C. Higher temperature washing gives cleaner and faster results because mismatched probes are unable to withstand the higher wash temperatures and are removed by the wash solution.
In addition to hybridizing probes as de-scribed above, a probe can be hybridized to target nucleic acid in solution. Moreover, the probe or target can be immobilized on a solid support and hybridized with target or probe, whichever one choses.
Hybridizatlon conditions may have varying degrees of stringency. Stringency is affected by temperature, ~5 probe length, ionic strength and other related fac-tors. Nevertheless, by using a probe of the present invention, as long as hybridization is achieved, with any hybridization protocol employed, unmatched and mismatched probes may be removed under conditions that would normally also lead to the loss of the hybridized probe not containing a nucleoside residue of the invention. Probes will often hybridize to nucleic acid sequences that are similar to the target sequence but that are not of int rest. Hybridization of this 2 ~
-18- 07-21(281)A
type is responsible for much of the background en-countered when using normal DNA probes lacklng a nucleoside residue of the inventlon. There are a variety of methods for removing non-hybridized probes from the hybridizatlon process. Such methods include gel filtration, enzymatic digestion of all single stranded nucleic acids, and washings consisting of buffer rinses at increased temperatures.
Probe detection methods include Northern blots, Southern blots and dot blots. Actual detection of hybridized probes is accomplished by measuring the label on the hybridized probes after removal of all non-hybridized and mismatched probes. The more common radioactive labels of choice include phosphorus 32 (32p), tritium (3H), carbon-14 (14C), sulfur 35 (35S), and fluorescent labels, chemiluminescent labels, and labels recognized by antibodies which are themselves labelled may also be used. The particular label used is a matter of choice. The manner in which a label is bound to the probe will vary depending upon the nature of the label chosen. In addition, a variety of methods are well known for introducing and measuring the various labels. Such methods include detection by radioscintillation counting, spectroscopy, such as fluorescence and luminescencé, by immunological means and by chemical means.
A probe of the invention may be used in primer extension proc~dures or in screening genomic libraries. Target sequences can be on the order of several hundred to several thousand nucleotides. A
probe of the invention is capable of hybridizing to target sequences of great length. As lon3 as the probe and target sequence are capable of hybxidizing to form a stable duplex, there is no target sequence too long to which a probe of the invention c~n be de-signed to hybridize. Likewise, probe length is only 2 ~
-19- 07-21(281)A
limited by the ability to hybridize and form a duplex to a target sequence. A suitable length of probe is about 15 to 50 bases in length with a preferred length belng about lS to 25 bases ln length. Methods for identifying target sequences and for preparing probe regions are well known. The target sequences can be from any DNA or RNA sequence, either procaryote or eucaryote includlng bacteria, yeast, plants and animals, these include sequences of infectious micro-organism, virus, or organism including parasites,human or non-human (animal) DNA or RNA sequences such as sequences characteristic of a genetic abnormality or other condition, and sequences derived from genetic engineering experiments such as total mR*~A or random fragments of whole cell DNA. Nucleic acid sequences may be obtained from a variety of sources including blood, tissue and cell samples, as well as DNA from the same sources amplified by such means as the polymerase chain reaction (PCR). Toxin producing microorganisms like Escherichia, Vibrio, Yersivea, Rlebsiella and Salmonella may be identified also.
Specific species include Haemophilis ducrei, Vibris cholerae, and E. coli. Other agents of clinical importance include Chlamydia trachomatous, genital Herpes virus, norwalk agent, Rotavirus, Campylabacter jejuni, ~eisseria gonorrhea, Herpes simplex virus, Brucella abor~us, Haemophilis influenza, ~ycobacterium, tuberculosis, Pseudomonas pseudomallei, Salmonella typAi, Sa~monella typhimurium, Neisseria meningitidis, Epstein-Barr virus, and human papilloma virus. In addition, the target se~uence can be complementary to a nucleic acid sequence which is characteristic of a class of human or non-human pathogens, like all enteric bacilli or all chlamydia. The target sequence can be complementary to a nucleic acid sequence which ~a~ 2 -20- 07-21(281)A
is characteristic of a host cell or vector used in the manufacture of recombinant DNA products, such as to detect the presence of such DNA or RNA contamlnants ln the product. Such detection may be useful in both diagnostics and therapeutics.
In some instances of probe use, lt is desired to differentiate between a single nucleotide mismatch of the target sequence and probe. A probe of the invention can be designed so that hybridlzation with a target sequence resulting ln a single nucleo-tide mismatch will be removed, leaving only the desired target sequence and probe hybridized molecule.
Since a substantial number of human genetic diseases are caused by single point mutations, the probes of the present invention can be used to determine geno-types and aid in the dia~nosis of genetic dlseases, even prenatally.
Sickle cell anemia and ~l-antitrypsin deficiency are examples of diseases arising from single point mutations. The following probe use, based on the se~uence disclosure in Conner et al., Proc._Natl. Acad. S l., 80:278, 1983, illustrates how a probe containing a nucleoside of the invention, 4-hydroxy-1-(2 deoxy-beta-D-erythro-pentofuranosyl) -2(lH)-pyridone (2), commonly named 2'-deoxy-3-deazauridine or DdU (when Rl, R2 and R3 are hydrogen in the nucleoside residue formula) can be used to detect the presence of a normal ~-globin gene and the defective ~--globin gene responsible for sickle cell anemia. Adenosine, thymidine, cytidine and guanosine are designated A, T, C and G respectively. A segment . of the normal gene for ~-globin has the following se quence:
-21- 07-21(281)A
5' CT CCT GAG GAG AAG TCT GC 3' 3' GA GGA CTC CTC TTC AGA CG 5' therefore examples of probes for the normal ~-globin gene may have the following sequences:
3' GA GGA CTY CTC TTC AGA CG S' GA GGA YTC CTC TTC AGA CG
GA GGA CTC YTC TTC AGA CG
GA GGA CTC CTY TTC AGA CG
Y = 2'-deoxy-3-deazauridine A defective ~-globin gene responsible for sickle cell disease has a single base mutation in the starred position of the normal gene sequence where the A
(starred) is changed to a T. Therefore, examples of probes designed to be complementary to the gene for sickle cell anemia have the following sequences:
3' GA GGA CAC CTY TTC AGA CG 5' GA GGA YAC CTC TTC AGA CG
GA GGA CAY CTC TTC AGA CG
GA GGA CAC YTC TTC AGA CG
Y - 2'-deoxy-3-deazauridine Furthermore, based on the sequence disclosure in Kidd et al., Nature, 304:230, 1983, a probe of the present invention can be designed to hybridize to the comple-mentary sequence in the gene containing the normal .
2~
-22- 07-21(281)A
sequence for ~l-antitrypsin. Also, a probe of the inventlon containlng 2'-deoxy-3-deazauridine can be designed to hybridize to the corresponding gene sequence responsible for ~l-antitrypsin deficiency. A
segment of the normal gene for al-antitrypsin has the following sequence:
5' ACC ATC GAC GAG AAA GGG A 3' 3' TGG TAG CT2 CTC TTT CCC T 5' Probes of the invention that can be designed comple-mentary to the normal gene segment have the following sequences:
3' TGG TAG CTG CTY TTT CCC T S' TGG TAG YTG CTC TTT CCC T
TGG TAG CTG YTC TTT CCC T
Y = 2'-deoxy-3-deazauridine Probes of the invention that can be designed to be complementary to the sequence responsible for al-antitrypsin deficiency, where the starred G in the no~mal gene sequence is changed to an A, have the following sequences:
3' TGG TAG CTG TTY TTT CCC T 5 ' TGG TAG YTG TTC TTT CCC T
Y = 2'-deoxy-3-deazauridine Point mutations may arise from a single base . substitution, insertion, or deletion in the DNA
~2~2 -~3- 07-21(281)A
sequence of a single gene. Using a probe of the invention to detect such sequences would eliminate the dependencies on restrictlon enzyme recognition si~e alteratlons, which have a low probabllity of occur-rence for any given point mutation. In addition,therapeutic uses for probes of the lnvention include insertion into various cells under conditions which allow them to hybridize to messenger RNA's (mRNA).
The ability to hybridize to mRNA may allow for the inactivation of undesired mRNA's such as viral and oncogene mRNA. Not only can the probes of the inven-tion be used to hybridize to mRNA in vivo, but the probes of the present invention may be used to hybri-dize, and therefore, inactivate mRNA during i~ vitro processes like in vitro translation.
DESCRIPTIONS OF THE PREFERRED EMBODIMENTS
The following examples illustrate specific embodiments of the invention described herein. As would be apparent to skilled artisans, various changes and modifications are possible and are contemplated within the scope of the invention described.
The following structures listed in Table II
are useful in the synthesiG involved in the Examples.
7~ ~3 ~
-24- 07-21(2al~A
TABLE 2.
OH
.,~
HO
HO OH
4-hydroxy-l-beta-D-erythro-pentofuranosyl-2(lH)-pyridone ( 1 ) OH
0~
HO
~0 .~ ~
4-hydroxy-1-(2-deoxy-beta-D-erythro-pentofuranosyl) -2(lH)-pyridone (2) ~2~2 -25- 07-21(281)A
OC(O)N(Ph)2 ~0 ~
HO OH
4-0-diphenylcarbamoyl-1-beta-D-erythro-pentofuranosyl-2(lH)-pyridone (3) OC~O)N(Ph)2 (~)2S~
~ Si O OH
.- 4 4-0-diphenylcarbamoyl-1-[3,5-0-[1,1,3,3-tetrakis (l-methylethyl)-1,3-disiloxanediyl~-beta-D-erythro-S pentofuranosyl]-2(lH)~pyridone (4) 3 ~ 2 -26- 07-21(281~A
oC(O)N(Ph)2 0~
1~
(iPr)-SI--O OC(S)OPIl 4-0-diphenylcarbamoyl~ 2-0-(phenoxythloxomethyl) -3,5-0-11,1,3,3-tetrakis~1-methylethyl)-1,3-dlsiloxanediyl]-be~a-D-erythro-pentofuranosyl]-2 (lH)-pyridone (5) oC(o)N(phk 0~ ,.
1~
" (~)2SI--O
4-O-diphenylcarbamoyl-1-[2-deoxy-3,5~0-~1,1,3,3 tetrakis(l-methyle~hyl)-1,3-disiloxanediyl]-beta-D-erytAro-pentofuranosyl]-2(lH)-pyridone (6) ' 2 -27- 07-21(281)A
oC(O)N(Ph~2 ~
HO ~o~/
)J
HO
?
4-O-diphenylcarbamoyl-1-(2-deoxy-beta-D-erythro-pento-furanosyl)-2(1H)-pyridone (7) OC(O~N(Ph)7 H3CO ~ ~\~
HO
4-O-diphenylcarbamoyl-1-[5-O-[bis(4~methoxyphenyl) phenylmethyl]-2-deoxy-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone (8) 25~32A~
-28- 07-21(281)A
OC(O)N(Ph)2 ~3~ ~
H3CO ~
o (l~pr~2~ OCH2CH2CN
4-O-diphenylcarbamoyl-1-[~5-O-[bls(4-methoxyphenyl) phenylmethyl]-3-0-~bls(l-methylethyl)amlno]-(2-cyanoethoxy)phosphino]-2-deoxy-be~a-D-erythro-pento-furanosyl~-2(lH)-pyridone (9) OC~O)N(Ph)2 h~CO ~ ~ y (I Pr)2N OCH3 LQ
4-O-diphenylcarbamoyl 1-[5-O-[bis(4-methox~phenyl) phenylmethyl]-3-O-~bis(l-methylethyl)amino]-methoxyphosphino~-2-deoxy-bet~-D-erythro-pentofuranosyl]-2(1H)-pyridone (10) % 3 2 ~
-29- 07-21(281)A
OCtO)N(Ph)2 CO ~ ~
HO OH
4-O-diphenylcarbamoyl-1-[5-O-~(4-methoxyphenyl)dlphenyl-methyl]-beta-D-eruth~o-pentofuranosyl-2(lH)-pyridone ( 1 1 ) OC(O)N~Ph)2 HO ~
HO OH
. 1~
4-O-diphenylcarbamoyl-3-iodo~ eta-D-erythro-pento-S furanosyl-2(lH)-pyridone (12) .. .~
2~2~
_30_ 07-21(281)A
oC(O)N~Ph)2 0~1 HO OH
4-O-diphenylcarbamoyl-5-iodo-1-beta-D-erythro-pento-furanosyl-2(lH)-pyridone (13) OC(O)N(Ph)2 H,CO~ ~
HO OH
." , "
4-O-diphenylcarbamoyl-3-iodo-1-[5-O-[(4-methoxyphenyl) diphenylmethyl]-beta-D-erythro-pentofuranosyl]-2(1H)-pyridone (14) 2~2~
-31- 07-21(281)A
OC(O)N(Ph)2 H,CO ~ ~
HO OH
4-O-diphenylcarbamoyl-5-iodo-1-[5-0-[4-methoxyphenyl) diphenylmethyl]-beta-D-erythro-pentofuranosyl]-2tlH)-pyridone (15) oC(o)~ph2 OCH~
HO OH
4-O-diphenylcarbamoyl-1-[5-O-[bis~4-methoxyphenyl) phenylmethyl]-beta-D-erythro-pentofuranosyl]-2(1H)-pyridone (16) .
2 ~ 2 ~
-32- 07-21(2~1~A
OC(O)N(Ph), H3CO ~ ~y HO OSl(l Pr)3 4-O-diphenylcarbamoyl-1-[[5-0-[(4-methoxyphenyl) diphenylmethyl]-2-O-tris(l-methylethyl)silyl]-beta-D-erytAro-pentofuranosyl]-1(2H)-pyridone (17) oC(O)N~Ph)2 H~CO ~ O ~
HO O Sl- (t~u) Me 4-O-diphenylcarbamoyl-1-[[5-0-[(4-methoxyphenyl~
S diphenylmethyl]-2-O-(1,l-methylethyl)dimethylsilyl]
-~eta-D-erythro-pentofuranosyl~-2(lH)-pyridone (18) 2~2~2 -33- 07-21(281)A
OC~O)N(Ph)2 H,CO ~ O ~
HO OSl(l Pr)3 4-O-diphenylcarbamoyl-3-iodo-1-[[5-0-[(4-methoxyphenyl) dlphenylmethyl]-2-0-tris(l-methylethyl)silyl]-be~a-D-erytAro-pentofuranosyl]-2(lH)-pyridone ~19) oC(O)N~Ph)2 H,CO ~ O ~
HO OSI(I-Pr~3 '`' iLQ ~
4-0-diphenylcarbamoyl-S-iodo-1-[[5-O-[(4-methoxyphenyl~
S diphenylmethyl)-2-O-tris(l-methylethyl)silyl]-beta-D-erytAro-pentofuranosyl]-2(lH)-pyridone (20) -34- 07-21~281)A
OC(O)N~Ph)2 H3C0 ~ ~y O OSI(I Pr)3 (I-Pr)2N OCH2CI l;~CN
~1 ,.
4-O-diphenylcarbamoyl-1-[[[5-0-[(4-methoxyphenyl) diphenylmethyl]-3-O-[bis(l-methylethyl)amino]
(2-cyanoethyl)phosphino]-2-O-tris(1-methylethyl)silyl]
-beta-D-erythro-pen~ofuranosyl]-2(lH)-pyridone (21) oc(o)N(ph)2 h3CO ~ ~ ~ /
o 0--Sl--(~ Bu) Me (I-Pr)2N OCH2CH2CN
.
4-O-diphenylcarbamoyl-1-[[[5-0-[(4-methoxyphenyl) diphenylmethyl]-3-O-[bis(1-methylethyl)amino](2-cyanoethyl)phosphino]-2-O-(l,l-methylethyl) dimethylsilyl]-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone (22) 2~2~
_35 07-21(281)A
OC(C))l`J(Ph)2 H,C0 ~ O ~y o ~:)SI(I~P~)3 (l-Pr)2N OCH2CH2CN
4-0-diphenylcarbamoyl-3-iodo-1-[[[5-0-[(4-methoxyphenyl) diphenylmethyl]-3-0-[bis(1-mPthylethyl)amino](2 cyanoethyl) phosphino]-2-0-tris(1-methylethyl)silyl]-beta-D-erythro-pentofuranosyl]-2(lH)-pyrldone ~23) OC(O)N(Ph)2 11,CO~O~
O OSI(I Pr)3 .~ (I-Pr)2N OCH2CH2CN
L~
4-0-diphenylcarbamoyl-5-iodo-1-[[[5~0-[(4-methoxyphenyl) diphenylmethyl]-3-0-[bis(l-methylethyl)amino](2-cyanoethyl) phosphino]-2-0-tris(1-methylethyl)silyl]-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone (24) 2 ~ 8 ~
-36- 07-21~281)A
OC(O)N(Ph)2 H,CO~ o~N
H--P--O `
O NH(E~)3~
4-O-diphenylcarbamoyl-1-[[5-O-[bis(4-methoxyphenyl) phenylmethyl]-3-O-(hydroxyphosphinyl)-2-deoxy]-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone trl-ethylammonium salt (25) OC(O)N(Ph)2 H3CO -~ o~h Pr)3 H--P = O
o NH~Et)3 ~ `
~
4-O-diphenylcarbamoyl-1-[[5-O [(4-methoxyphenyl) diphenylmethyl]-3-O-(hydroxyphosphinyl)-2-O-tris-(l-methylethyl)silyl]-beta-D-erytAro-pentofuranosyl]-2(lH)-pyridone triethylammonium salt (26) c~s~ ~ 82 -37- 07-21(281)A
OC(O)N(Ph)i of~
H3CO ~ ~ Me o` o sl-(tau) H--P--O Me o NH(Et)3~
4-O-diphenylcarbamoyl 1-[~5-O-[(4-methoxyphenyl) diphenylmethyl]-3-O-(hydroxyphosphinyl)-2-O-(1,1-dimethylethyl)dimethylsilyl]-beta-D-erythro-pento-furanosyl]-2(lH)-pyridone triethylammonium salt (27) OC(O)N(Ph)2 1~
(~),SI ~ ~
" (iPr)2SI--O
4-O-diphenylcarbamoyl-3-iodo-1-[2-deoxy-3,5 O-[l,1, 3,3-tetrakis(l-methylethyl)-1,3-disiloxanediyl]-~eta-D-erythro-pentofuranosyl]-2(lH)-pyridone (28) . , ~ , ~ , , ;
292~2 -38- 07-21(281)A
o~(o)N(ph)2 ~1 ,,,.. :''' (iP~),SI--O
2~
4-O-diphenylcarbamoyl-5-iodo-1-[2-deoxy-3,5-0-[1,1, 3,3-tetrakis(l-methylethyl)-1,3-disiloxanediyl]-beta-D-erythro-pentofuranosyl]-2(1H)-pyridone (29) OC(O)N(Ph)2 1~3 ' O
.~ :
~Q
4-O-diphenylcarbamoyl-3-iodo-1-t2-deoxy-beta-D-erythro-pentofuranosyl)-2(1H)-pyridone ~30) .
-: . :
~2~2 -39- 07-21(281)A
C)C(O)N(Ph)2 0~~
HO ~/
OH
4-0-diphenylcarbamoyl-5-iodo-l-(2-deoxy-beta-D-erythro-pentofuranosyl)-2.~1H)-pyridone (31) OC:(O)N(Ph)2 H3CO I ~3 H3CO ~ O ~
OH
4-O-diphenylcarbamoyl-3-iodo-1-[5~0-[bis(4-methoxyphenyl)phenylmethyl]-2-deoxy-beta-D-S erythro-pentofuranosyl]-2(1H)-pyridone (32) 2~2~
-40- 07-21(281)A
OC~O)N(Ph)a ~13(~ ~1 H3CO ~
OH
4-O-diphenylcarbamoyl-5-iodo-l-[5-0-[bis(4-methoxyphenyl)phenylmethyl] 2-deoxy-beta-D-erythro-pentofuranosyl3-2(lH)-pyridone (33) OC(O)N(Phk H3CO I ;~ 3 H3CO ~ ~
o \
-Pr)2N OcH2cH2c N
~. `
4-0-diphenylcarbamoyl-3-iodo-1-[5-0-[bis (4-methoxyphenyl)phenylmethyl]-[3-0-[bis(1-methylethyl)amino](2-cyanoethoxy)phosphino]-2-deoxy-beta~D-ery~hro-pentofuranosyl]-2(1H)-pyridone (34) 2~2~ 2 -41- 07-21(281)A
OC(O~N(Ph)2 H3CO f ~, H3CO ~
o (I-Pr)2N OCH2CH2CN
4-O-diphenylcarbamoyl-5-iodo-1-[5-O-[bis(4-methoxyphenyl)phenylmethyl]-[3-O-[bis(l-methylethyl)amino](2-cyanoethoxy)phosphino]-2-deoxy-beta-D-eryt~ro-pentofuranosyl]-2(1H)-pyridone (35) OC~O)N(Ph)2 ~ ,.
(iPr)~SI
(iPr)2SI--O
4-O-diphenylcarbamoyl-3-ethenyl-1[2-deoxy-3, 5-0-[1,1,3,3-tetraki~(l-methylethyl)-1,3-disiloxanediyl]-bet a-_-erythro pentofuranosyl]-~(lH)-pyridone (36) .
2 ~
-42- 07-21(281)A
OC(O)N(Ph)2 HO
. OH
4-O-diphenylcarbamoyl-3-ethenyl-1(2-deoxy-beta-D-erytAro-pentofuranosyl)-2(lH)-pyrldone (37) VC(O)N~Ph~2 H3CO ~ O
OH
~ .
4-O-diphenylcarbamoyl-3-ethenyl-l-[5~G-[bis (4-methoxyphenyl)phenylmethyl]-2-deoxy-beta-D-S erytAro-pentofuranosyl]-2(lH)-pyridone (38) .
', . .
2~2~2 -43- 07-21(281)A
OC(O)N(Ph)2 H3CO ~3 H3CO ~
OY
~l~pr)2N OCH~CH2CN
4-O-diphenylcarbamoyl-3-ethenyl-1-[5-O-[bis(4-methoxyphenyl)phenylmethyl]-[3-0-[bis(l-methylethyl) amino](2-cyanoethoxy)phosphino]-2-deoxy beta-D-erytAro-pentofuranosyl]-2(lH)-pyridone (39) oC(O)N~Ph)2 of~
(iPr),SI~O~
~)2SI O~H3 4-O-diphenylcarbamoyl-1-[[3,5-0-[1,1,3,3-tetrakis (1-methylethyl)-1,3-disiloxanediyl]-2-0-methyl]-beta-D-ery~hro-pentofuranosyl]-2(1H)-pyridone (40) ?J~2~82 -44- 07-21(2al)A
OC(O)N(Ph)2 oJ~
HO~/
4-0-diphenylcarbamoyl-1-(2-0-methyl-beta-D-erythro-pentofuranosyl)-2(lH)-pyrldone (41) OC(O)N(Phk H3CO f ~3 H3CO ~ O ~
HO OCHa 9LL ' 4-0-diphenylcarbamoyl-1-[5-O~[bis-(4-methoxyphenyl)phenylmethyl]-2-O-methyl-beta-D-erythro-pentofuranosyl] 2(lH)-pyridone (42) J~ ~2 -45- 07-21(281)A
oC(03N(Ph)2 H3CO ~3 H3CO ~ ~
OCI'13 (l~Pr)2N OCt~2t:H2CN
4-O-diphenylcarbamoyl-1-[5-O-[bis(4-methoxyphenyl) phenylmethyl]-3-O-[bis(1-methylethyl)amino(2-cyanoethoxy)phosphino]-2-O-methyl-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone (43) OC(O)~PI12 1~
(i~)2 Sl ~ ~
(i~)~SI - O OH
4-O-diphenylcarbamoyl-3-iodc-1-[3,5-O-[1,1,3,3-tetrakis(l-methylethyl)-1,3-dis1loxanediyl]-beta-D-erythro-pentofuranosyl]~2(lH)-pyridone (44) ~,~S~4~2 -46- 07-21(281)A
OC(O)NPh2 Os[~l .
(iPr)~SI~O~
I iPr)~SI--O OH
~ `
4-O-diphenylcarbamoyl-5-iodo-1-[3,5-0-[1,1,3,3-tetrakis(l-methylethyl)-1,3-disiloxanediyl]-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone (45) -47- 07-21(281)A
4-hydroxy-1-beta-D-erythro-pentofuranosyl-2(1H)-pyridone (1), commonly called 3-deazauridine (Slgma, st. Louis, MO) is converted to the 4-O-diphenyl-carbamoyl-1-~eta-D-erythro-pentofuranosyl-2(lH)-pyri-done (3), in the following manner. 15.0 gm. of 3-dea-zauridlne are dried in a vacuum desiccator containing phosphorous pentoxide ( P4 01 o) to obtain 14.7 gm. (6.04 X 10 2 mole). The dried starting uridine is placed in a round bottom flask and 140 mL acetonitrile (CH3CN) and 50 mL dimethylformamide (DMF) added along with 16.1 mL (9.24 X 10 2 mole) dlisopropyl ethylamine.
The mixture is stirred under argon and diphenylcar-bamoyl chloride (22 gm., 9.50 X 10 2 moles) in 60 mLs CH3CN is added dropwise over about 30 minutes at room temperature. The reaction is stirred overnight result-ing in a pale yellow solution. The volatiles are removed under reduced pressure on a rotary evaporator to obtain a thick syrup. The syrup is dissolved in toluene ~50 mL) and concentrated under reduced pres-sure (thls is repeated twlce) and the residue is triturated with ethyl acetate ~EtOAc) to obtain a white solid which is filtered, washed with EtOAc and ethanol tEtOH) and dried to give 16.8 gm. of pale yellow powder. The washings are concentrated and the re~idue purified by flash chromatography (Merck Silica) using EtOAc: methan~l (MeOH) (3%): triethylamine (Et3N) (0.5%). The fractions containing product are evapo-rated and triturated with ~tOAc, filtered and dried to give another 6.5 gm. of desired product, 4-O-diphenyl-carbamoyl-l-beta-D-erythro-pentofuranosyl-2(1H)-pyridone (3), (86% isolated yield). m.p. 167-68C; Rf C~2Cl2:
- MeOH 9:1) = 0.4 1HNMR, ~(~MSO-d6): 8.06 (d, 1~, J=7.66 Hz,H-6), 7.44-7.42 (m, 10H, aromatic), 6.29 ~dd 1~, J=2.88, 7.66Hz, H-5), 6.20 (d, lH, J = 5.~9 Hz, H-1'), 2 ~ 2 -48- 07-21(281)A
5.99 (d, lH, J = 2.88 Hz, H-3), 5.42 (d, lH, J = 5.18 Hz, 2'-OH), 5.13 (t, lH J = 5.05 Hz, 5' OH), 5.03 (d, lH, J = 5.36 Hz, 3' -OH), 3.96 & 3.88 (m 3H, H-2', 3' & 4'), 3.70 (m, lH, H-5'), 3.60 (m, lH, H-S'I); mass spectrum (FAB) m/z (relative lntensity) 439 (M+Li) (80), 307 (100), 196 (75); Elemental Analysis calculated for C23H22N2O7 : C, 63.01; H, 5.02; N, 6.54 Found C, 63.17; H, 5.15 N, 6.29.
To 15.0 gm. (O.0343 mole) of 4-0-dlphenyl-carbamoyl-1 -be ta-D-erythro-pentofuranosyl-2(lH)-pyridone (3), dissolved in 150 mL dry DMF (Aldrich) is added 30.1 mL (0.150 mole) diisopropylethylamlne. The solution is stirred under an Argon atmosphere and 11.9 mL
(11.9 gm, O.0381 mole) dichlorotetraisopropylsiloxane dissolved in 50 mL dry DMF is added in dropwise fashion over about 1 hour. The reaction is carried out at room temperature. TLC (M~rck 60 - p.254, 1:1 hexane:EtOAc) shows good conversion to product after several hours. The volatiles are removed under ~acuum and worked up by dissolving the residue in methylene chloride (CH2Cl2) and water. The organic layer is separated, washed with 0.5 M hydrochloric acid (HCl), and aqueous sodium bicarbonate (saturated) and dried with anhydrou~ sodium sulphate. The oil obtained upon removal of solvent is purified by preparative liquid chromatography (Waters Prep 500, silica gel, 3:1 hexane: ethyl acetate) to obtain 16.5 gm. (71% iso-lated yield) of light yellow product, 4-O-diphenyl-carbamoyl-l-[3,5-O-1l,1,3,3-tetrakis(l-methylethyl)-1, 3-disiloxanediyl]-beta-D--ery~hro-pentofuranosyl-2 (lH)-pyridone (4), RF 0.36 (EtOAC : ~e~ane 1:1);
H NMR, ~(CDCl~): 7.78 ~d, 1~, J = 7.8 H-2 H-6, . 7.4-7.26 (M, 10H, Aromatic) 6.31 (d, ~, J - 2.44 Hz, H-3), 6.~0 _(dd, lH, J = 7.8, 2.~3 Hæ, ~-5), 4.32-3.99 (m, 5H, H-2', 3', 4', 5' & 5" ), 3.03 (d, lH, J - 1.1 ~?,4'1~2 _~9_ 07-21(281)A
Hz 2'-OH), 1.1-0.96 (m, 28H, 2x S1-i-propyl); mass spectrum (FAB) m/z 687 (M~Li) i HR FAB MS : Calculated for C3~H48N2Si2O~Li, 687.3109 - ~ound 687.3099.
To 6.5 gm. (9.5 X 10 3 mole) 4-O-diphenyl-carbamoyl-1-[3,5-0-[1,1,3,3-tetrakis(l-methylethyl) -1,3-disiloxanedlyl]-heta-D-erythro-pentofuranosyl]-2(1H)-pyridone (4), dlssolved in 65 mL CH2Cl2 is added 1.75 gm (1.43 X 10 2 mole) 4-dimethylaminopyi~
dine and 1.45 mL (1.81 gm, 1.05 X 10 2 mole) phenoxy-thionocarbonyl chloride. The reaction mixt~re is stirred overnight at room temperature under argon. The volatiles are removed under vacuum and the orange semi-solid residue taken up in EtOAc. The organic layer is washed with water, 0.25 N HCl and saturated aqueous NaHCO3. The organic layer is dried with magnesium sulphate (MgSO9), filtered, and concentrated to about 25 mL. Purification is achleved by silica gel chromatography on a Waters Prep 500 using 3:1 hexane: EtOAc. A yellowish product is obtained which if carried forward at this stage does not reduce well.
A second pass over silica ~el is carried out ~o obtain 6.9 gm. of light yellow amorphous product (90% iso-lated yield), 4-O-diphenylcarbamoyl1-~2-O-(phenoxy-thioxomethyl)-3,5-O-[l,l, 3,3-tetrakis(l-methylethyl) -1,3-disiloxanediyl]-~eta-D-erythro-pentofuranosyl3-2(lH)-pyridone ~5). M.p. 117C (dcomp). RF 0-5 ?
(EtOAc : Hexane l ~ HNMR, ~(CDCl3) : 7.79 (d, lH, J = 7.8 Hz, H-6), 7.43 7.11 (m, 15H, aromatic), 6.32 (d,lR, J= 2.44 Hz, ~-3); 6.19 (dd, lH, J = 2.44, 7.8 Hz, H-5), 5.97 (d, l~, J = 4.69 Hz, ~-1), 4.5 (dd, lH, J = 4.7, 9.4 ~z, H-3), 4.28 (m, 1~, H-4'), 4.15 (m, lH, H-5'), 4.05 (m, l~, H-5" ), 1.55-0.96 (N, 28~, 2x-Si-(i-propyl)2); mass spectrum (FAB) m/z (relative intensity~ 824 (M+Li)+ (20), 664 (100), 307 (40); ~R
FAB MS : Calculated for C42Hs2N2Si2SogLi 823.3092.
c~ ~ ~2 ~
-50- 07-21(281)A
Found 823.3084. Elemental analysis : calculated for C42H5~N2Si2SOg; C,61.76; H,6.37; N,3.43. ~ound C,61.82 ; H,6.37 ; N, 3.32.
To 6.0 gm. (7.3 X 10 3 mole) 4-O-diphenyl-S carbamoyl-1-[2'-O-(phenoxythioxomethyl) -3',5'-O-[1,1, 3,3-tetrakis(l-methylethyl)-1,3-disiloxanediyl]-beta-D-ery~hro-pentofuranosyl]-2(lH)-pyridone (5) in 75 mL
toluene is added 2.94 mL tri-n-butyl-tin hydride (1.0 X 10 mole) and 0.24g AIBN (a~obisisobutyronitrile, 1.5 X 10-3 mole). The reaction mixture is stirred under argon atmosphere at 85C for 16 hours. The toluene is removed under re~uced pressure and the residue purified by flash chromatography on silica gel using 3:1 EtOAc:hexane as eluent. Appropriate frac-tions are combined and dried under vacuum to give 4.1 gm 84% isolated yield of the desired product as a syrup 4-O-diphenylcarbamoyl-1-[2-deoxy-3,5-0-[1,1,3,3-tetrakis (l-methylethyl)-1,3-disiloxanediyl]-beta-D-erythro-pento-furanosyl ]-2(1H)-pyridone (6), Rf 0.52 (EtOAc :
Hexane 1 ~ HNMR ~(CDCl3): 7.84 (d, lH, J = 7.83 Hz, H-5), 7.4-7.24 (m, 10H, Aromatic), 6.31 (d, lH J =
2.09 Hz, H-3), 6.22 (m, 2H, H-5, H-1'), 4.4 (m, lH, H-3'), 4.14 (m, lH, H-4'),4.05 (m, lH, ~-5'), 3.81 (m, lH, H-5"), 2.60 (m, lH, H-2'), 2.25 (m, lH, H~
1.09-0.98 ~m, 28H, 2x- Si-(i-propyl)2); mass spectrum (FAB) m/z (relative intensity) 672 (M+Li) (80), 482 (10), 313 (100). ER FAB MS : Calculated for C35H48O7N3Si3Li, 671.3160. Fou~d 671.3171.
To 3.0 gm (4.5 X 10 3 mole) 4-0-diphenylcar-bamoyl-1-~2-deoxy-3,5-O-[1,1,3,3-tetrakis(l-methyl~
ethyl)-1,3-disiloxanediyl]-~eta-D-erythro-pentofuranosyl]
-2(1H)-pyridone (6), in 30 mL CH3CN at 0C is added . O.62 gm l2.0 X 10 3 mole) tetrabutylammonium fluoride trihydrate (Aldrich, Milwaukee, WI) with stirring.
The reaction mixture is stirred at 0C for 30 minutes, 2~2~
-51- 07-21(281~A
then stlrred for 30 minutes at room temperature.
Solvent is removed under reduced pressure and the deslred product isolated by silica gel flash chroma-tography using ~:1 CH2Cl2:MeOH containing 0.5% Et3N.
S A total of 1.5 gm. ~79% isolated yield) of desired 4-O-diphenylcarbamoyl-1-(2-deoxy-beta-D-erythro-pento-furanosyl)-~(lH~-pyridone (7~, is obtained. The product is crystallized from EtOAc; mp 147 - 48C. Rf 0~4(CH2Cl2 : MeOH 9:1) 1HNMR, ~(DMSO-d6) 7.98 (d, lH, J = 7.77~z, H-6), 7.43-7.3 (m, lOH, aromatic), 6.30 (m, 2H, H-5, H-l'), 6.18 (d, lH, J = 2.56 Hz, H-3), 5.23 (d, lH J = 4.18 H~, 2'-OH), 5.01 (t, lH, 5'- OH), 4.20 (m, lH, 3'-H), 3.84 (m, lH, H-4'), 3.6 (m, 2H, H-5' &
5"), 2.25 (m, lH, H-2'), 1.95 (m, lH, H-2); mass spectrum FAB m/z (relative intensity) 423 (M + Li) (70), 312 (90), 242 (100); Analysis calculated for:
C23H22N2G6 0-5H20 : C, 65.03 : H, 5.34 N, 6.49. Found:
C, 65.04 : H, 5.21i N, 6.57.
To 1.1 gm (2.61 x 10 3 mole) 4-O-diphenyl-carbamoyl-1-(2-deoxy-be~a-D~erythro-pentofuranosyl)-2 (lH)-pyridone (7), in 15 mL dry pyridine is added 1.3 gm (3.8 x 10 3 mole) dimethoxytrityl chloride, 0.6~ mL
(3.9 x 10 3 mole) diisopropylethylamine and 30 mg 4-N,N-dimethylaminopyridine (2.5 x 10 4 mole). The reaction mixture is stirred at room temperature under argon for 4 hours. Volatiles are removed under reduced pressure and excess pyridine is removed by repeated (3 x 20 mL) distillation of toluene under reduced pressure. The residue is purified by silica gel flash chromatography using 2% MeOH in C~2C12 containing 0.5% triekhylamine. 1.1 ~m of a yellow amorphous solid is obtained which is rechromatographed ` (same solvent) to give 0.9 gm (1.44 x 10 3 mole) desired product as a white amorphous ma~erial, 4-O-di-phenylcarbamoyl-1-[5-O-[bis(4-methoxyphenyl~phenyl-~2~ 2 -52- 07-21(281)A
methyl]-2'-deoxy-beta-D~erythro-pentofuranosyl] -2~1H)-pyridone (8). RF 0.56 (CH2Cl2:MeOH, 9:1); 1HNMR, ~(CDCl3): 7.92 (d, lH, J = 7.89 Hz, H-6), 7.42 - 7.22 (m, l9H, aromatic), 6.84 (2s, 4H, aromatic), 6.39 (dd, lH, J = 5.9 Hz, H-1'), 6.~5 (d, lH, J = 2.52 Hz, H-3), 5.97 (dd, lH, J = 2.44, 7.89 Hz; H-5), 4.47 (m, lH, H-4'), 4.03 (m, lH, H-4'), 3.77 (s, 6H, 2-OCH3), 3.5 ~m, lH, H-5'), 3.42 (m, lH, H-5"), 2.60 (m, lH, H-2'), 2.2 (m, lH, H-2"); mass spectrum ~FAB) m/z (relative intensity) 731 (m+Li) (25), 427 (100); HR FAB MS;
C44H~oN~O8 : 731.2945, found ; 731.2939.
To 0.47 gm (6.43 x 10 4 mole) 4-0-dlphenylcar-bamoyl-l-[5-O-[bis(4^methoxyphenyl)phenylmethylJ-2-deoxy-~eta-D-erythro-pentofuranosyl]-2(1H)-pyridone (8), in 8 mL CH2Cl2 is added 0.45 mL (2.5 x 10 3 mole) N,N-diisopropylethylamine. 0.3 gm ~1.27 x 10 3 mole) ~-cyanoethyloxy- N,N-diisopropyl chlorophosphine (ABN) in 1 mL CH2Cl2 is added dropwise over about 15 minutes. The reaction mixture is stirred under argon atmosphere at room temperature for an additional hour.
The solution is concentrated to dryness using a rotary evaporator, dissolved in EtOAc and washed with saturat-ed aqueous NaHCO3 (3 x Z0 mL), distilled water and dried with (MgSO4). The yellow solution is evaporated to dryness and dried under vacuum to give 07.4 gm of viscous product that is dissolved in EtOAc and precip-itated by the addition of hexane. The mixture is cooled, solvents decanted and the residue washed with cold hexane and dried under vacuum to give 0.55 gm (6.0 x 10 4 mole, 79% isolated yield) of 4-O~diphenyl-carbamoyl-l-[[5-O-[bis~4-methoxyphenyl)phenylmethyl]-3-O-tbis(l-methylethyl)amino~-(2-cyanoethoxy~phosphino]
.~ -2-deoxy-beta-D-erythro-pentofuranosyl]-2(1~)-pyridone (9), TLC conditions; Merck silica gel 60 p254, EtOAc :
Hexane, 1:1; RF = 0.27.1HNMR, ~tcDcl3) 7.99, ~.92 (2d 2 ~ 2 ~
-53- 07-21(281)A
lH, J = 7.73 Hz, H-6), 7.42-7.22 (m, l9H aromatic), 6.84 (m, 4H, aromatlc), 6.42 (dd, lH, J = 6.27 Hz, H-l), 6.26 (d, lH, J =2.44 Hz,H-3), 5.89 (dd lH, J
=2.43, 7.8 Hz, H-5), 4.6 (m, lH, H-3'), 4.15 (m, lH, H-4'), 3.76 (s, 6H, 2x-OCH3), 3.74-3.35 (m, 4H, H-5', 5", 2x CH(Me)2), 2.65 (m, lH, H-2'), 2.6 (t, 2H, J =
6.34 Hz, CH2CN), 2.4 (T, 2H, J= 6.3 Hz, CH2), 2.2 (m, lH, H-2"), 1.28-1.05 (m, 12H, 2x-N-CH (CH3)2); mass spectrum (FAB) m/z (relative lntensity) : 931 (m+Li) (100), 627 (15); HR FAB MS: calculated for Cs3 H57 N4 Og PLi, 931.4023; Found 931.4015.
4-O-diphenylcarbamoyl-l-[S-O-[bis(4-methoxy-phenyl)phenylmethyl]-3-O[bis(1-methylethyl)amino]-methoxyphosphino]-2-deoxy-beta-D-erythro-pentofuranosyl]
-2(1H)-pyridone (10), is synthesized from 4-O-diphenyl-carbamoyl-1-[5-O-[bis(4-methoxyphenyl) phenylmethyl]-2-deoxy-beta-D-erythro-pentofuranosyl]-2(1H)-pyridone (8), and N,N-diisopropylmethylphosphonamidic chloride in substantial accordance with the teaching in Example 1.
The yield of desired product after silica gel chromatog-raphy is 68%. 31-P NMR (CDCl3, d, 149.2, 148.8 vs. 5%
H3PO4).
4-O-diphenylcarbamoyl-l-beta-D-eryt~ro-pentofuranosyl-2(lH)-pyridone (3), prepared in sub-stantial accordance with the teaching of Example 1, can be converted to 4-O-diphenylcarbamoyl-1~5-0-[~4-methoxyphenyl) diphenylmethyl]-beta-D-e~ythro-pento furanosyl]-2(1H)-pyridone (11), in the following manner. To 15.0 gm (3.43 x 10- 2 mole~ of 4-O-di-phenylcarbamoyl-1-beta-D-erytAro-pentofuranosyl-2(lH)-2~32~T~
-54- 07-21(281)A
pyridone (3), dlssolved in dry pyridine is added 10.5 mL (6 x lO- 2 mole) dlisopropylethylamine and 30 mg (2.5 X 10-4 mole) 4-N,N-dimethylaminopyridine. To this is added li.7 gm (3.9 x 10- 2 mol~) monomethoxy-triphenylmethyl chloride. The reaction mixture isstirred at room temperature under argon atmosphere until complete. Volatiles are removed in vacuo and reduced pyridine is removed by repeated (3x) distilla-tion of toluene under reduced pressure. The recidue is purified by silica gel flash chromatography using a suitable solvent system made slightly basic with 0.5%
triethylamine. In this manner the desired product, 4-O-diphenylcarbamoyl-1-5-0-[(4-methoxyphenyl)diphenyl-methyl] -beta-D-ery~hro-pentofuranosyl]-2 (lH)-pyridone (ll), can be obtained.
4-O-diphenylcarbamoyl-3-iodo-1-be~a-D-eryt~ro-pentofuranosyl -2(lH)-pyridone (12), and 4-O-diphenyl-carbamoyl-5-iodo-1-beta-D-erythro-pentofuranosyl-2(1H)-pyridone (13), can be converted to 4-O-diphenylcar-bamoyl-3-iodo-l-[5-0-[(4-methoxyphenyl)diphenylmethyl]-be~a-D-erytAro-pentofuranosyl]-2(1H)-pyridone (14), and 4-O-diphenylcarbamoyl-5~iodo-1-[5~0-[4~methoxy-phenyl) diphenylmethyl]-beta-D-eryth~o-pentofuranosyl]
-2(1H)-pyridone (lS), respectively, in the same manner. 4-O-diphenylcarbamoyl-1-[5-O-[bis ~4-methoxy-phenyl) phenylmethyl]-beta-D-erythro-pentofuranosyl]-2 (lH)-pyridone (16), can be obtained in a similar manner by substituting dimethoxytriphenylmethyl chloride ~or monomethoxytriphenylmethyl chloride.
To 4.7 sm (7.9 x 10-3 mole~ 4-O-diphenyl-~` carbamoyl-1-[5-0-[~4-methoxyphenyl)diphenylmethyl]-2 ~ o ~
-55- 07-21(281)A
beta-D-erythrO-pentofUranosyl]-2(1H)-pyridone (11) in anhydrous DMF under argon atmosphere is added 1~18 gm (1.73 x 10- 3 mole) imidazole and 3.38 mLs (1.58 x 10 2 mole) triisopropylsilyl chloride while stirring.
S The reaction is allowed to proceed until complete, quenched with aqueous saturated NaHC03, and concen-trated in vacuo. The resulting residue is co-evap-orated with toluene, dissolved in CH2Cl2 and washed with water. The CH2C12 layer is dried with MgSO9 (anhydrous) and concentrated in vacuo . The desired product, 4-0-diphenylcarbamoyl-1-[[5-0-[(4-methoxy-phenyl)diphenylmethyl]-2-0-tris(1-methylethyl)silyl]-beta-D-erythro-pentofuranosyl]-1(2H)~pyridone (17), can be obtained by silica gel chromatography in a fashion similar to that employed by N. Usman, K. K. Ogilvie, M.Y. Jiang, and R.J. Cedergren, J. Am. Chem. Soc., 1987, 109, 7845-7854.
By substitutin~ tert-butyldimethylsilyl chloride for triisopropylsllyl chloride, one can obtain 4-0-diphenylcarbamoyl-1-[~5-0-[(4-methoxyphe-nyl)diphenylmethyl]-2-0-(1,l-methylethyl)dimethylsil-yl~-beta-D-erythro-pentofuranosyl]-2llH)-pyridone (18). 4-0-diphenylcarbamoyl-3-iodo-1-[5-0-[(4-methoxy-phenyl) diphenylmethyl]-be t a-D-erytAro-pentofuranosyl]-2 (lH)-pyridone (14), and 4-0-diphenylcarbamoyl-5-iodo-1-[5-0-[4-methoxyphenyl)diphenylmethyl] -beta-D-erythro-pentofuranosyl]-2 (lH)-pyridone (15), can be converted to 4-0-diphenylcarbamoyl-3-iodo-1-[[5-0-[(4-methoxy-phenyl) diphenylmethyl]-2-0-tris(l-methylethyl)~ilyl]-beta-~-erythro-pentofuranosyl]-2(1~)-pyridone (19), and 4-0-diphenylcarbamoyl-5-iodo-1-[[5-0-[(4-me~hoxy-ph~nyl)diphenylmethyl]-2-0-tris(l-methylethyl) silyl]-beta-D-erythro-pentofuranosyl]-2(1H)~pyridone (20), respectively, in a similar manner.
2~2~
-56- 07-21(281)A
To a stirred dry tetrahydrofuran (THF) solution of 7.0 mL (4 x 10- 2 mole) dll opropylethyl-amine, 3.08 gm (1.3 x 10-2 mole) (N,N-diisopropyl)-~-cyanoethoxyphosphonamldlc chloride and a catalytic amount (215 mg, 1.75 x 10- 3 mole) of 4-N,N-dimethylaminopyrl-dine is added dropwise to a solution of 8.66 gm (1 x 10-2 mole) 4-Q-diphenylcarbamoyl-1 [[5-0-[(4-methoxy-phenyl)diphenylmethyl]-2-0-tris(l-methylethyl)sllyl]-beta-D-erythro-pentofuranosyl]-1(2H)-pyrldone (17), in dry THF. The reaction is allowed to go to completion and worked-up by adding EtOAc and washing with saturated aqueous NaHCO3. The organic layer is dried and solvent removed in vacuo. The desired product, 4-0-di-phenylcarbamoyl-1-[[[5-0-[(4-methoxyphenyl)diphenyl-methyl]-3-0-[bis(l-methylethyl)amino](2-cyanoethyl) phosphino]-2-0-tris(1-methylethyl)sllyl]-beta-D-erythr~-pentofuranosyl]-2(1H)-pyridone (21), can be obtained in substantially pure form by silica gel flash chroma-tography.
In a similar fashion, 4-0-diphenylcarbamoyl-1~[[[5-0-[(4-methoxyphenyl)diphenylmethyl]-3-0-[bis (l~methylethyl)amino](2-cyanoethyl)phosphino]-2-0-(1, l-methylethyl)dimethylsilyl]-beta-D-erythro-pentofur-anosyl]-2(1H)-pyridone (~2), can be obtained by using 4-0-diphenylcarbamoyl-1-[[5-0-[(4-methoxyphenyl) diphenylmethyl]-2-0-(1,1-methylethyl)dimethylsilyl]-~eta-D-erythro-pentofuranosyl]-2(1R)-pyridone (18).
In the same manner, 4-0-diphenylcarbamoyl-3-iodo-1-[[5-0-[(4-methoxyphenyl) diphenylmethyl]-2-0-tris(1-methylethyl)silyl]-beta-D-ery~hro-pentofuranosyl]-2[1~)-pyridone (19), and ~-0-diphenylcarbamoyl-5-iodo-1-' [f5-0-[(4-methoxyphenyl)diphenylmethyl~-2~0-tris(1-methylethyl) silyl] -be ta-D~pentofuranosyl]-2 (lH)-pyridone (20), can be converted to 4-O~diphenylcar-, . . - . .. . . .
2 ~ 2 -57- 07-21~281)A
bamoyl-3-iodo-l-1[(5-0-[(4-methoxyphenyl)diphenylmethyl~
-3-0-[bis(l-methylethyl)amino](2-cyanoethyl) pnosphlno]~
2-0-tris(l-methylethyl)silyl]-beta-D-erythro-pento-furanosyl]-2 (lH)-pyrldone (23), and 4-0-diphenylcar-bamoyl-5-iodo-1-[[~5-0-~(4-methoxyphenyl) diphenylme-thyl]-3-0-[bis (l-methylethyl)amino](2-cyanoethyl) phosphino]-2-0-tris(l-methylethyl) sllyl]-beta-D-erytAro-pentofuranosyl]-2 (lH)-pyridone (24), re-spectively.
4-0-diphenylcarbamoyl-1-[5-0-[bis(4-meth-oxyphenyl)phenylmethyl]-2-deoxy-beta-D-erythro-pento-furanosyl]-2(lH)-pyridone ~8), can be converted to 4-0-diphenylcarbamoyl-1-[[5-0-[bis~4-methoxyphenyl)phenyl-methyl]-3-0-(hydroxyphosphinyl)-2-deoxy]-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone triethylammonium salt (25), in substantial accordance with the procedure of B.C.
Froehler, P.G. Ng, and M.D. Matteucci, Nuc. Acids Res., 14,5399-5407 ~1986). Thus, to a stirred solution of phosphorous trichloride (PCl3) (1.03 gm, 7.5 x lO- 3 mole) and N-methylmorpholine (7.59 gm, 7.5 x lO- 2 mole) in 750 mL anhydrous CH2Cl2 is added 1,2,4-tri-azole (1.73 gm, 2.5 x 10-2 mole) at room temperature.
After 30 minutes the reaction mixture is cooled to 0C and 4-0-diphenylcarbamoyl~ 5-0-[bis(4-methoxy-phenyl)phenylmethyl]-2-deoxy-beta-D-erythro-pento-furanosyl]-2 (lH)-pyridone (8), (0.94 gm, 1.5 x 10-3 mole) in 200 mL anhydrous CH2C12 is added dropwise over 20 minutes, stirred for 10 minutes, poured into 600 mL of l.OM aqueous triethylammonium bicarbonate ( TEAB, p~ 8.5), shaken and separated. The aqueous ,- phase is extracted with 200 mL C~2Cl2 and ~he combined organic phase dried over Na2SO~ and evaporated to a foam. The desired 4-0-diphenylcarbamoyl-1-[[S-O-[bis (4-methoxyphenyl)phenylmethyl]~3-0-(hydroxyphosphinyl)--58- 07- 1(281)A
2 deoxy]-beta~D-erythro-pentofuranosyl]-2(lH)-pyridone triethylammonlum salt (25), can be obtalned by silica gel flash chromatography followed by TEAB extractlon and evaporatlon~ 4-O-dlphenylcarbamoyl~ 5-O-[(4-methoxyphenyl)dlphenylmethyl~-3 -O- ( hydrOXyphOSphlnyl ) -2-O-tris-(l-methylethyl~sllyl]-~eta-D-erythro-pento-furanosyl]-2(1~)-pyridone trlethylammonium salt (26), and 4-O-diphenylcarbamoyl-1-[15-O-[(4-methoxy-phenyl)dlphenylmethyl]-3-O-(hydroxyphosphinyl)-2-O-(1,l-dimethylethyl)dimethylsilyl]-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone triethylammonium salt (27), can be obtained in the same manner.
A mlxture of 4-O-diphenylcarbamoyl-1-~2-de-QXy-3, 5-0- ~ , 3, 3-tetrakis(l-methylethyl)-1,3-disi-loxanediyl]-beta-D-erythro-pentofuranosyl]-2(lH)-pyri-done, (6), (3.9g, 0.00S9mol), N-iodosuccinimide (1.4g, 0.0062 mol) in acetonitrile (40mL) containing di-chloroacetic acid (0.18g, 0.0014 mol) is heated at 60C for 4 hours under axgon atmosphere. The result-ing dark brown solution is concentrated under reduced pressure and the residue dissolved in 125 mL of CH2Cl2. This solution is washed with saturated agueous NaHCO3 (3 x 25 mL) followed by water (2 x 50 mL), dried (Na2SO9) and concentrated in vacuo. The resulting dark brown residue is purified by silica gel .
flash chromatography using EtOAc:~exane (1:4 v/v) as the eluent. ~he 3-iodo derivative is eluted first followed by the 5-iodo derivative. The fractions containing the 3-iodo derivative are combined, con-centrated under reduced pressure and the residue `` crystallized from MeOH to give 4-O-diphenylcarbamoyl-3-iodo-1-[2-deoxy-3,5-0-[1,1,3,3-tetrakis(1-methyl-ethyl)-1,3-disiloxanediyl] ^beta-D-erythro-pentofur-anosyl]-2(1~)-pyridone (28), (3.7g, 80%) as shiny 2 ~ 2 ~ 2 -59- 07-21(281)A
white flakes. m.p. 132-33C. Rf 0.57 (EtOAc:Hexane 1:1). IHNMR (300MHz, CDCl3) ~:7.9 (d, J = 7.8Hz,l,H6), 7.44-7.2 ~m, 10, aromatic), 6.32 (d, J - 7.8Hz, 1, H5), 6.17 (d, J = 6.73, 1, Hl'), 4.39 ~m, 1, H3'), 4.18 ~m, 1, H5'), 4.04 (m, 1, H5"), 3.8 (m, 1, H4'), 2.59 (m, 1, H2'), 2.26 (m, 1, H2"), 1.1 - 0.9 (m, 28.0[Si(iPr)2]2); FAB mass spectrum (m/z) 797 (M+Li), 439 (M+Li-sugar)i Analysis calculated for C35H~7IN207 Si2:C, 53.16i H, 5.95; N, 3.54. Found: C,52.82;
H, 5.95; N, 3.47. Fractions containing 4-0-diphenyl-carbamoyl-5-iodo-1-[2-deoxy-3,5-0-[1,1,3,3-tetrakis (l-methylethyl)-1,3-disiloxanediyl] -beta-D-ery~Aro-pentofuranosyl]-2(lH)-pyridone (29) are combined, c~ncentrated under reduced pressure and dried under high vacuum to give 0.25g (5%), as an amorphous solid.
Rf 0.48 (EtOAc: Hexane l:i). 1HNMR (300 MHz, CDCl3) ~:8.01 (s, 1, H6), 7.4 - 7.2 (m, 10, aromatic), 6.52 (s, 1, H3), 6.13 (dxd, 1, H1'), 4.36 (m, 1, H3'), 4.15 (m, 1, H5'), 4.04 (m, 1, H5"~, 3.8 (m, 1, ~4') 2.55 (m, 1, H2'), 2.25 (m, 1, H2"), 1.2 - 0.95 (m, 28 O[Si(i-Pr~2]2). FAB mass spectrum (m/z) 797 (~+Li), 439 (M~Li-sugar), HRFABMS observed (M~Li) 797.2134 calculated for C3sH4~IN2O~Si2 797.2127.
A mixture of 4-O-diphenylcarbamoyl-3-iodo-1-[2-deoxy-3,5~O~[1,1,3,3-tetrakis(1-methylethyl)-1,3~ ,' disilo~anediyl]-beta-D-erytAro-pentofuranosyl]-2~
pyridone (28), (lg, 0.0013 mL) and tetrabutylammonium fluoride trihydrate (0.25g, 0.0008 mL) in acetonitrile (20 mL) is stirred at 0C for 30 minutes followed by stirring at room temperature for 30 minute~. The solution is concentrated under reduced pressure and the residue purified by silica gel ~lash chromatography using C~2Cl2-MeO~ (4%) containing 0~5% Et3N as the eluent. Fractions containing 4-O-diphenylcarbamoyl-3-iodo-l-(2-deoxy-be ta-D-erythro -pentofuranosyl) 2(1~-2~24~2 -60~ 07-21(281~A
pyrldone (30), [indicated by TLC uslng CH2Cl2-MeOH(5%)]
are combined, concentrated under reduced pressure and dried on a desiccator under vacuum to glve C.5g (72%) of product. Rf 0.49 (CH2Cl2-MeOH 9~ HNMR (300 MHz, DMSO-d6) ~: 8.07 (d, J = 7.7Hz, 1, H6), 7.47 - 7.3 (m, 10, aromatic) 6.48 (d, J = ~.7 ~z, 1, H5), 6.26 (dxd, J = 6.3Hz, 1, H1'), 5.28 (d, J = 4.2Hz, 1, 2'-OH) 5.08 (t, J = 5.22 Hz, 1, 5'-OH), 4.23 (m, 1, H3'), 3.87 ~m, 1, H4') 3.61 (m, 2, H5' x 5"), 3.33 (s, 6, 2x-OCH3), 2.29 (m, 1, H2'), 1.99 (m, 1, H2"); FAB mass spectrum (m/z) 549 (M + H), 433 (m-sugar); HRFABMS observed (M+Li) 555.0602, calculated or C23H~1N~O6ILi 555.0604.
4-O-diphenylcarbamoyl-5-iodo-1-(2-deoxy-beta-D-ery~hro-pentofuranosyl)-2(lH)-pyridone (31), is obtained under similar reaction conditions as the 3-isomer, using tetraethylammonium fluoride, yield 63%. Rf 0.47 (CH2Cl2-MeOH 9:1), 1HNMR ~300 MHz, DMSQ-d6) ~8.4 (s, 1, H6), 7.6 - 7.2 (m, 10, axomatic), 6.44 (s, 1, H3), 6.24 (dxd, J = 6.3Hz, 1, ~ , 5.24 (d, J = 4.41 Hz, 1, 3'-OH), 5.14 (t, 1, 5'-OH), 4.23 (m, 1, H3'), 3.85 (m, 1, H4'), 3.59 (m, 2, ~5' x 5"), 2.49 (m, 1, H2'), 2.02 (m, 1, H2")i FAB mass spectrum (m/z) 555 (M+Li), 439 (M+Li-sugar).
A mixture of 4-O-diphenylcarbamoyl-3-iodo-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-2(1~)-pyri-done (30), (0.22g, 0.0006 mol), dimethoxytritylchloride (O.27g, 0.0008 mol), dimethylaminopyridine (DMAP) (O.025g) and diisopropylethylamine (O.14 mL, 0.0008 mol), in dry pyridine (5 mL) is stirred at room temperature for 6 hours and left overnight at 0C.
Pyridine is distilled under reduc~d pressure and the residue purified by silica gel flash chromatography using EtOAc-Hexane containing 0.5~ Et3N. The appro-priate fractions (visualized under W lamp) are combined, concentrated under reduced pressure and the -61- 07-21(2~1)A
resulting residue dried under high vacuum to give O.26g (76%) of ~-O-diphenylcarbamoyl-3-iodo-1-[5-O-[bis(4-methoxyphenyl)phenylmethyl]-2-deoxy-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone ~32), as an S amorphous material (Homogenous on TLC). Rf = 0.76 [CH2Cl2-MeOH (10%)]. IHNMR (CDCl3), ~:7.8 (d, J =
7.7Hz, 1, H6), 7.4 - 6.82 (m, 23, aromatic), 6.35 (d, J = 7.7Hz, H5), 6.2 (dxd, 1, H') 4.5 (m, l, H3'), 4.05 (m, l, H4'), 3.9 ~m, 2, H5'x 5") 3.8 (s, 6, 2X-OCH3), 2.50 (m, 1, H2'), 2.35 (m, 1, H2"); FAB mass spectrum `
(m/z): 857 ~M+Li), 533 (M+Li-DMT); HRFABMS observed 857.1904, calculated for C44~39IN2O8Li 857.1911.
4-O-~iphenylcarbamoyl-5-iodo-1-[5-O-[bis (4-methoxyphenyl)phenylmethyl]-2-deoxy-beta-D-erythro-pentofuranosyl]-2(1H)-pyridone (33), is prepared under similar reaction conditions. Yield 65%. R- 0.73 [CH2Cl~-MeOH (10%)]; 1HNMR (DMSO-d6) ~: 8.07 (s, l, H6), 7.46 - 6.8 (m, 23 aromatic), 6.51 (s, 1, ~3), 6.25 (dxd, l, J = 6.8Hz, 1, Hl'), 5.31 (d, J = 4.35 Hz, 1, 3'-OH), 4.19 (m, 1, H3'), 3.98 (m, 1, H4'), 3.22 (m, 2, H5' x 5"), 2.3 (m, l, E2~), 2.1 (m, l, H2"); FAB mass spectrum (m/z) 857(M+Li), 553 (M~Li-DMT).
To a chilled (ice bath) solution, 4-O-diphe-nylcarbamoyl-3-iodo-1-[5-O-[bis(4-methoxyphenyl)phe-nylmethyl]-2-deoxy-beta-D-erythro-pentofuranosyl]-2(1~) -pyridone ~32), (0.24g, 0.00028 mol) in dichloromethane (5 mL) containing diisopropylethylamine (0.1 mL, 0.00057 mol), is added dropwise a solution of ~-cyano-ethoxy-N,N-diisopropylamine chlorophosphine (0.lg, O.00042 mol), in dichloromethane (1 mL). The reaction mixture is stirred at room temperature for 2 hours.
TLC (EtOAc) reveals completion of ~he reaction. The solution is concentrated to dryness, the residue dissolved in EtOAc (10 mL), and washed with 10%
-62- 07-21(281)A
NaHCO3 (3 x 10 mL), followed by water (3 x 10 mL).
The organlc layer ls dried over anhydrous Na2 S04, concentrated under reduced pressure and the resulting syrupy residue purified by silica gel flash chroma-tography using EtOAc-Hexane (1:1) as the eluent to give 0.2g (63%) of 4-O-diphenylcarbamoyl-3-iodo-1-[5 -O-[bis(4-methoxyphenyl)phenylmethyl]-[3-O-[bis(l-methylethyl)amino](2-cyanQethoxy)phosphino]-2-deoxy-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone (34), as a white amorphous solid. IH, NMR (300 MHz, CDCl3) ~:8.20 (d, J = 7.6 Hz, H6), 8.05 (d, J = 7.6 Hz, H6), 7.4 - 6.8 (m, 23, aromatic), 6.35 (m, 1, H1'~, 6.2 x 6.02 (d, J = 7.6 Hz, 1, a5), 4.65 x 4.55 (m, 1, H3'), (4.15 (m, 1, H4'), 3.77 (2s, 6~ 2x -OCH3), 3.85 - 3.3 (m, 4, H5' x 5", -CH2-0-P-), 2.75 x 2.25 (m, 2, H2' x H2"), 2.6 x 2.4 (2t, 2, -CH2CN), 1.4 - 1.05 [(m, 14, N(i-Pr)2]; FAB mass spectrum (m/z) 1057 (M~Li), 753 (M+Li-DMT); HRFABMS: observed 1057.2999, calculated for Cs3H56IN~Og PLi 1057.2990.
4-O-diphenylcarbamoyl-5-iodo~ 5-O-[bis (4-methoxyphenyl)phenylmethyl]-[3-O-[bis(l-methyl-ethyl)amino](2-cyanoethoxy) phosphino]-2l-deoxy]-beta-D-erythro-pentofuranQsyl)-2(1H)-pyridone (35) is prepared as described for the 3-iodo derivative. Yield 74%; lENMR
(CDCl3) ~: 8.19 x 8.15 ~2s, 1, H6~, 7.5 - 6.8 (m, 23, aromatic), 6.51 (s, 1, 3H), 6.37 (d x d, 1, ~1'), 4.5 (m, 1, ~3') 4.19 (m, 1, H~'), 3.78 (2s, 6, 2 x -OCH3), 3.7 - 3.2 (m, 4, ~5' x 5" x -C~2-0-P), 2.75 x 2.15 (m, 2, ~2' x H2'l), 2.6 x 2.4 (2t, 2, -CH2CN) 1.35 - 1.05 lm, 14-N (i~Pr)2]. FAB mass spectrum (m/z) 1057 (M+Li), 753 (M+Li~DMT), ERFABMS observed 1057.
3035, calculated for Cs3~50lN4Og P 1057.2990.
. .
~2~
-63- 07-21(281)A
A mixture of 4-O-dlphenylcarbamoyl-3-io-do-1-~2-deoxy 3,5 O-[1,1,3,3-tetrakls(1-methyleth-yl)-1,3-dislloxanediyl]-beta-D-erythro-pentofuranosyl]-2(1H)-pyridone (28), (1.2g, 0.0015 mol), vinyl tri-n-butyltin (O.8 mL, 0.0027 mol) and palladium chloride bistriphenylphosphlne (0.lg) in dry aceto-nitrile (20 mL) ls heated at 60~C for 16 hours, under argon atmosphere. The solutlon is concentrated under reduced pressure and excess vinyl tri-n-butyltin ls removed by repeated (3 times) distillation with DMF.
The resulting viscous oil is dissolved in ethylace~ate (15 mL) filtered through celite and the flltrate concentrated to give a syrupy residue. This residue is purified by flash chromatography using EtOAc Hexane (1:4) as the eluent. The fractlons containing the desired product (visualized under W ) are combined, concentrated under reduced pressure and the resulting residue dried under high vacuum in a desicator to give 0.85g (82%) of 4-O-diphenylcarbamoyl-3-ethenyl-1 [2-deoxy-3,5-0-[1,1,3, 3-tetrakis(l-methyl ethyl)-1,3-disiloxanediyl]-beta-D-ery~hro-pentofuranosyl]-2(1H)-pyridone (36), as a pale yellow amorphous material.
Rf = 0.63 (EtOAc-Hexane 1~ HNMR 1300 MHz, CDCl3) ~:7.79 (d, J = 7.69Hz, 1, ~6), 7.42 - 7.2 (m, 10, aromatic), 6.45 (dxd, J = 17.7~z & 11.85~z, 1, CH=CH2) 6.35 (d, J = 7.69Hz, 1, H5), 6.2 (m, 2, Hl' x CH=CH2), 5.38 (dxd, J = 2.5 x 11.8~2)i 4.39 (m, 1, H3'), 4.2 (m, 2, ~5' x 5"), 3.8 (m, 1, ~4'), 2.6 (m, 1, ~2'), 2.26 (m, 1, H2"), 1.15 o 0.85 (m, 28, O~Si-(i-Pr)2)2;
FAB mass spectrum (m/z) 697 (M+Li), 339 (~+Li-sugar);
,. H~FABMS observed (N~Li) 697.3321, calculated for C3,H50N2O7 Si2Li 697.3317.
2 ~ 2 ~
-64- 07-21(281)A
To a chllled (ice bath) solution of 4-O-di-phenylcarbamoyl-3-ethenyl-1[2-deoxy-3,5-O-[1,1,3,3-tetrakls (l-methylethyl)-1,3-disiloxanediyl]-beta-D-erythro~ pentofuranosyl]-2(1H)-pyridone (36) (0.39, 0.00063 mol) in acetonitrile (5 mL) is added tetraethyl-ammonium fluoride (O.035g, 0.0002 mol). The reaction mixture is stirred at 0C for 30 minutes and at room temperature for 1 hour. During this period, desily-ation is complete as revealed by TLC of the reaction mixture. The solution is concentrated under reduced pressure and the residue purified by flash chroma-tography using CH2Cl2-MeOH (5%) containing 0.5% Et3N
to give 0.18g (94%) of 4 0-diphenylcarbamoyl-3-ethenyl-1(2-deoxy-~eta-D-erythro-pentofuranosyl)-2(lH)-pyridone (37). Rf = 0.26 (CH2Cl2-MeOH, 5%); lHNMR (300MHz, DMSO-d6) ~: 7.98 (d, J = 7.70Hz, H6), 7.55 - 7.2 ~m, 10, aromatic), 6.5 (m, 2, H5 & CH-C~2), 6.49 (d, J =
7.7Hz, 1, H5), 6.39 (dxd, J = 6.4Hz, 1, Hl'), 5.35 (dxd, 1, J = 3.01Hz, ll.9Hz), 5.24 (d, 1, J = 4.25 Hz, 2'-OH), 5.04 (t, 1, J = 5.14 Hz, 5'-OH), 4.23 (m, 1, H3'), 3.86 ~m, 1, H4'), 3.62 (m, 2, H5' x 5"), 2.35 (m, 1, H2'), 2.00 (m, 1, H2"), FAB mass spectrum (m/z) 4.49 (M~H), 333; HRF~BMS observed (M+Li) 455.1795 calculated for C2sH24N~o6Li 455.1794.
A mlxture of 4-_-diphenylcarbamoyl-3-ethe-nyl-1~2-deoxy-beta-D-erythro-pentofuranosyl)-2~lH)-pyri-done (37), (0.lg, 0.00022 mol), dimethoxytritylchloride (O.llg, 0.00032 mol) and diisopropylethylamine (O.08 mL, 0.00046 mol) in anhydrous pyridine ~3 mL) is stirred for 24 hours at xoo~ temperature under argon atmosphere. Pyridine is distilled off under reduced pressure and this residue purified by flach chroma-` tography using EtOAc-~exane (1:1) containing 0.5%
Et3N. Fractions containing the desired product (visualized under W lamp) are combined, concentrated 2~2~2 -65- 07-21(281)A
and this residue dried in a dessicator under high vacuum to afford 4-O-diphenylcarbamoyl-3-ethenyl-1-[5-O ~bis(4-methoxyphenyl)phenylmethyl]-2-deoxy-beta-D-ery~hro-pentofuranosyl]-2(lH)-pyridone (38), (0.13g, 78%) as white amorphous material. Rf 0.6 [CH2Cl2-MeOH (5%)]. 1HNMR (DMSO-d6) 7.8? (d, J =
7.7Hz, 1, H6), 7.46 - 6.8 (m, 23, aromatic) 6.5 (dxd, 1, -CH=CH2), 6.3 (m, 3, Hl', H5' x CH=CH2), 5.35 (m, 2, 2'-OH x -CH=CH2), 4.27 (m, 1, H3'), 3.99 (m, 1, H4'), 3.72 Is, 6, 2x-OCH3), 3.2~ (m, 2, H5' x H5"), 2.4 (m, 1, H2'), 2.05 ~m, 1, H2"); FAB mass spectrum (m/z): 757 (M+Li), 453.
To an ice cooled solution of 4-O-diphenyl-carbamoyl-3-ethenyl-1-[5-O-[bis(4-methoxyphenyl)phenyl-methyl]-2-deoxy-be~a-D-erythro-pentofuranosyl]-2(1H)-pyridone (38), (O.llg, 0.00015 mol) in dry dichloro-methane (5 mL) containing diisopropylamine (O.05 mL, 0.00029 mol), is added dropwise a solution of N,N,-diisopropylamine-~-cyanoethoxychlorophosphine (0.052g, 0.0002 mol) in dichloromethane (1 mL). The resulting mixture is stirred at 0C for 1 hour and at room temperature for 3 hours. TLC (EtOAC) revealed complete conversion to the product. The reaction mixture is concentrated under reduced pressure, the residue is dissolved in ethyl acetate (10 mL) and washed with 10%
NaHCO~ (2 x 10 mL) followed by water (2 x 10 mL).
The organic layer is dried (Na~S04) and concentrated under reduced pressure to give a syrupy material.
This syrupy material is purified by flash chromato-graphy using EtOAc containing 0.5% Et3N. The frac-tions containing 4-O-diphenylcarbamoyl-3-e~henyl-1-[5-O-[bis(4-methoxyphenyl~phenylmethyl]-3-O-~bis(l-methylethyl) amino] ~2~cyanoethoxy~phosphino]-2-de-oxy-beta-D-erythro-pentofuranosyl]-2(1~)-pyridone 2~2~
-66- 07-21(281)A
(39) are pooled, concentrated ~nd dried in a desicator under vacuum to afford (0.13g, 87%) as a white amorphous material. lHNMR (CDCl3) ~:7.93 x 7.8 (2d, J = 7.7Hz, 1, H6), 17.43 - 6.8 (m, 23, aromatic~ 6.55 - 6.3 (m, S 2, H1' x CH=CH2), 6.25 (m, 2, HS' x CH=CH2), 5.35 (m, 1, -CH=CH~), 4.50 (m, 1, H3'), 4.20 ~m, 1, H4'), 3.76 (s, 6, 2x-OCH3), 3.45 [m, 4, HS' x 5" & -N-lCH(CH3)2]2, 2.60 x 2.40 (2 (-5, -CH2-CH2CN x H2'), 2.25 (m, 1, H2"), 1.35 - 1.0 lm, 12,-N-[CH(CH3)2]2; FAB mass spectrum (m/z): 957 (M+Li), 733 & 653.
EXAMpr~ g To 5.0 ~m (7.3 x 10- 3 mole) 4-0-diphenylcar-bamoyl-l-[3,5-0-[1,1,3,3-tetrakis(l-methyl-ethyl)-1,3-disiloxanediyl]-beta-D-erythro-pentofurano-syl]-2(1H)-pyridone (4), in 30 mLs dry DMF can be added 0.24 gm (1 x 10- 2 mole) NaH while stirring.
To this is added 1.26 gm (1 x 10-2 mole) dimethyl sulfate. The reaction mixture is kept under argon until complete and then volatiles are removed under reduced pressure. The residue is taken up in EtOAc and the organic layer washed with saturated aqueous NaHCO3 and distilled water, then dried (MgSO4), filtered and solvent removed. Purification by silica gel chromatography can be used to give the desired product, 4-O-diphenylcar~amoyl~ [3,5-0-[1,1,3,3-tetra-kis(l-methylethyl)-1,3-disiloxanediyl]-2'-O-methyl]-~e~a-D-ery~hro-pentofuranosyl ]-2~lH)-pyridone (40).
To 3.1 gm (4.5 x 10-3 mole) 4-O-diphenylcarbamoyl-1-[~3, 5-0-[1,1,3,3-tetrakis(l-methylethyl)-1,3-disiloxanediyl]
-2-0-methyl ] -~eta-D-erythro-pentofuranosyl ] -2 ( lH) -pyridone (40) in 30 mL CH3CN at 0C is added 9.65- gm ( 2 . O x 10- 3 mole) tetraethylammonium fluoride trihydrate (Aldrich, Milwaukee, WI) while stirring. The reaction -67- 07-21(281)A
mixture is kept at OC for 30 minutes, warmed to room temperature and kept at room temperature for 30 mlnutes. Solvent is removed under reduced pressure and the desired product can be isolated by silica gel flash chromatography to give 4-O-diphenylcarbamoyl-l-(2-O-methyl -be ta-D-erythro-pentofuranosyl) 2(lH)-pyridone (41). To 1.4 gm (3.2 x 10-~ mole) 4-O-diphenylcarbamoyl-l-(2'-O-methyl-~eta-D-erythro-pentofuranosyl)-2(1~)-pyridone (41), in 15 mL dry pyridine is added 1.3 gm (3.95 x 10- 3 mole) diiso-propylethylamine and 30 mg 4-N,N-dimethylamino-pyridine (2.5 x 10-4 mole) and 1.2 gm (3.5 x 10-3 mole) dimethoxy tritylchloride. The reaction mixture is stirred at room temperature for 4 hours or until the reaction is substantially complete. Vola-tiles are removed and excess pyridine is removed by repeated (3 x 20mL) distillation of toluene under reduced pressure. The residue is purified by silica gel chromatography and 4-O-diphenylcarbamoyl-1-[5-O-~bis-(4-methoxyphenyl)phenylmethyl]-2'-O-methyl-beta-D-erytAro-pentofuranosyl~-2(lH)-pyridone (42), can be obtained. To 0.55 gm (7.3 x 10- 4 mole) 4-O-diphenyl-carbamoyl-1-[5'-O- fbis- (4-methoxyphenyl)phenylmethyl]-2'-O-methyl-beta-D-erythro pentofuranosyl]-2(lH~-pyridone (42), in C~2C12 is added 0.45 mL (2.5 x 10-3 mole) N, N-diisopropylethylamine. To this is added, dropwise over about 15 minutes 0.19 gm (8.0 x 10-~ mole~ ~-cyanoethyloxy-N,N-diisopropyl chloro-phosphine (American Bio~etics, Inc., ~ayward, CA) in 5 mL CR2Cl2. The reaction mixture is stirred and kept under an argon atmosphere at room temperature for an additional hour. The solution is concentrated to .~ dryness under vacuum usi~g a ro~ary evaporator and the residue taken up in EtOAc, washed with saturated aqueous NaHCO3 (3 x 20 mL) and distilled water, then dried (MgSO4~. The solution is concentrated to dryness and dried under vacuum to give a product 2 ~ 8 2 -6~- 07-21(281)A
which can be isolated by taklng lt up ln EtOAc and precipitating with hexane to give substantially pure 4-O-diphenylcarbamoyl-l-(5-O-~bis (4-methoxy phenyl) phenylmethyl]-3-O-[bis~l-methylethyl)amino (2-cyano-ethoxy)phosphino~-2-O-methyl-beta-D-eryt~ro~pento-furanosyl]-2(lH)-pyridone (43).
A mixture of 4-O-diphenylcarbamoyl-1-[3,5-O-[1,1,3,3-tetrakls(1-methylethyl)-1,3-disiloxanediyl]-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone (4), (6.8 gm, 1 x 10-2 mole), N-iodosuccinimide (2.7 gm, 1.2 x 10-2 mole) in dry DMF containing dichloroacetic acid (0.36 gm, 2.8 x 10- 3 mole) is heated at 60C
under argon atmosphere until the reaction is complete.
The resulting mixture is concentrated in vacuo and the residue can be purified by silica gel flash chroma-tography to obtain as a major product 4-O diphenylcar-bamoyl-3 iodo-l-[3,5-O-[1,1,3,3-tetrakis (l-methyl-ethyl)-1,3-disiloxanediyl]-beta-D-erythro-pento-furanosyl ]-2(1H)-pyridone (44) and as a minor product 4-O-diphenylcarbamoyl-5-iodo-1~[3,5-O-[1,1,3, 3-tetrakis~1-methylethyl)-1,3-disiloxanediyl]-~eta-D-erythro-pentofuranosyl]-2(lH)-pyridone (45).
A mixture of 3.7 gm ~4.6 x 10-3 mole) of 4-O-diphenylcarbamoyl-3-iodo-1-[3,5-O-[1,1,3,3-tetra-kis(l-methylethyl)-1,3-disiloxanediyl] -beta-D-erythro-pentofuranosyl]-2(lH)-pyridone (44), and 0.88 gms (2.8 x 10-3 mole) tetrabutylammonium fluoride tri-hydrate in acetoni~rile is stirred a~ 0C for 30 minutes followed by stirring at room temperature for 30 minutes. The solution is concentrated in vacuo and 2~2~2 -69- 07-21(281)A
the residue can be purified by silica gel flash chromatography to obtain 4-O-diphenylcarbamoyl-3-iodo-l-beta-D-erythro-pentofuranosyl-2(1H)-pyridone (12).
In a similar fashion, the 5-iodo isomer, 4-O-diphenylcarbamoyl-5-iodo-1-beta-D-erythro-pento-furanosyl-2 (lH)-pyridone (13) can be obtained from 4~0-diphenylcarbamoyl-5-iodo 1-[3,5-0-[1,1,3,3-tetrakis ~l-methylethyl)-1,3-dlsiloxanediyl]-beta-D-erythro-pentofuranosyl]-2(lH~-pyridone (45).
EXAMPLE l?
All DNA oligonucleotides of the invention are synthesized on an ABI 380B synthesizer (Applied Biosystems, Inc., Foster City, CA) on a 1 micromole scale. Purification is achieved by polyacrylamide gel electrophoresis (PAGE). Oligonucleotides are labelled at their S' end by transfer of 32p phosphate from g~mma-32P-ATP using T4 polynucleotide kinase as described in T. Maniatis, E. F. Fritsch, and J.
Sambrook, "Molecular Cloning", Cold Spring Harbor Laboratory, (1982). Radiolabelled probe is separated from the reaction mixture by Sephadex G-50 chromato-graph (eluted with water). About 1 x 106 cpm/filter of labelled probe is included in the hybridization solution. After overnight hybridization, blots are washed in 6 x SSC (Standard Saline Citrate) at the appropriate temperature for 5 minutes. Autoradiograms are obtained using Kodak XAR-5 film with one intensi-fying screen (24 hours).
The 0.8 Kb DNA fragment encoding the small subunit of soybean ribulose-l, 5-bisphosphate (RuBP) carboxylase (S.C. Berry-Low et al., J. Mol. and_A~lied 2~2~
-70- 07-21(281)A
Gen., 1:483 (1982)) is obtained from plasmid pSRS 0.8 using the following conditlons. 6.8 uL (400 ug) of pSRS o.8 plasmid solution ls combined with 8 ~L of lOX
restriction buffer [SO mM NaCl, 10 mM tris Cl(pH=
7.5), 10 mM MgCl~, 1 mM dithiothreitol], 63 ~L deionized water, and 2 ~L EcoRI (20 units/~L) restriction enzyme. To obtain the homologous wheat RuBP fragment (R. Broglie, Biotechnology, 1:55 (1983)) 3 uL pw9 (1.3 mg/mL) plasmid is combined with 8 ~L of lOX restriction buffer, 67 ~L deionized water and 2 ~L PST (20 units/~L) restriction enzyme. Both reactions are incubated at 37C for l hour and subsequently heated to 70C for 5 minutes to denature the enzymes. The DNA in 20 ~L of each reaction mixture is separated by electrophoresis on a 1.2% agarose gel using lX TBE (Tris, Borate, EDTA) as buffer. After electrophoresis at 30V, overnight, the gel is washed 2X with 25PmM HC1 for 15 minutes, then washed (2X water) and the DNA denatured by soaking the gel in 1.5M NaCl, 0.5M NaOH (2 X 15 minutes). The denatured gel is washed twice with water and neutralized with 3.OM sodium chloride (NaCl), 0.5M tris HCl pH = 7.4 (2 X 20 mins). The DNA
fragments are transferred onto nitrocellulose paper according to Southern (E.M. Southern, J. Mol. Bio., 98:503 (1975)). Panels are prehybridized in 6 X SSC, lO X Denhardt's solution (0.2% BSA. 0.2% Ficell, and 0.2% polyvinylpyrolide) containing 100 ~g/mL yea~t tRNA at room temperature for 1-2 hours. Hybridization is carried out at 52-54C for 16-18 hours.
, A nitrocellulose filter is prepared with both soy and wheat RuBP carboxylase DNA in substantial accordance with the teaching of Example 13 and a single -71- 07-21(281)A
stranded oligonucleotide probe complementary to a target sequence on the 0.8 kb EcoRI fragment of soy RuBP is synthesi7ed. The target portion of the approxlmate 800bp DNA fragment has the sequence:
*
3'-CCGTCGAAGGTGTACCAGGT-5' The 20-base probe has the se~uence:
5'-GGCAGCTTCCACATGGTCCA-3' The sequence for wheat RuBP carboxylase DNA is the same except that an A is substltuted for C in the starred position. The effects of the G-A mismatch pairing when soy probe is hybridized with wheat target is determined by comparing the melting temperatures (Tm), the temperature at which 50% of the probe dissociates. The melting temperature of soy probe without 2'-deoxy-3-deazauridine hybridized to soy RuBP
DNA and soy probe hybridized to wheat RuBP DNA where there is a single base mismatch present is measured.
The hybridization panel is pretreated with about 15 mLs of 6 x SSC buffer (0.9 M NaCl, 0.09 M
sodium citrate), lOX Denhardt's solution and 110 ~g/mL yeast tRNA for about 3 hours at 5~C.
Normal oligonucleotide probes containing native bases and probes in which 2'-deoxy-3-deazauri-dine replaces cytidine in ~he nonmal seguence axe labelled at the 5' end with y_32p to a specific activity of about 3.9 x 106 cpm/pmole. About 0.15 pmole of labelled soy probe i added to fre~h hybrid~
ization ~uffer (10 mL~ and the panel is heated in the buffer at about 54C for 12 hour~. The panel is washed at 58C in 6 x SSC for 5 minutes and a~ auto-,'i:
~2~ 8~
72- 07-21(281)A
radiogram exposure (24 hours) obtained using a Kodak XAR-5 film with one intensifying screen. Soy RuBP DNA
is stron~ly exposed on the autoradiogram while wheat RuBP DNA is only weakly detectable. The panel is then washed at 60C for S mlnutes. The soy DNA signal remains strong and no wheat DNA signal is detected.
The panel is then washed at 63C and the soy signal lS
still visible but only to the extent of about half that visible at 60C. Thus, the melting temperature (Tm) of the soy RuBP probe and soy target DNA is about 63C and the Tm for the soy RuBP complement and wheat target DNA is estimated to be about 58C. The Tm for the probes listed in Example 18, Table III, were determined in a similar manner.
A soy RuBP probe is constructed in substan-tial accordance with the teaching of Example 14, in-cluding the same sequence, however, the cytidine at position 9 from the 5' end is replaced with 2'-deoxy-3-deazauridine. This probe is 5' labelled in substan-tial accordance with the teaching of Example 12 and has the following sequence:
5'-GGCAGCTTYCACATGGTCCA-3' `!' Y = 2'-deoxy-3-deazauridine ~ freshly prepared nitrocellulose panel is employed and the 2'-deo~y3-deazauridine containing probe is allowed to hybridiæe, and then washed in sub-stantial accordance with the teaching of Example 14.
.. Washes are conducted at 60, 70, 80 and 90C. The soy specific probes hybridize only to the soy RuBP DNA
and are distinctly visible throughout the wash cycle 2 ~
-73- 07-21(281)A
with wheat DNA weakly vlsible. At 90C the intensity of the 3NA band in the autoradiogram was about 25% of the intensity observed at 70C. (Intensity at 70C
is about equal to 60C).
In a separate experiment the soy probe contalning 2'-deoxy-3-deazauridine is resynthesized, labelled with 32p and a freshly prepared nitrocellu-lose panel is probed. Washes are conducted at temp-eratures of 64~, 66 and 70C. Again excellent selectivity and detectability are maintained. A 5 minute wash at 100C in 0.1 x SSC is accomplished with about one-half of the original (64C) concentration of probe remaining hybridized to the soy RuBP DNA.
The oligonucleotide probe complemen~ary to wheat RuBP DNA is synthesized in substantial accor-dance with the teaching of Example 12, with 2'-deoxy-3-deazauridine replacing cytidine at position 9 from the 5' end. The 20-base probe has the following sequence:
5'-GGC AGC TTY CAC ATA GTC CA-3' Y = 2'-deoxy-3-deazauridine A freshly prepared nitrocellulose panel is treated with the 2'-deoxy-3-deazauridine containing wheat probe in substan~ial accordance with the teach-ing o~ E~ample 13. Washes are conducted at 70, 80 and 90C. In this instance, the wheat probe detects only the wheat target D~A. At 90C about 50% of the original signal remains on ~he autoradiogram.
.
EXAMPLE l?
Three 17-base pair probes to the gene for Bacillus thuringiensis toxin (B.t.t.) are synthesized ~ ~ 2 ~
_74_ 07-21(281)A
ln substantial accordance with the teaching of Example 12. One wlth 2'-deoxy-3-deazauridine replacing cytidine at pOSltion 8 from the 5' end (Btt-8), another with 2'-deoxy-3-deazauridlne replacing cyti-dine at position 12 from the 5' end (Btt-12) and the normal probe without 2'-deoxy-3-deazauridine (Btt-N).
The probes have the following sequences:
5'-ATG M T CCG AAC AAT CG-3' (Btt-N) 5'-ATG AAT CYG AAC M T CG-3' Y = 2'-deoxy-3-deazauridine ~Btt-8) 5'- ATG AAT CCG AAY AAT CG-3' Y = 2'-deoxy-3-deazauridine (Btt-12) These probes are 32P-labeled in substantial accordance with the teaching of Example 12 and used to identify E. coli colonies containing the Btt gene.
Seventeen colonies are screened. Three of ~he ~. coli colonies contain the correct Btt gene, the remainder contain only a portion of the Btt gene and do not contain the portion to which the probe is designed to hybridize. Three duplicate colony filters are prepared in which the DNA from the E. coli colonies growing in a culture dish is transferred to the filter (ICN Bio-chemicals, "Protocols for DN~ and RNA Transfer, DNA
Electrotransfer and Protein Transfer to Biotrans~
Nylon Membran~s," 1985, Pall Corporation). One filter is probed with the Btt-N probe; ~he ~econd filter is probed with Btt-8 and the third probed with Btt-12.
The hybridization is carried out at about 40-42C for 20 hours. Each filter is subsequently washed in 1 X
SSC 1% SDS (sodium dodecyl sulfate) for 3 minutes at ~Ja~4l8~, 75- 07-21(281)A
45C. This wash is repeated and the filters are exposed to x-ray film for 4 hours. All three probes hybridize to the same colonles, all of which contain the correct Btt gene.
The filters are then washed at 65C for 5 minutes ln 1 X SSC 1% SDS. The normal probe, Btt-N, lS completely removed at this temperature, however, both Btt-8 and Btt-12 containing 2'-deoxy-3-deazauri-dine remain hybridized to the colonles containlng the Btt gene. The two 2'-deoxy-3-deazauridine containing probe filters are washed for 1 hour in 50% formamide containing about 10 mM sodium phosphate (Na~PO4), pH
6.5 at 65C. Both Btt-8 and Btt-12 are still hybri-dized after treatment and easily detected by auto-radiography.
Single-stranded oligonucleotide probes are synthesized in accordance with Example 12, without 2'-deoxy-3-deazauridine and with 2'-deoxy-3-deazauri-dine (indicated by "Y") substltuted for a cytidine.
The probes are hybrldized with complementary target DNA, and the corresponding melting temperatures are compared as ~hown in Table III. The hybrid molecules generated with probes containing 2'-deo~y-3-deazauri-dine have considerably higher apparent melting tempera-tures than hybrid molecules containing probes without 2'-deoxy-3-deazauridine, Table III lists probes of the invention complementary to soybean ribulose bis-phosphate (RuBP) carbo~ylase small subunit DNA (soy), wheat RuBP carboxylase small subuni~ DNA (wheat), the gene for Bacillus ~hurin~iensis toxin ~ ~t) and human ,. cytomegalovirus gene (HCV). Control probes (not shown) have the same sequences as the probes shown in Table III, however, control probes have a cytidine in place of "Y".
2~2~2 -76~ 07-21(281~A
_able III
Probe Sequence Tm*
Tm* Probes Probe Control of the Target Probe Sequence (5'-3') Length HT~ Probes Invention soy AGCT TCC AYA TGG TCC 16 Mer 40 56 >90C
soy AGCT TCC AYA TGG TCC A 17 mer 45 ~58 >90C
soy GCA GCT TYC ACA TGG TCC l8 mer 45 60 >90C
soy GGCA GCT TYC ACA TGG TCC A 20 mer 52 63 >90C
wheat GGC AGC TTY CAC ATA GTC CA 20 mer 52 ~65 >90C
Bt~ ATG M T CYG AAC AAT CG 17 mer 4l ~65 >90C
Btt ATG AAT CCG M Y AAT CG 17 mer 4l <65 >90C
HCV TAG CGG CGA CGY ACG TAC AC 20 mer 52 ND >90C
HC~ CTC TAG GYT GTC GGC CAG CC 20 mer 52 ND >90C .
soy TAG GCA GCT TCC AYA TGG TCC , AGT AGC 27 mer 40 76 >gOC
Y = 2'-deoxy-3-deazauridine * = Temperature at which approximately l/2 the signal of labeled probe remains *~ = Hybridization temperature C
2~32~
-77- 07-21(281)A
.
A soy RuBP probe is constructed in substan-tial accordance with the teaching of Example 14, including the same probe sequence, however, the cytidine at position 19 ~from the 5'-end) is replaced with 2'-deoxy~3-deazauridine:
5'-GGC AGC TTC CAC ATG GTC YA - 3' Y = 2'-deoxy~3-deazauridine This probe is 32-P labelled in substantial accordance with the teaching of Example 12 and employed in a Southern experiment using a freshly prepared nitro-cellulose blot in substantial accordance with Example 14. The extent of hybridizatlon of the probe is about equal to both soy and wheat DNA at 60C. Other washes are conducted at 63C, 70C and 83C. After the 83C
wash, about 15% of the concentration distinguishable at 63C still remains distinguishable for both soy and wheat RuBP DNA seguences.
A soy RuBP probe is constructed in substan-tial accordance wi~h the teaching of Example 14, including the same seguence, however, the cytidine at position 9 (from the 5' end) is replaced with 4-O-di-phenylcarbamoyl-3-iodo-1-(2-deoxy-beta-D-erythro-pento-furanosyl)-2(1~)-pyridone (30), which becomes 3-iodo-4-hydroxy-l-(2-deoxy-~eta-D-ery~hro-pentofuranosyl)-2(1~) pyridone, after depxotection. This probe is 32-P
labelled in substantial accordance with the teaching of Example 12 and has the following sequence:
.
S ' -GGC AGC T~X CAC ATG GTC CA - 3 ' X = 3-iodo-4-hydroxy-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-2(lH)-pyridone.
2 ~ 2 -78 07~ ~(2~ ~A) ' A freshly prepared nitrocellulose panel is employed and the 3-iodo-4-hydroxy-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-2(lH)-pyridone (12) containing probe allowed to hybridize in substantial accordance with the teaching of Example 12, however, ln this case 6 picomole of 32-P radlolabelled probe is employed.
Washes are accomplished in substantial accordance with the teaching of Example 21. At 80C about 50% of the concentration visible at 60C stlll remains visible.
Therefore, the apparent Tm (at which 50% of the signal remains) is about 80C.
A soy RuBP probe is constructed in substan-tial accordance with Example 20, however, X = 5-iodo-4-hydroxy-1-(2-deoxy-beta-D-eryt~ro-pentofuranosyl)-2 (lH)-pyridone, which is 4-0-diphenylcarbamoyl-5-iodo-1-(2-deoxy-beta~D-erythro-pentofuranosyl)-2(lH)-pyridone (31) prior to deprotection. A freshly prepared nitro-cellulose panel is employed and hybridization carried out at 52C wi~h 3 picomoles of 32-P labelled probe.
Washes are conducted at 58, 63, 70, 80 and 100C
in 6 x SSC for 5 minutes. At 100C approximately 50%
of the signal visible at 63C (the Tm of the probe without 5-iodo-4-hydroxy-1-(2-deoxy-~eta D~e~y~hro-pentofuranosyl)-2 (lH)-pyridone (13)) still remains visible. Therefore, the apparent Tm (at which 50% of the signal remains) is about 100C. Both soy and wheat RuBP DN~ are visible to an equal extent. When the hybridization is carried out at 37C, and the panel washed at 80C about 25% of ~he concentration visible at 60C still remains visible.
. .
A soy RuBP probe is constructed in substan-tial accordance with the teaching of Example 20, -79- 07-21(281)A
however, X = 4-O-dlphenylcarbamoyl-3-ethenyl~ 2-deoxy-beta-D-erythro-pentofuranosyl)-2(lH)-pyridone (37).
A freshly prepared nitrocellulose panel is employed and hybridizatlon carried out at 53C with 32-P
labelled probe. In this case, the autoradlographlc signal for both soy and wheat sequences were equally intense after washing at 63C for 5 minutes. Further washes were conducted at 69, 80 and 100C. About 75% of the signal visible at 63C (the Tm of the probe without 4-O-diphenylcarbamoyl-3-ethenyl-1 (2-deoxy-beta-D-erythro-pentofuranosyl)-2(1H)-pyridone (37)) remains at 80C for both soy and wheat D~A.
About 10% of the signal visible at 63C remains after the 100C wash. Therefore, the apparent Tm (at which 50% of the signal remains) is about 80C.
A soy RuBP probe is constructed in substan-tial accordance with the teaching of Example 14, however, the probe (Soy 27) is extended to include 27 bases of soy sequence:
5' TAG GCA GCT TCC ACA TGG TCC AGT AGC 3' (Soy 27) and the cytidine at position 14 from the 5' end is replaced with 2'-deoxy -3-deazauridine:
5' TAG GCA GCT TCC AYA TGG TCC AGT AGC 3' Y = 2'-deoxy-3-deazauridine.
The probe is 32P-labelled and hybridized to a freshly prepared panel in substantial accord~nce with Example ,. 14, except the hybridization temperature is 4aC.
.
'~ ~s~ 2 -80- 07-21(281)A
Washes are carried out at 66, 75, 79, 90 and 100C. Both soy and wheat DNA are visualized to an equal extent throughout the wash cycle. At 90C about 25% of the DNA visible at 79C still remains visible.
S At 100C the slgnal is weak but still easily discern-ible.
Although the invention has been described with respect to specific modifications, the details thereof are not to be construed as limitations, for it will be apparent that various equivalents, changes and modifications may be resorted to without departing from the spirit and scope thereof and it is understood that such equivalent embodiments are to be included herein.
TABLE 1.
OC~O)N(Ph)2 H,CO ~ O
(I Pr)2N ~OCI12CH2CN
4-O-diphenylcarbamoyl-l-~ f s-o- [bls(4-methoxyphenyl) phenylmethyl]-3-O-[bis(l-methylethyl)amlno~-(2-cyanoethoxy)phosphino]-2-deoxy-beta-D-erythro-pento-S furanosyl]-2(1~)-pyridone (9) OC~O)N(Phk H~CO 0~ o ~
~I Pr)2N ~ OCH3 '` !9 4-O-diphenylcarbamoyl-1-15-O-[bis(4-methoxyphenyl) phenylmethyl]-3-O-[bis(l-methylethyl~amino]-methoxy-phosphino]-2-deoxy-beta-~-erythro-pentofuranosyl]-2(1H)-pyridone (10~
, - , ` , .
2~2~2 _9- 07-21~281)A
OC(o)N(ph)2 '~3C0 ~ _yyf ~3 O OSI~I-Pr)3 ~I Pr)2N OCH2CH2CN
Ll 4-O-diphenylcarbamoyl-1-[[[5-O-[(4-methoxyphenyl) diphenylmethyl]-3-O-[bis(l-methylethyl)amino~
(2-cyanoethyl)phosphino]-2-O-tris(l-methylethyl)silyl]
-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone (21) OC(O)N(PI~)2 II~c2~0 ~
/
oO - Sl~ u) IU~
~P
t~Pr)2N OCH2CH2CN
, 4-O-diphenylcarbamoyl-1-[[[5-0-[(4-methoxyphenyl) diphenylmethyl]-3-O-[bis(1-methylethyl)amino~(2-cyanoethyl)phosphino)-2-O-(l,l methylethyl) dimethylsilyl]-beta-D-erythro-pentofuranosyl]-2(1H)~
pyridone (22) ~2~2 -10- 07-21~281)A
OC(O)N(PI~)2 H,CO {~ O ~
O OSI(I Pr)3 (I P~)2N OCH,CH2CN
2~
4-O-diphenylcarbamoyl-3-iodo-1-[[[5-0-[(4-methoxyphenyl) diphenylmethyl]-3-0-[bis(l-methylethyl)amino](2-cyanoethyl) phosphino]-2-O-trls(l-methylethyl)sllyl]-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone ~23) o~(o)N~ph)2 ~3 0~
H,CO~ ~
O OSI(I-Pr)3 ~P~)2N OCH2CH2CN
.- ~
4-O-diphenylcarbamoyl-5-iodo-1-[[[S-O-[(~-methoxyphenyl) diphenylmethyl]-3-O-[bis(l-methylethyl3amino]t2-cyanoethyl) phosphino]-2-O-tris(l-methylethyl)silyl]-beta-D-eryt~ro-pentofuranosyl]-2(1~) pyridone (24) :
.
2~2~
~ 07-21~281)A
oC~O)N~Ph)2 H3CO ~ O
H--P=O
O HN(Et)3-4-O-diphenylcarbamoyl-l-[ E 5 -O- [bis(4-methoxyphenyl) phenylmethyl]-3-0- ~ hydroxyphosphinyl ) -2-deoxy]-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone trl-ethylammonium salt (25).
Oc(o)N(ph)a H~CO ~ O ~
o OS~ Pr)3 H--P =O
~ HN(E~)3~
4-O-diphenylcarbamoyl-1-[[5-0-[(4-methox~phenyl) diphenylmethyl]-3-O-(hydroxyphosphinyl)-2-0-tris-(l-methylethyl)silyl]-bet a-D-erythro -pentofuranosyl]-2(lH~-pyridone triethylammonium salt (26~
.
.
æ~2~-8~
-12- 07-21(281)~
OC~O)N~Ph)2 ~3CO ~ o ~
O - Sl ~ U) \ Me H--P = o O HN~Et)~-~
4-O-diphenylcarbamoyl-1-[[5-0-[(4-methoxyphenyl) diphenylmethyl]-3-O-(hydroxyphosphinyl)-2-O-(l,l-dlmethylethyl)dlmethylsllyl]-beta-D-erythro-pento-furanosyi]-2(lH)-pyrldone trlethylammonium salt (27 OC(O)N(Ph)2 _ N
~:0~ ~
(I Pr)2N OCHaCH2t:~J
s g-O-diphenylcarbamoyl-3-iodo-1-[S-O-tbis (4-methoxyphenyl)phenylmethyl]-[3~0-[bis(1-methylethyl)amino](2-cyanoethoxy)phosphino]-2-deoxy-~eta-D-erythro-pentofuranosyl]-2(lH)-pyridone ~34) 2~2~
-13- 07-21(281)A
oC~o)p~(ph~2 H3CO f ~ I
~,CO~O~
(I Pr)2N ocH2~l~2cN
4-O-diphenylcarbamoyl-5-iodo-1-[S-O-[bis(4-methoxyphenyl)phenylmethyl]-[3-O-[bis(l-methylethyl)amlno](2-cyanoethoxy)phosphino]-2-deoxy-beta-D-erythro~pentofuranosyl]-2tlH)-S pyridone ~35) OC(O)N~PI1)2 Ha~
H~CO~ o~/
(I Pr)2N C)CH2CH2CN
~2 ~
4-O-diphenylcarbamoyl-3-ethenyl-1-[5-O-[bis(4-methoxyphenyl)phenylme~hyl]-[3-0-[bis(1-methylethyl) amino](2-cyanoethoxy)phosphino]-2-deoxy-beta~D-erythro-pentofuranosyl]-2(1~)-pyridone (39) 2~)2~
-14- 07-21(281)A
oC(O)N(Ph~2 D,~3 N
HO ~
-0~1 ~7 4-O-diphenylcarbamoyl-3-ethenyl-1(2-deoxy-beta-D-erythro-pentofuranosyl)-2(lH)-pyrldone (37) OC(O)N(Ph)2 H3CO ~3 H3CO ~ ~
OCH, )2N OCH20H2CN
. 4~
4-O-diphenylcarbamoyl-l-[S-O-[bis(4-methoxyphenyl) phenylmethyl]-3-O-[bis(l-methylethyl)amino(2~
S cyanoethoxy)phosphino~-2-O-methyl-be~a-D-erythro-pentofuranosyl]-2(1~)-pyridone (43) , 2 -15- 07-21(281)A
Typically, the ratio of the extent of hybrldization of perfect and mlsmatched duplexes ls critically dependent upon the temperatures at which hybridization and washing are carried out. Such critical dependence on temperature leads to compromise between the higher temperatures requlred to achieve dissociation of mismatched duplexes and the lower temperatures needed to give the greatest degree of binding and, therefore, the highest sensitivity.
However, when hybridization takes place between a probe of the invention and a complementary nucleic acid sequence, the resulting hybrid molecule exhibits enhanced stabilization resulting in higher melting temperatures, sometimes 25C higher, or more, than a corresponding hybrid molecule formed without a probe of the invention. Therefore, after hybridization, uncomplexed probe and mismatched complexes will be washed off at higher stringency conditions, thereby virtually eliminating all background readings and improving the ease and accuracy of probe use.
In order to incorporate a nucleoside residue of the invention into a probe, in place of one or more cytidine, the nucleoside is converted to a phosphor-amidite derivative that is appropriately protected for DNA synthesis. See S. P. Adams et al., J. Am. Chem.
Soc., 105:661 (1983), L. J. McBride and M. H. Caruthers;
Tetrahedron Lett., 24:245 (19~3), and N. D. Sinha et al., Nucleic Acids Res., 12:4539 (1984). It i~
anticipated that all protecting groups will serve the same function of appropriately protecti~g the various groups of the nucleoside. Examples of appropriate protecting groups include triisopropylsilyloxy, t-butyl-2 ~ 2 ~
-16~ 07-21(281)A
dlmethylsilyloxy, methoxyl, dimethoxytrlphenylmethyl, triphenylmethyl (trltyl), monomethoxytriphenylmethyl, trimethoxytriphenylmethyl, plxyl, N,N-dllsopropylamlne, morpholino, N,N-diethylamlno, N,N-dimethylamine, methyl, ~-cyanoethyl, hydrogen phosphonate, dl-o-anisyl-l-napthylmethyl, and p-anisyl-l-napthylphenylbutyl.
An example of such a phosphoramidite deriva-tive is the 2'-deoxy-3-deazaurldine derivatlve, 4-O-di-phenylcarbamoyl-l-[[5-O-[bis(4-methoxyphenyl)phenylmeth-yl]-3-O-[bis(l-methylethyl)amino]-(2-cyanoethoxy)phos-phino]-2-deoxy-beta-D-erythro-pentofuranosyl] -2(lH)-pyridone (9). The nucleic acids that may be used for purposes of practicing the invention include both the naturally occurring sugar-phosphate backbones as well as modified backbones as generally illustrated by Miller and T'so, Ann. Reports Med. Chem., 23:295 (19~8) and Moran et al., Nuc. Acids Res., 14:5019 (1987). Likewise, the ribo~e forms of the nucleoside of the invention are also converted to the phosphorami-dite form with appropriate protection of the 2'hydroxyl group before being placed into a nucleic acid probe. Methods for incorporating the phosphoramidite into a nucleic acid strand include the use of solid phase, solution phase triesters and ~-phosphonate intermediates as generally illustrated by Froehler et al., Nuc. Acids Res., 14:5399 (lg86), Narang et al., Methods En~., 68:90 (1979) and Ogilvie, K. K. et al., Proc. Natl Acad. Sci. USA, 85:5764 (1988).
After making a probe of the invention, the probe may be used to detect a target sequence. The particular hybridization technique for locating a ~' target sequence is not essential to the inYention.
Typically, hybridization with a normal probe proceeds at about 5C to 10C below the melting temperature (Tm) of a normal phosphodiester probe. Usually, hybridi-2 ~
-17- 07-21(281)A
zation ls carried out in a buffered solution between about pH 6 to 8. Optimal hybrldlzatlon conditions may be determined empirically. Hybrldization for a probe of the invention is carried out in a similar manner.
An lmportant advantage of the probes of this invention is that the resulting hybrid molecules have significantly higher melting temperatures than corres-ponding hybrid molecules which contain no probes of the invention. This higher meltlng temperature permits higher wash temperatures than can be employed for probes without a nucleoside residue of the inven-tion, for example, 50-100C, preferably between about 65-85C. Higher temperature washing gives cleaner and faster results because mismatched probes are unable to withstand the higher wash temperatures and are removed by the wash solution.
In addition to hybridizing probes as de-scribed above, a probe can be hybridized to target nucleic acid in solution. Moreover, the probe or target can be immobilized on a solid support and hybridized with target or probe, whichever one choses.
Hybridizatlon conditions may have varying degrees of stringency. Stringency is affected by temperature, ~5 probe length, ionic strength and other related fac-tors. Nevertheless, by using a probe of the present invention, as long as hybridization is achieved, with any hybridization protocol employed, unmatched and mismatched probes may be removed under conditions that would normally also lead to the loss of the hybridized probe not containing a nucleoside residue of the invention. Probes will often hybridize to nucleic acid sequences that are similar to the target sequence but that are not of int rest. Hybridization of this 2 ~
-18- 07-21(281)A
type is responsible for much of the background en-countered when using normal DNA probes lacklng a nucleoside residue of the inventlon. There are a variety of methods for removing non-hybridized probes from the hybridizatlon process. Such methods include gel filtration, enzymatic digestion of all single stranded nucleic acids, and washings consisting of buffer rinses at increased temperatures.
Probe detection methods include Northern blots, Southern blots and dot blots. Actual detection of hybridized probes is accomplished by measuring the label on the hybridized probes after removal of all non-hybridized and mismatched probes. The more common radioactive labels of choice include phosphorus 32 (32p), tritium (3H), carbon-14 (14C), sulfur 35 (35S), and fluorescent labels, chemiluminescent labels, and labels recognized by antibodies which are themselves labelled may also be used. The particular label used is a matter of choice. The manner in which a label is bound to the probe will vary depending upon the nature of the label chosen. In addition, a variety of methods are well known for introducing and measuring the various labels. Such methods include detection by radioscintillation counting, spectroscopy, such as fluorescence and luminescencé, by immunological means and by chemical means.
A probe of the invention may be used in primer extension proc~dures or in screening genomic libraries. Target sequences can be on the order of several hundred to several thousand nucleotides. A
probe of the invention is capable of hybridizing to target sequences of great length. As lon3 as the probe and target sequence are capable of hybxidizing to form a stable duplex, there is no target sequence too long to which a probe of the invention c~n be de-signed to hybridize. Likewise, probe length is only 2 ~
-19- 07-21(281)A
limited by the ability to hybridize and form a duplex to a target sequence. A suitable length of probe is about 15 to 50 bases in length with a preferred length belng about lS to 25 bases ln length. Methods for identifying target sequences and for preparing probe regions are well known. The target sequences can be from any DNA or RNA sequence, either procaryote or eucaryote includlng bacteria, yeast, plants and animals, these include sequences of infectious micro-organism, virus, or organism including parasites,human or non-human (animal) DNA or RNA sequences such as sequences characteristic of a genetic abnormality or other condition, and sequences derived from genetic engineering experiments such as total mR*~A or random fragments of whole cell DNA. Nucleic acid sequences may be obtained from a variety of sources including blood, tissue and cell samples, as well as DNA from the same sources amplified by such means as the polymerase chain reaction (PCR). Toxin producing microorganisms like Escherichia, Vibrio, Yersivea, Rlebsiella and Salmonella may be identified also.
Specific species include Haemophilis ducrei, Vibris cholerae, and E. coli. Other agents of clinical importance include Chlamydia trachomatous, genital Herpes virus, norwalk agent, Rotavirus, Campylabacter jejuni, ~eisseria gonorrhea, Herpes simplex virus, Brucella abor~us, Haemophilis influenza, ~ycobacterium, tuberculosis, Pseudomonas pseudomallei, Salmonella typAi, Sa~monella typhimurium, Neisseria meningitidis, Epstein-Barr virus, and human papilloma virus. In addition, the target se~uence can be complementary to a nucleic acid sequence which is characteristic of a class of human or non-human pathogens, like all enteric bacilli or all chlamydia. The target sequence can be complementary to a nucleic acid sequence which ~a~ 2 -20- 07-21(281)A
is characteristic of a host cell or vector used in the manufacture of recombinant DNA products, such as to detect the presence of such DNA or RNA contamlnants ln the product. Such detection may be useful in both diagnostics and therapeutics.
In some instances of probe use, lt is desired to differentiate between a single nucleotide mismatch of the target sequence and probe. A probe of the invention can be designed so that hybridlzation with a target sequence resulting ln a single nucleo-tide mismatch will be removed, leaving only the desired target sequence and probe hybridized molecule.
Since a substantial number of human genetic diseases are caused by single point mutations, the probes of the present invention can be used to determine geno-types and aid in the dia~nosis of genetic dlseases, even prenatally.
Sickle cell anemia and ~l-antitrypsin deficiency are examples of diseases arising from single point mutations. The following probe use, based on the se~uence disclosure in Conner et al., Proc._Natl. Acad. S l., 80:278, 1983, illustrates how a probe containing a nucleoside of the invention, 4-hydroxy-1-(2 deoxy-beta-D-erythro-pentofuranosyl) -2(lH)-pyridone (2), commonly named 2'-deoxy-3-deazauridine or DdU (when Rl, R2 and R3 are hydrogen in the nucleoside residue formula) can be used to detect the presence of a normal ~-globin gene and the defective ~--globin gene responsible for sickle cell anemia. Adenosine, thymidine, cytidine and guanosine are designated A, T, C and G respectively. A segment . of the normal gene for ~-globin has the following se quence:
-21- 07-21(281)A
5' CT CCT GAG GAG AAG TCT GC 3' 3' GA GGA CTC CTC TTC AGA CG 5' therefore examples of probes for the normal ~-globin gene may have the following sequences:
3' GA GGA CTY CTC TTC AGA CG S' GA GGA YTC CTC TTC AGA CG
GA GGA CTC YTC TTC AGA CG
GA GGA CTC CTY TTC AGA CG
Y = 2'-deoxy-3-deazauridine A defective ~-globin gene responsible for sickle cell disease has a single base mutation in the starred position of the normal gene sequence where the A
(starred) is changed to a T. Therefore, examples of probes designed to be complementary to the gene for sickle cell anemia have the following sequences:
3' GA GGA CAC CTY TTC AGA CG 5' GA GGA YAC CTC TTC AGA CG
GA GGA CAY CTC TTC AGA CG
GA GGA CAC YTC TTC AGA CG
Y - 2'-deoxy-3-deazauridine Furthermore, based on the sequence disclosure in Kidd et al., Nature, 304:230, 1983, a probe of the present invention can be designed to hybridize to the comple-mentary sequence in the gene containing the normal .
2~
-22- 07-21(281)A
sequence for ~l-antitrypsin. Also, a probe of the inventlon containlng 2'-deoxy-3-deazauridine can be designed to hybridize to the corresponding gene sequence responsible for ~l-antitrypsin deficiency. A
segment of the normal gene for al-antitrypsin has the following sequence:
5' ACC ATC GAC GAG AAA GGG A 3' 3' TGG TAG CT2 CTC TTT CCC T 5' Probes of the invention that can be designed comple-mentary to the normal gene segment have the following sequences:
3' TGG TAG CTG CTY TTT CCC T S' TGG TAG YTG CTC TTT CCC T
TGG TAG CTG YTC TTT CCC T
Y = 2'-deoxy-3-deazauridine Probes of the invention that can be designed to be complementary to the sequence responsible for al-antitrypsin deficiency, where the starred G in the no~mal gene sequence is changed to an A, have the following sequences:
3' TGG TAG CTG TTY TTT CCC T 5 ' TGG TAG YTG TTC TTT CCC T
Y = 2'-deoxy-3-deazauridine Point mutations may arise from a single base . substitution, insertion, or deletion in the DNA
~2~2 -~3- 07-21(281)A
sequence of a single gene. Using a probe of the invention to detect such sequences would eliminate the dependencies on restrictlon enzyme recognition si~e alteratlons, which have a low probabllity of occur-rence for any given point mutation. In addition,therapeutic uses for probes of the lnvention include insertion into various cells under conditions which allow them to hybridize to messenger RNA's (mRNA).
The ability to hybridize to mRNA may allow for the inactivation of undesired mRNA's such as viral and oncogene mRNA. Not only can the probes of the inven-tion be used to hybridize to mRNA in vivo, but the probes of the present invention may be used to hybri-dize, and therefore, inactivate mRNA during i~ vitro processes like in vitro translation.
DESCRIPTIONS OF THE PREFERRED EMBODIMENTS
The following examples illustrate specific embodiments of the invention described herein. As would be apparent to skilled artisans, various changes and modifications are possible and are contemplated within the scope of the invention described.
The following structures listed in Table II
are useful in the synthesiG involved in the Examples.
7~ ~3 ~
-24- 07-21(2al~A
TABLE 2.
OH
.,~
HO
HO OH
4-hydroxy-l-beta-D-erythro-pentofuranosyl-2(lH)-pyridone ( 1 ) OH
0~
HO
~0 .~ ~
4-hydroxy-1-(2-deoxy-beta-D-erythro-pentofuranosyl) -2(lH)-pyridone (2) ~2~2 -25- 07-21(281)A
OC(O)N(Ph)2 ~0 ~
HO OH
4-0-diphenylcarbamoyl-1-beta-D-erythro-pentofuranosyl-2(lH)-pyridone (3) OC~O)N(Ph)2 (~)2S~
~ Si O OH
.- 4 4-0-diphenylcarbamoyl-1-[3,5-0-[1,1,3,3-tetrakis (l-methylethyl)-1,3-disiloxanediyl~-beta-D-erythro-S pentofuranosyl]-2(lH)~pyridone (4) 3 ~ 2 -26- 07-21(281~A
oC(O)N(Ph)2 0~
1~
(iPr)-SI--O OC(S)OPIl 4-0-diphenylcarbamoyl~ 2-0-(phenoxythloxomethyl) -3,5-0-11,1,3,3-tetrakis~1-methylethyl)-1,3-dlsiloxanediyl]-be~a-D-erythro-pentofuranosyl]-2 (lH)-pyridone (5) oC(o)N(phk 0~ ,.
1~
" (~)2SI--O
4-O-diphenylcarbamoyl-1-[2-deoxy-3,5~0-~1,1,3,3 tetrakis(l-methyle~hyl)-1,3-disiloxanediyl]-beta-D-erytAro-pentofuranosyl]-2(lH)-pyridone (6) ' 2 -27- 07-21(281)A
oC(O)N(Ph~2 ~
HO ~o~/
)J
HO
?
4-O-diphenylcarbamoyl-1-(2-deoxy-beta-D-erythro-pento-furanosyl)-2(1H)-pyridone (7) OC(O~N(Ph)7 H3CO ~ ~\~
HO
4-O-diphenylcarbamoyl-1-[5-O-[bis(4~methoxyphenyl) phenylmethyl]-2-deoxy-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone (8) 25~32A~
-28- 07-21(281)A
OC(O)N(Ph)2 ~3~ ~
H3CO ~
o (l~pr~2~ OCH2CH2CN
4-O-diphenylcarbamoyl-1-[~5-O-[bls(4-methoxyphenyl) phenylmethyl]-3-0-~bls(l-methylethyl)amlno]-(2-cyanoethoxy)phosphino]-2-deoxy-be~a-D-erythro-pento-furanosyl~-2(lH)-pyridone (9) OC~O)N(Ph)2 h~CO ~ ~ y (I Pr)2N OCH3 LQ
4-O-diphenylcarbamoyl 1-[5-O-[bis(4-methox~phenyl) phenylmethyl]-3-O-~bis(l-methylethyl)amino]-methoxyphosphino~-2-deoxy-bet~-D-erythro-pentofuranosyl]-2(1H)-pyridone (10) % 3 2 ~
-29- 07-21(281)A
OCtO)N(Ph)2 CO ~ ~
HO OH
4-O-diphenylcarbamoyl-1-[5-O-~(4-methoxyphenyl)dlphenyl-methyl]-beta-D-eruth~o-pentofuranosyl-2(lH)-pyridone ( 1 1 ) OC(O)N~Ph)2 HO ~
HO OH
. 1~
4-O-diphenylcarbamoyl-3-iodo~ eta-D-erythro-pento-S furanosyl-2(lH)-pyridone (12) .. .~
2~2~
_30_ 07-21(281)A
oC(O)N~Ph)2 0~1 HO OH
4-O-diphenylcarbamoyl-5-iodo-1-beta-D-erythro-pento-furanosyl-2(lH)-pyridone (13) OC(O)N(Ph)2 H,CO~ ~
HO OH
." , "
4-O-diphenylcarbamoyl-3-iodo-1-[5-O-[(4-methoxyphenyl) diphenylmethyl]-beta-D-erythro-pentofuranosyl]-2(1H)-pyridone (14) 2~2~
-31- 07-21(281)A
OC(O)N(Ph)2 H,CO ~ ~
HO OH
4-O-diphenylcarbamoyl-5-iodo-1-[5-0-[4-methoxyphenyl) diphenylmethyl]-beta-D-erythro-pentofuranosyl]-2tlH)-pyridone (15) oC(o)~ph2 OCH~
HO OH
4-O-diphenylcarbamoyl-1-[5-O-[bis~4-methoxyphenyl) phenylmethyl]-beta-D-erythro-pentofuranosyl]-2(1H)-pyridone (16) .
2 ~ 2 ~
-32- 07-21(2~1~A
OC(O)N(Ph), H3CO ~ ~y HO OSl(l Pr)3 4-O-diphenylcarbamoyl-1-[[5-0-[(4-methoxyphenyl) diphenylmethyl]-2-O-tris(l-methylethyl)silyl]-beta-D-erytAro-pentofuranosyl]-1(2H)-pyridone (17) oC(O)N~Ph)2 H~CO ~ O ~
HO O Sl- (t~u) Me 4-O-diphenylcarbamoyl-1-[[5-0-[(4-methoxyphenyl~
S diphenylmethyl]-2-O-(1,l-methylethyl)dimethylsilyl]
-~eta-D-erythro-pentofuranosyl~-2(lH)-pyridone (18) 2~2~2 -33- 07-21(281)A
OC~O)N(Ph)2 H,CO ~ O ~
HO OSl(l Pr)3 4-O-diphenylcarbamoyl-3-iodo-1-[[5-0-[(4-methoxyphenyl) dlphenylmethyl]-2-0-tris(l-methylethyl)silyl]-be~a-D-erytAro-pentofuranosyl]-2(lH)-pyridone ~19) oC(O)N~Ph)2 H,CO ~ O ~
HO OSI(I-Pr~3 '`' iLQ ~
4-0-diphenylcarbamoyl-S-iodo-1-[[5-O-[(4-methoxyphenyl~
S diphenylmethyl)-2-O-tris(l-methylethyl)silyl]-beta-D-erytAro-pentofuranosyl]-2(lH)-pyridone (20) -34- 07-21~281)A
OC(O)N~Ph)2 H3C0 ~ ~y O OSI(I Pr)3 (I-Pr)2N OCH2CI l;~CN
~1 ,.
4-O-diphenylcarbamoyl-1-[[[5-0-[(4-methoxyphenyl) diphenylmethyl]-3-O-[bis(l-methylethyl)amino]
(2-cyanoethyl)phosphino]-2-O-tris(1-methylethyl)silyl]
-beta-D-erythro-pen~ofuranosyl]-2(lH)-pyridone (21) oc(o)N(ph)2 h3CO ~ ~ ~ /
o 0--Sl--(~ Bu) Me (I-Pr)2N OCH2CH2CN
.
4-O-diphenylcarbamoyl-1-[[[5-0-[(4-methoxyphenyl) diphenylmethyl]-3-O-[bis(1-methylethyl)amino](2-cyanoethyl)phosphino]-2-O-(l,l-methylethyl) dimethylsilyl]-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone (22) 2~2~
_35 07-21(281)A
OC(C))l`J(Ph)2 H,C0 ~ O ~y o ~:)SI(I~P~)3 (l-Pr)2N OCH2CH2CN
4-0-diphenylcarbamoyl-3-iodo-1-[[[5-0-[(4-methoxyphenyl) diphenylmethyl]-3-0-[bis(1-mPthylethyl)amino](2 cyanoethyl) phosphino]-2-0-tris(1-methylethyl)silyl]-beta-D-erythro-pentofuranosyl]-2(lH)-pyrldone ~23) OC(O)N(Ph)2 11,CO~O~
O OSI(I Pr)3 .~ (I-Pr)2N OCH2CH2CN
L~
4-0-diphenylcarbamoyl-5-iodo-1-[[[5~0-[(4-methoxyphenyl) diphenylmethyl]-3-0-[bis(l-methylethyl)amino](2-cyanoethyl) phosphino]-2-0-tris(1-methylethyl)silyl]-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone (24) 2 ~ 8 ~
-36- 07-21~281)A
OC(O)N(Ph)2 H,CO~ o~N
H--P--O `
O NH(E~)3~
4-O-diphenylcarbamoyl-1-[[5-O-[bis(4-methoxyphenyl) phenylmethyl]-3-O-(hydroxyphosphinyl)-2-deoxy]-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone trl-ethylammonium salt (25) OC(O)N(Ph)2 H3CO -~ o~h Pr)3 H--P = O
o NH~Et)3 ~ `
~
4-O-diphenylcarbamoyl-1-[[5-O [(4-methoxyphenyl) diphenylmethyl]-3-O-(hydroxyphosphinyl)-2-O-tris-(l-methylethyl)silyl]-beta-D-erytAro-pentofuranosyl]-2(lH)-pyridone triethylammonium salt (26) c~s~ ~ 82 -37- 07-21(281)A
OC(O)N(Ph)i of~
H3CO ~ ~ Me o` o sl-(tau) H--P--O Me o NH(Et)3~
4-O-diphenylcarbamoyl 1-[~5-O-[(4-methoxyphenyl) diphenylmethyl]-3-O-(hydroxyphosphinyl)-2-O-(1,1-dimethylethyl)dimethylsilyl]-beta-D-erythro-pento-furanosyl]-2(lH)-pyridone triethylammonium salt (27) OC(O)N(Ph)2 1~
(~),SI ~ ~
" (iPr)2SI--O
4-O-diphenylcarbamoyl-3-iodo-1-[2-deoxy-3,5 O-[l,1, 3,3-tetrakis(l-methylethyl)-1,3-disiloxanediyl]-~eta-D-erythro-pentofuranosyl]-2(lH)-pyridone (28) . , ~ , ~ , , ;
292~2 -38- 07-21(281)A
o~(o)N(ph)2 ~1 ,,,.. :''' (iP~),SI--O
2~
4-O-diphenylcarbamoyl-5-iodo-1-[2-deoxy-3,5-0-[1,1, 3,3-tetrakis(l-methylethyl)-1,3-disiloxanediyl]-beta-D-erythro-pentofuranosyl]-2(1H)-pyridone (29) OC(O)N(Ph)2 1~3 ' O
.~ :
~Q
4-O-diphenylcarbamoyl-3-iodo-1-t2-deoxy-beta-D-erythro-pentofuranosyl)-2(1H)-pyridone ~30) .
-: . :
~2~2 -39- 07-21(281)A
C)C(O)N(Ph)2 0~~
HO ~/
OH
4-0-diphenylcarbamoyl-5-iodo-l-(2-deoxy-beta-D-erythro-pentofuranosyl)-2.~1H)-pyridone (31) OC:(O)N(Ph)2 H3CO I ~3 H3CO ~ O ~
OH
4-O-diphenylcarbamoyl-3-iodo-1-[5~0-[bis(4-methoxyphenyl)phenylmethyl]-2-deoxy-beta-D-S erythro-pentofuranosyl]-2(1H)-pyridone (32) 2~2~
-40- 07-21(281)A
OC~O)N(Ph)a ~13(~ ~1 H3CO ~
OH
4-O-diphenylcarbamoyl-5-iodo-l-[5-0-[bis(4-methoxyphenyl)phenylmethyl] 2-deoxy-beta-D-erythro-pentofuranosyl3-2(lH)-pyridone (33) OC(O)N(Phk H3CO I ;~ 3 H3CO ~ ~
o \
-Pr)2N OcH2cH2c N
~. `
4-0-diphenylcarbamoyl-3-iodo-1-[5-0-[bis (4-methoxyphenyl)phenylmethyl]-[3-0-[bis(1-methylethyl)amino](2-cyanoethoxy)phosphino]-2-deoxy-beta~D-ery~hro-pentofuranosyl]-2(1H)-pyridone (34) 2~2~ 2 -41- 07-21(281)A
OC(O~N(Ph)2 H3CO f ~, H3CO ~
o (I-Pr)2N OCH2CH2CN
4-O-diphenylcarbamoyl-5-iodo-1-[5-O-[bis(4-methoxyphenyl)phenylmethyl]-[3-O-[bis(l-methylethyl)amino](2-cyanoethoxy)phosphino]-2-deoxy-beta-D-eryt~ro-pentofuranosyl]-2(1H)-pyridone (35) OC~O)N(Ph)2 ~ ,.
(iPr)~SI
(iPr)2SI--O
4-O-diphenylcarbamoyl-3-ethenyl-1[2-deoxy-3, 5-0-[1,1,3,3-tetraki~(l-methylethyl)-1,3-disiloxanediyl]-bet a-_-erythro pentofuranosyl]-~(lH)-pyridone (36) .
2 ~
-42- 07-21(281)A
OC(O)N(Ph)2 HO
. OH
4-O-diphenylcarbamoyl-3-ethenyl-1(2-deoxy-beta-D-erytAro-pentofuranosyl)-2(lH)-pyrldone (37) VC(O)N~Ph~2 H3CO ~ O
OH
~ .
4-O-diphenylcarbamoyl-3-ethenyl-l-[5~G-[bis (4-methoxyphenyl)phenylmethyl]-2-deoxy-beta-D-S erytAro-pentofuranosyl]-2(lH)-pyridone (38) .
', . .
2~2~2 -43- 07-21(281)A
OC(O)N(Ph)2 H3CO ~3 H3CO ~
OY
~l~pr)2N OCH~CH2CN
4-O-diphenylcarbamoyl-3-ethenyl-1-[5-O-[bis(4-methoxyphenyl)phenylmethyl]-[3-0-[bis(l-methylethyl) amino](2-cyanoethoxy)phosphino]-2-deoxy beta-D-erytAro-pentofuranosyl]-2(lH)-pyridone (39) oC(O)N~Ph)2 of~
(iPr),SI~O~
~)2SI O~H3 4-O-diphenylcarbamoyl-1-[[3,5-0-[1,1,3,3-tetrakis (1-methylethyl)-1,3-disiloxanediyl]-2-0-methyl]-beta-D-ery~hro-pentofuranosyl]-2(1H)-pyridone (40) ?J~2~82 -44- 07-21(2al)A
OC(O)N(Ph)2 oJ~
HO~/
4-0-diphenylcarbamoyl-1-(2-0-methyl-beta-D-erythro-pentofuranosyl)-2(lH)-pyrldone (41) OC(O)N(Phk H3CO f ~3 H3CO ~ O ~
HO OCHa 9LL ' 4-0-diphenylcarbamoyl-1-[5-O~[bis-(4-methoxyphenyl)phenylmethyl]-2-O-methyl-beta-D-erythro-pentofuranosyl] 2(lH)-pyridone (42) J~ ~2 -45- 07-21(281)A
oC(03N(Ph)2 H3CO ~3 H3CO ~ ~
OCI'13 (l~Pr)2N OCt~2t:H2CN
4-O-diphenylcarbamoyl-1-[5-O-[bis(4-methoxyphenyl) phenylmethyl]-3-O-[bis(1-methylethyl)amino(2-cyanoethoxy)phosphino]-2-O-methyl-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone (43) OC(O)~PI12 1~
(i~)2 Sl ~ ~
(i~)~SI - O OH
4-O-diphenylcarbamoyl-3-iodc-1-[3,5-O-[1,1,3,3-tetrakis(l-methylethyl)-1,3-dis1loxanediyl]-beta-D-erythro-pentofuranosyl]~2(lH)-pyridone (44) ~,~S~4~2 -46- 07-21(281)A
OC(O)NPh2 Os[~l .
(iPr)~SI~O~
I iPr)~SI--O OH
~ `
4-O-diphenylcarbamoyl-5-iodo-1-[3,5-0-[1,1,3,3-tetrakis(l-methylethyl)-1,3-disiloxanediyl]-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone (45) -47- 07-21(281)A
4-hydroxy-1-beta-D-erythro-pentofuranosyl-2(1H)-pyridone (1), commonly called 3-deazauridine (Slgma, st. Louis, MO) is converted to the 4-O-diphenyl-carbamoyl-1-~eta-D-erythro-pentofuranosyl-2(lH)-pyri-done (3), in the following manner. 15.0 gm. of 3-dea-zauridlne are dried in a vacuum desiccator containing phosphorous pentoxide ( P4 01 o) to obtain 14.7 gm. (6.04 X 10 2 mole). The dried starting uridine is placed in a round bottom flask and 140 mL acetonitrile (CH3CN) and 50 mL dimethylformamide (DMF) added along with 16.1 mL (9.24 X 10 2 mole) dlisopropyl ethylamine.
The mixture is stirred under argon and diphenylcar-bamoyl chloride (22 gm., 9.50 X 10 2 moles) in 60 mLs CH3CN is added dropwise over about 30 minutes at room temperature. The reaction is stirred overnight result-ing in a pale yellow solution. The volatiles are removed under reduced pressure on a rotary evaporator to obtain a thick syrup. The syrup is dissolved in toluene ~50 mL) and concentrated under reduced pres-sure (thls is repeated twlce) and the residue is triturated with ethyl acetate ~EtOAc) to obtain a white solid which is filtered, washed with EtOAc and ethanol tEtOH) and dried to give 16.8 gm. of pale yellow powder. The washings are concentrated and the re~idue purified by flash chromatography (Merck Silica) using EtOAc: methan~l (MeOH) (3%): triethylamine (Et3N) (0.5%). The fractions containing product are evapo-rated and triturated with ~tOAc, filtered and dried to give another 6.5 gm. of desired product, 4-O-diphenyl-carbamoyl-l-beta-D-erythro-pentofuranosyl-2(1H)-pyridone (3), (86% isolated yield). m.p. 167-68C; Rf C~2Cl2:
- MeOH 9:1) = 0.4 1HNMR, ~(~MSO-d6): 8.06 (d, 1~, J=7.66 Hz,H-6), 7.44-7.42 (m, 10H, aromatic), 6.29 ~dd 1~, J=2.88, 7.66Hz, H-5), 6.20 (d, lH, J = 5.~9 Hz, H-1'), 2 ~ 2 -48- 07-21(281)A
5.99 (d, lH, J = 2.88 Hz, H-3), 5.42 (d, lH, J = 5.18 Hz, 2'-OH), 5.13 (t, lH J = 5.05 Hz, 5' OH), 5.03 (d, lH, J = 5.36 Hz, 3' -OH), 3.96 & 3.88 (m 3H, H-2', 3' & 4'), 3.70 (m, lH, H-5'), 3.60 (m, lH, H-S'I); mass spectrum (FAB) m/z (relative lntensity) 439 (M+Li) (80), 307 (100), 196 (75); Elemental Analysis calculated for C23H22N2O7 : C, 63.01; H, 5.02; N, 6.54 Found C, 63.17; H, 5.15 N, 6.29.
To 15.0 gm. (O.0343 mole) of 4-0-dlphenyl-carbamoyl-1 -be ta-D-erythro-pentofuranosyl-2(lH)-pyridone (3), dissolved in 150 mL dry DMF (Aldrich) is added 30.1 mL (0.150 mole) diisopropylethylamlne. The solution is stirred under an Argon atmosphere and 11.9 mL
(11.9 gm, O.0381 mole) dichlorotetraisopropylsiloxane dissolved in 50 mL dry DMF is added in dropwise fashion over about 1 hour. The reaction is carried out at room temperature. TLC (M~rck 60 - p.254, 1:1 hexane:EtOAc) shows good conversion to product after several hours. The volatiles are removed under ~acuum and worked up by dissolving the residue in methylene chloride (CH2Cl2) and water. The organic layer is separated, washed with 0.5 M hydrochloric acid (HCl), and aqueous sodium bicarbonate (saturated) and dried with anhydrou~ sodium sulphate. The oil obtained upon removal of solvent is purified by preparative liquid chromatography (Waters Prep 500, silica gel, 3:1 hexane: ethyl acetate) to obtain 16.5 gm. (71% iso-lated yield) of light yellow product, 4-O-diphenyl-carbamoyl-l-[3,5-O-1l,1,3,3-tetrakis(l-methylethyl)-1, 3-disiloxanediyl]-beta-D--ery~hro-pentofuranosyl-2 (lH)-pyridone (4), RF 0.36 (EtOAC : ~e~ane 1:1);
H NMR, ~(CDCl~): 7.78 ~d, 1~, J = 7.8 H-2 H-6, . 7.4-7.26 (M, 10H, Aromatic) 6.31 (d, ~, J - 2.44 Hz, H-3), 6.~0 _(dd, lH, J = 7.8, 2.~3 Hæ, ~-5), 4.32-3.99 (m, 5H, H-2', 3', 4', 5' & 5" ), 3.03 (d, lH, J - 1.1 ~?,4'1~2 _~9_ 07-21(281)A
Hz 2'-OH), 1.1-0.96 (m, 28H, 2x S1-i-propyl); mass spectrum (FAB) m/z 687 (M~Li) i HR FAB MS : Calculated for C3~H48N2Si2O~Li, 687.3109 - ~ound 687.3099.
To 6.5 gm. (9.5 X 10 3 mole) 4-O-diphenyl-carbamoyl-1-[3,5-0-[1,1,3,3-tetrakis(l-methylethyl) -1,3-disiloxanedlyl]-heta-D-erythro-pentofuranosyl]-2(1H)-pyridone (4), dlssolved in 65 mL CH2Cl2 is added 1.75 gm (1.43 X 10 2 mole) 4-dimethylaminopyi~
dine and 1.45 mL (1.81 gm, 1.05 X 10 2 mole) phenoxy-thionocarbonyl chloride. The reaction mixt~re is stirred overnight at room temperature under argon. The volatiles are removed under vacuum and the orange semi-solid residue taken up in EtOAc. The organic layer is washed with water, 0.25 N HCl and saturated aqueous NaHCO3. The organic layer is dried with magnesium sulphate (MgSO9), filtered, and concentrated to about 25 mL. Purification is achleved by silica gel chromatography on a Waters Prep 500 using 3:1 hexane: EtOAc. A yellowish product is obtained which if carried forward at this stage does not reduce well.
A second pass over silica ~el is carried out ~o obtain 6.9 gm. of light yellow amorphous product (90% iso-lated yield), 4-O-diphenylcarbamoyl1-~2-O-(phenoxy-thioxomethyl)-3,5-O-[l,l, 3,3-tetrakis(l-methylethyl) -1,3-disiloxanediyl]-~eta-D-erythro-pentofuranosyl3-2(lH)-pyridone ~5). M.p. 117C (dcomp). RF 0-5 ?
(EtOAc : Hexane l ~ HNMR, ~(CDCl3) : 7.79 (d, lH, J = 7.8 Hz, H-6), 7.43 7.11 (m, 15H, aromatic), 6.32 (d,lR, J= 2.44 Hz, ~-3); 6.19 (dd, lH, J = 2.44, 7.8 Hz, H-5), 5.97 (d, l~, J = 4.69 Hz, ~-1), 4.5 (dd, lH, J = 4.7, 9.4 ~z, H-3), 4.28 (m, 1~, H-4'), 4.15 (m, lH, H-5'), 4.05 (m, l~, H-5" ), 1.55-0.96 (N, 28~, 2x-Si-(i-propyl)2); mass spectrum (FAB) m/z (relative intensity~ 824 (M+Li)+ (20), 664 (100), 307 (40); ~R
FAB MS : Calculated for C42Hs2N2Si2SogLi 823.3092.
c~ ~ ~2 ~
-50- 07-21(281)A
Found 823.3084. Elemental analysis : calculated for C42H5~N2Si2SOg; C,61.76; H,6.37; N,3.43. ~ound C,61.82 ; H,6.37 ; N, 3.32.
To 6.0 gm. (7.3 X 10 3 mole) 4-O-diphenyl-S carbamoyl-1-[2'-O-(phenoxythioxomethyl) -3',5'-O-[1,1, 3,3-tetrakis(l-methylethyl)-1,3-disiloxanediyl]-beta-D-ery~hro-pentofuranosyl]-2(lH)-pyridone (5) in 75 mL
toluene is added 2.94 mL tri-n-butyl-tin hydride (1.0 X 10 mole) and 0.24g AIBN (a~obisisobutyronitrile, 1.5 X 10-3 mole). The reaction mixture is stirred under argon atmosphere at 85C for 16 hours. The toluene is removed under re~uced pressure and the residue purified by flash chromatography on silica gel using 3:1 EtOAc:hexane as eluent. Appropriate frac-tions are combined and dried under vacuum to give 4.1 gm 84% isolated yield of the desired product as a syrup 4-O-diphenylcarbamoyl-1-[2-deoxy-3,5-0-[1,1,3,3-tetrakis (l-methylethyl)-1,3-disiloxanediyl]-beta-D-erythro-pento-furanosyl ]-2(1H)-pyridone (6), Rf 0.52 (EtOAc :
Hexane 1 ~ HNMR ~(CDCl3): 7.84 (d, lH, J = 7.83 Hz, H-5), 7.4-7.24 (m, 10H, Aromatic), 6.31 (d, lH J =
2.09 Hz, H-3), 6.22 (m, 2H, H-5, H-1'), 4.4 (m, lH, H-3'), 4.14 (m, lH, H-4'),4.05 (m, lH, ~-5'), 3.81 (m, lH, H-5"), 2.60 (m, lH, H-2'), 2.25 (m, lH, H~
1.09-0.98 ~m, 28H, 2x- Si-(i-propyl)2); mass spectrum (FAB) m/z (relative intensity) 672 (M+Li) (80), 482 (10), 313 (100). ER FAB MS : Calculated for C35H48O7N3Si3Li, 671.3160. Fou~d 671.3171.
To 3.0 gm (4.5 X 10 3 mole) 4-0-diphenylcar-bamoyl-1-~2-deoxy-3,5-O-[1,1,3,3-tetrakis(l-methyl~
ethyl)-1,3-disiloxanediyl]-~eta-D-erythro-pentofuranosyl]
-2(1H)-pyridone (6), in 30 mL CH3CN at 0C is added . O.62 gm l2.0 X 10 3 mole) tetrabutylammonium fluoride trihydrate (Aldrich, Milwaukee, WI) with stirring.
The reaction mixture is stirred at 0C for 30 minutes, 2~2~
-51- 07-21(281~A
then stlrred for 30 minutes at room temperature.
Solvent is removed under reduced pressure and the deslred product isolated by silica gel flash chroma-tography using ~:1 CH2Cl2:MeOH containing 0.5% Et3N.
S A total of 1.5 gm. ~79% isolated yield) of desired 4-O-diphenylcarbamoyl-1-(2-deoxy-beta-D-erythro-pento-furanosyl)-~(lH~-pyridone (7~, is obtained. The product is crystallized from EtOAc; mp 147 - 48C. Rf 0~4(CH2Cl2 : MeOH 9:1) 1HNMR, ~(DMSO-d6) 7.98 (d, lH, J = 7.77~z, H-6), 7.43-7.3 (m, lOH, aromatic), 6.30 (m, 2H, H-5, H-l'), 6.18 (d, lH, J = 2.56 Hz, H-3), 5.23 (d, lH J = 4.18 H~, 2'-OH), 5.01 (t, lH, 5'- OH), 4.20 (m, lH, 3'-H), 3.84 (m, lH, H-4'), 3.6 (m, 2H, H-5' &
5"), 2.25 (m, lH, H-2'), 1.95 (m, lH, H-2); mass spectrum FAB m/z (relative intensity) 423 (M + Li) (70), 312 (90), 242 (100); Analysis calculated for:
C23H22N2G6 0-5H20 : C, 65.03 : H, 5.34 N, 6.49. Found:
C, 65.04 : H, 5.21i N, 6.57.
To 1.1 gm (2.61 x 10 3 mole) 4-O-diphenyl-carbamoyl-1-(2-deoxy-be~a-D~erythro-pentofuranosyl)-2 (lH)-pyridone (7), in 15 mL dry pyridine is added 1.3 gm (3.8 x 10 3 mole) dimethoxytrityl chloride, 0.6~ mL
(3.9 x 10 3 mole) diisopropylethylamine and 30 mg 4-N,N-dimethylaminopyridine (2.5 x 10 4 mole). The reaction mixture is stirred at room temperature under argon for 4 hours. Volatiles are removed under reduced pressure and excess pyridine is removed by repeated (3 x 20 mL) distillation of toluene under reduced pressure. The residue is purified by silica gel flash chromatography using 2% MeOH in C~2C12 containing 0.5% triekhylamine. 1.1 ~m of a yellow amorphous solid is obtained which is rechromatographed ` (same solvent) to give 0.9 gm (1.44 x 10 3 mole) desired product as a white amorphous ma~erial, 4-O-di-phenylcarbamoyl-1-[5-O-[bis(4-methoxyphenyl~phenyl-~2~ 2 -52- 07-21(281)A
methyl]-2'-deoxy-beta-D~erythro-pentofuranosyl] -2~1H)-pyridone (8). RF 0.56 (CH2Cl2:MeOH, 9:1); 1HNMR, ~(CDCl3): 7.92 (d, lH, J = 7.89 Hz, H-6), 7.42 - 7.22 (m, l9H, aromatic), 6.84 (2s, 4H, aromatic), 6.39 (dd, lH, J = 5.9 Hz, H-1'), 6.~5 (d, lH, J = 2.52 Hz, H-3), 5.97 (dd, lH, J = 2.44, 7.89 Hz; H-5), 4.47 (m, lH, H-4'), 4.03 (m, lH, H-4'), 3.77 (s, 6H, 2-OCH3), 3.5 ~m, lH, H-5'), 3.42 (m, lH, H-5"), 2.60 (m, lH, H-2'), 2.2 (m, lH, H-2"); mass spectrum ~FAB) m/z (relative intensity) 731 (m+Li) (25), 427 (100); HR FAB MS;
C44H~oN~O8 : 731.2945, found ; 731.2939.
To 0.47 gm (6.43 x 10 4 mole) 4-0-dlphenylcar-bamoyl-l-[5-O-[bis(4^methoxyphenyl)phenylmethylJ-2-deoxy-~eta-D-erythro-pentofuranosyl]-2(1H)-pyridone (8), in 8 mL CH2Cl2 is added 0.45 mL (2.5 x 10 3 mole) N,N-diisopropylethylamine. 0.3 gm ~1.27 x 10 3 mole) ~-cyanoethyloxy- N,N-diisopropyl chlorophosphine (ABN) in 1 mL CH2Cl2 is added dropwise over about 15 minutes. The reaction mixture is stirred under argon atmosphere at room temperature for an additional hour.
The solution is concentrated to dryness using a rotary evaporator, dissolved in EtOAc and washed with saturat-ed aqueous NaHCO3 (3 x Z0 mL), distilled water and dried with (MgSO4). The yellow solution is evaporated to dryness and dried under vacuum to give 07.4 gm of viscous product that is dissolved in EtOAc and precip-itated by the addition of hexane. The mixture is cooled, solvents decanted and the residue washed with cold hexane and dried under vacuum to give 0.55 gm (6.0 x 10 4 mole, 79% isolated yield) of 4-O~diphenyl-carbamoyl-l-[[5-O-[bis~4-methoxyphenyl)phenylmethyl]-3-O-tbis(l-methylethyl)amino~-(2-cyanoethoxy~phosphino]
.~ -2-deoxy-beta-D-erythro-pentofuranosyl]-2(1~)-pyridone (9), TLC conditions; Merck silica gel 60 p254, EtOAc :
Hexane, 1:1; RF = 0.27.1HNMR, ~tcDcl3) 7.99, ~.92 (2d 2 ~ 2 ~
-53- 07-21(281)A
lH, J = 7.73 Hz, H-6), 7.42-7.22 (m, l9H aromatic), 6.84 (m, 4H, aromatlc), 6.42 (dd, lH, J = 6.27 Hz, H-l), 6.26 (d, lH, J =2.44 Hz,H-3), 5.89 (dd lH, J
=2.43, 7.8 Hz, H-5), 4.6 (m, lH, H-3'), 4.15 (m, lH, H-4'), 3.76 (s, 6H, 2x-OCH3), 3.74-3.35 (m, 4H, H-5', 5", 2x CH(Me)2), 2.65 (m, lH, H-2'), 2.6 (t, 2H, J =
6.34 Hz, CH2CN), 2.4 (T, 2H, J= 6.3 Hz, CH2), 2.2 (m, lH, H-2"), 1.28-1.05 (m, 12H, 2x-N-CH (CH3)2); mass spectrum (FAB) m/z (relative lntensity) : 931 (m+Li) (100), 627 (15); HR FAB MS: calculated for Cs3 H57 N4 Og PLi, 931.4023; Found 931.4015.
4-O-diphenylcarbamoyl-l-[S-O-[bis(4-methoxy-phenyl)phenylmethyl]-3-O[bis(1-methylethyl)amino]-methoxyphosphino]-2-deoxy-beta-D-erythro-pentofuranosyl]
-2(1H)-pyridone (10), is synthesized from 4-O-diphenyl-carbamoyl-1-[5-O-[bis(4-methoxyphenyl) phenylmethyl]-2-deoxy-beta-D-erythro-pentofuranosyl]-2(1H)-pyridone (8), and N,N-diisopropylmethylphosphonamidic chloride in substantial accordance with the teaching in Example 1.
The yield of desired product after silica gel chromatog-raphy is 68%. 31-P NMR (CDCl3, d, 149.2, 148.8 vs. 5%
H3PO4).
4-O-diphenylcarbamoyl-l-beta-D-eryt~ro-pentofuranosyl-2(lH)-pyridone (3), prepared in sub-stantial accordance with the teaching of Example 1, can be converted to 4-O-diphenylcarbamoyl-1~5-0-[~4-methoxyphenyl) diphenylmethyl]-beta-D-e~ythro-pento furanosyl]-2(1H)-pyridone (11), in the following manner. To 15.0 gm (3.43 x 10- 2 mole~ of 4-O-di-phenylcarbamoyl-1-beta-D-erytAro-pentofuranosyl-2(lH)-2~32~T~
-54- 07-21(281)A
pyridone (3), dlssolved in dry pyridine is added 10.5 mL (6 x lO- 2 mole) dlisopropylethylamine and 30 mg (2.5 X 10-4 mole) 4-N,N-dimethylaminopyridine. To this is added li.7 gm (3.9 x 10- 2 mol~) monomethoxy-triphenylmethyl chloride. The reaction mixture isstirred at room temperature under argon atmosphere until complete. Volatiles are removed in vacuo and reduced pyridine is removed by repeated (3x) distilla-tion of toluene under reduced pressure. The recidue is purified by silica gel flash chromatography using a suitable solvent system made slightly basic with 0.5%
triethylamine. In this manner the desired product, 4-O-diphenylcarbamoyl-1-5-0-[(4-methoxyphenyl)diphenyl-methyl] -beta-D-ery~hro-pentofuranosyl]-2 (lH)-pyridone (ll), can be obtained.
4-O-diphenylcarbamoyl-3-iodo-1-be~a-D-eryt~ro-pentofuranosyl -2(lH)-pyridone (12), and 4-O-diphenyl-carbamoyl-5-iodo-1-beta-D-erythro-pentofuranosyl-2(1H)-pyridone (13), can be converted to 4-O-diphenylcar-bamoyl-3-iodo-l-[5-0-[(4-methoxyphenyl)diphenylmethyl]-be~a-D-erytAro-pentofuranosyl]-2(1H)-pyridone (14), and 4-O-diphenylcarbamoyl-5~iodo-1-[5~0-[4~methoxy-phenyl) diphenylmethyl]-beta-D-eryth~o-pentofuranosyl]
-2(1H)-pyridone (lS), respectively, in the same manner. 4-O-diphenylcarbamoyl-1-[5-O-[bis ~4-methoxy-phenyl) phenylmethyl]-beta-D-erythro-pentofuranosyl]-2 (lH)-pyridone (16), can be obtained in a similar manner by substituting dimethoxytriphenylmethyl chloride ~or monomethoxytriphenylmethyl chloride.
To 4.7 sm (7.9 x 10-3 mole~ 4-O-diphenyl-~` carbamoyl-1-[5-0-[~4-methoxyphenyl)diphenylmethyl]-2 ~ o ~
-55- 07-21(281)A
beta-D-erythrO-pentofUranosyl]-2(1H)-pyridone (11) in anhydrous DMF under argon atmosphere is added 1~18 gm (1.73 x 10- 3 mole) imidazole and 3.38 mLs (1.58 x 10 2 mole) triisopropylsilyl chloride while stirring.
S The reaction is allowed to proceed until complete, quenched with aqueous saturated NaHC03, and concen-trated in vacuo. The resulting residue is co-evap-orated with toluene, dissolved in CH2Cl2 and washed with water. The CH2C12 layer is dried with MgSO9 (anhydrous) and concentrated in vacuo . The desired product, 4-0-diphenylcarbamoyl-1-[[5-0-[(4-methoxy-phenyl)diphenylmethyl]-2-0-tris(1-methylethyl)silyl]-beta-D-erythro-pentofuranosyl]-1(2H)~pyridone (17), can be obtained by silica gel chromatography in a fashion similar to that employed by N. Usman, K. K. Ogilvie, M.Y. Jiang, and R.J. Cedergren, J. Am. Chem. Soc., 1987, 109, 7845-7854.
By substitutin~ tert-butyldimethylsilyl chloride for triisopropylsllyl chloride, one can obtain 4-0-diphenylcarbamoyl-1-[~5-0-[(4-methoxyphe-nyl)diphenylmethyl]-2-0-(1,l-methylethyl)dimethylsil-yl~-beta-D-erythro-pentofuranosyl]-2llH)-pyridone (18). 4-0-diphenylcarbamoyl-3-iodo-1-[5-0-[(4-methoxy-phenyl) diphenylmethyl]-be t a-D-erytAro-pentofuranosyl]-2 (lH)-pyridone (14), and 4-0-diphenylcarbamoyl-5-iodo-1-[5-0-[4-methoxyphenyl)diphenylmethyl] -beta-D-erythro-pentofuranosyl]-2 (lH)-pyridone (15), can be converted to 4-0-diphenylcarbamoyl-3-iodo-1-[[5-0-[(4-methoxy-phenyl) diphenylmethyl]-2-0-tris(l-methylethyl)~ilyl]-beta-~-erythro-pentofuranosyl]-2(1~)-pyridone (19), and 4-0-diphenylcarbamoyl-5-iodo-1-[[5-0-[(4-me~hoxy-ph~nyl)diphenylmethyl]-2-0-tris(l-methylethyl) silyl]-beta-D-erythro-pentofuranosyl]-2(1H)~pyridone (20), respectively, in a similar manner.
2~2~
-56- 07-21(281)A
To a stirred dry tetrahydrofuran (THF) solution of 7.0 mL (4 x 10- 2 mole) dll opropylethyl-amine, 3.08 gm (1.3 x 10-2 mole) (N,N-diisopropyl)-~-cyanoethoxyphosphonamldlc chloride and a catalytic amount (215 mg, 1.75 x 10- 3 mole) of 4-N,N-dimethylaminopyrl-dine is added dropwise to a solution of 8.66 gm (1 x 10-2 mole) 4-Q-diphenylcarbamoyl-1 [[5-0-[(4-methoxy-phenyl)diphenylmethyl]-2-0-tris(l-methylethyl)sllyl]-beta-D-erythro-pentofuranosyl]-1(2H)-pyrldone (17), in dry THF. The reaction is allowed to go to completion and worked-up by adding EtOAc and washing with saturated aqueous NaHCO3. The organic layer is dried and solvent removed in vacuo. The desired product, 4-0-di-phenylcarbamoyl-1-[[[5-0-[(4-methoxyphenyl)diphenyl-methyl]-3-0-[bis(l-methylethyl)amino](2-cyanoethyl) phosphino]-2-0-tris(1-methylethyl)sllyl]-beta-D-erythr~-pentofuranosyl]-2(1H)-pyridone (21), can be obtained in substantially pure form by silica gel flash chroma-tography.
In a similar fashion, 4-0-diphenylcarbamoyl-1~[[[5-0-[(4-methoxyphenyl)diphenylmethyl]-3-0-[bis (l~methylethyl)amino](2-cyanoethyl)phosphino]-2-0-(1, l-methylethyl)dimethylsilyl]-beta-D-erythro-pentofur-anosyl]-2(1H)-pyridone (~2), can be obtained by using 4-0-diphenylcarbamoyl-1-[[5-0-[(4-methoxyphenyl) diphenylmethyl]-2-0-(1,1-methylethyl)dimethylsilyl]-~eta-D-erythro-pentofuranosyl]-2(1R)-pyridone (18).
In the same manner, 4-0-diphenylcarbamoyl-3-iodo-1-[[5-0-[(4-methoxyphenyl) diphenylmethyl]-2-0-tris(1-methylethyl)silyl]-beta-D-ery~hro-pentofuranosyl]-2[1~)-pyridone (19), and ~-0-diphenylcarbamoyl-5-iodo-1-' [f5-0-[(4-methoxyphenyl)diphenylmethyl~-2~0-tris(1-methylethyl) silyl] -be ta-D~pentofuranosyl]-2 (lH)-pyridone (20), can be converted to 4-O~diphenylcar-, . . - . .. . . .
2 ~ 2 -57- 07-21~281)A
bamoyl-3-iodo-l-1[(5-0-[(4-methoxyphenyl)diphenylmethyl~
-3-0-[bis(l-methylethyl)amino](2-cyanoethyl) pnosphlno]~
2-0-tris(l-methylethyl)silyl]-beta-D-erythro-pento-furanosyl]-2 (lH)-pyrldone (23), and 4-0-diphenylcar-bamoyl-5-iodo-1-[[~5-0-~(4-methoxyphenyl) diphenylme-thyl]-3-0-[bis (l-methylethyl)amino](2-cyanoethyl) phosphino]-2-0-tris(l-methylethyl) sllyl]-beta-D-erytAro-pentofuranosyl]-2 (lH)-pyridone (24), re-spectively.
4-0-diphenylcarbamoyl-1-[5-0-[bis(4-meth-oxyphenyl)phenylmethyl]-2-deoxy-beta-D-erythro-pento-furanosyl]-2(lH)-pyridone ~8), can be converted to 4-0-diphenylcarbamoyl-1-[[5-0-[bis~4-methoxyphenyl)phenyl-methyl]-3-0-(hydroxyphosphinyl)-2-deoxy]-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone triethylammonium salt (25), in substantial accordance with the procedure of B.C.
Froehler, P.G. Ng, and M.D. Matteucci, Nuc. Acids Res., 14,5399-5407 ~1986). Thus, to a stirred solution of phosphorous trichloride (PCl3) (1.03 gm, 7.5 x lO- 3 mole) and N-methylmorpholine (7.59 gm, 7.5 x lO- 2 mole) in 750 mL anhydrous CH2Cl2 is added 1,2,4-tri-azole (1.73 gm, 2.5 x 10-2 mole) at room temperature.
After 30 minutes the reaction mixture is cooled to 0C and 4-0-diphenylcarbamoyl~ 5-0-[bis(4-methoxy-phenyl)phenylmethyl]-2-deoxy-beta-D-erythro-pento-furanosyl]-2 (lH)-pyridone (8), (0.94 gm, 1.5 x 10-3 mole) in 200 mL anhydrous CH2C12 is added dropwise over 20 minutes, stirred for 10 minutes, poured into 600 mL of l.OM aqueous triethylammonium bicarbonate ( TEAB, p~ 8.5), shaken and separated. The aqueous ,- phase is extracted with 200 mL C~2Cl2 and ~he combined organic phase dried over Na2SO~ and evaporated to a foam. The desired 4-0-diphenylcarbamoyl-1-[[S-O-[bis (4-methoxyphenyl)phenylmethyl]~3-0-(hydroxyphosphinyl)--58- 07- 1(281)A
2 deoxy]-beta~D-erythro-pentofuranosyl]-2(lH)-pyridone triethylammonlum salt (25), can be obtalned by silica gel flash chromatography followed by TEAB extractlon and evaporatlon~ 4-O-dlphenylcarbamoyl~ 5-O-[(4-methoxyphenyl)dlphenylmethyl~-3 -O- ( hydrOXyphOSphlnyl ) -2-O-tris-(l-methylethyl~sllyl]-~eta-D-erythro-pento-furanosyl]-2(1~)-pyridone trlethylammonium salt (26), and 4-O-diphenylcarbamoyl-1-[15-O-[(4-methoxy-phenyl)dlphenylmethyl]-3-O-(hydroxyphosphinyl)-2-O-(1,l-dimethylethyl)dimethylsilyl]-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone triethylammonium salt (27), can be obtained in the same manner.
A mlxture of 4-O-diphenylcarbamoyl-1-~2-de-QXy-3, 5-0- ~ , 3, 3-tetrakis(l-methylethyl)-1,3-disi-loxanediyl]-beta-D-erythro-pentofuranosyl]-2(lH)-pyri-done, (6), (3.9g, 0.00S9mol), N-iodosuccinimide (1.4g, 0.0062 mol) in acetonitrile (40mL) containing di-chloroacetic acid (0.18g, 0.0014 mol) is heated at 60C for 4 hours under axgon atmosphere. The result-ing dark brown solution is concentrated under reduced pressure and the residue dissolved in 125 mL of CH2Cl2. This solution is washed with saturated agueous NaHCO3 (3 x 25 mL) followed by water (2 x 50 mL), dried (Na2SO9) and concentrated in vacuo. The resulting dark brown residue is purified by silica gel .
flash chromatography using EtOAc:~exane (1:4 v/v) as the eluent. ~he 3-iodo derivative is eluted first followed by the 5-iodo derivative. The fractions containing the 3-iodo derivative are combined, con-centrated under reduced pressure and the residue `` crystallized from MeOH to give 4-O-diphenylcarbamoyl-3-iodo-1-[2-deoxy-3,5-0-[1,1,3,3-tetrakis(1-methyl-ethyl)-1,3-disiloxanediyl] ^beta-D-erythro-pentofur-anosyl]-2(1~)-pyridone (28), (3.7g, 80%) as shiny 2 ~ 2 ~ 2 -59- 07-21(281)A
white flakes. m.p. 132-33C. Rf 0.57 (EtOAc:Hexane 1:1). IHNMR (300MHz, CDCl3) ~:7.9 (d, J = 7.8Hz,l,H6), 7.44-7.2 ~m, 10, aromatic), 6.32 (d, J - 7.8Hz, 1, H5), 6.17 (d, J = 6.73, 1, Hl'), 4.39 ~m, 1, H3'), 4.18 ~m, 1, H5'), 4.04 (m, 1, H5"), 3.8 (m, 1, H4'), 2.59 (m, 1, H2'), 2.26 (m, 1, H2"), 1.1 - 0.9 (m, 28.0[Si(iPr)2]2); FAB mass spectrum (m/z) 797 (M+Li), 439 (M+Li-sugar)i Analysis calculated for C35H~7IN207 Si2:C, 53.16i H, 5.95; N, 3.54. Found: C,52.82;
H, 5.95; N, 3.47. Fractions containing 4-0-diphenyl-carbamoyl-5-iodo-1-[2-deoxy-3,5-0-[1,1,3,3-tetrakis (l-methylethyl)-1,3-disiloxanediyl] -beta-D-ery~Aro-pentofuranosyl]-2(lH)-pyridone (29) are combined, c~ncentrated under reduced pressure and dried under high vacuum to give 0.25g (5%), as an amorphous solid.
Rf 0.48 (EtOAc: Hexane l:i). 1HNMR (300 MHz, CDCl3) ~:8.01 (s, 1, H6), 7.4 - 7.2 (m, 10, aromatic), 6.52 (s, 1, H3), 6.13 (dxd, 1, H1'), 4.36 (m, 1, H3'), 4.15 (m, 1, H5'), 4.04 (m, 1, H5"~, 3.8 (m, 1, ~4') 2.55 (m, 1, H2'), 2.25 (m, 1, H2"), 1.2 - 0.95 (m, 28 O[Si(i-Pr~2]2). FAB mass spectrum (m/z) 797 (~+Li), 439 (M~Li-sugar), HRFABMS observed (M~Li) 797.2134 calculated for C3sH4~IN2O~Si2 797.2127.
A mixture of 4-O-diphenylcarbamoyl-3-iodo-1-[2-deoxy-3,5~O~[1,1,3,3-tetrakis(1-methylethyl)-1,3~ ,' disilo~anediyl]-beta-D-erytAro-pentofuranosyl]-2~
pyridone (28), (lg, 0.0013 mL) and tetrabutylammonium fluoride trihydrate (0.25g, 0.0008 mL) in acetonitrile (20 mL) is stirred at 0C for 30 minutes followed by stirring at room temperature for 30 minute~. The solution is concentrated under reduced pressure and the residue purified by silica gel ~lash chromatography using C~2Cl2-MeO~ (4%) containing 0~5% Et3N as the eluent. Fractions containing 4-O-diphenylcarbamoyl-3-iodo-l-(2-deoxy-be ta-D-erythro -pentofuranosyl) 2(1~-2~24~2 -60~ 07-21(281~A
pyrldone (30), [indicated by TLC uslng CH2Cl2-MeOH(5%)]
are combined, concentrated under reduced pressure and dried on a desiccator under vacuum to glve C.5g (72%) of product. Rf 0.49 (CH2Cl2-MeOH 9~ HNMR (300 MHz, DMSO-d6) ~: 8.07 (d, J = 7.7Hz, 1, H6), 7.47 - 7.3 (m, 10, aromatic) 6.48 (d, J = ~.7 ~z, 1, H5), 6.26 (dxd, J = 6.3Hz, 1, H1'), 5.28 (d, J = 4.2Hz, 1, 2'-OH) 5.08 (t, J = 5.22 Hz, 1, 5'-OH), 4.23 (m, 1, H3'), 3.87 ~m, 1, H4') 3.61 (m, 2, H5' x 5"), 3.33 (s, 6, 2x-OCH3), 2.29 (m, 1, H2'), 1.99 (m, 1, H2"); FAB mass spectrum (m/z) 549 (M + H), 433 (m-sugar); HRFABMS observed (M+Li) 555.0602, calculated or C23H~1N~O6ILi 555.0604.
4-O-diphenylcarbamoyl-5-iodo-1-(2-deoxy-beta-D-ery~hro-pentofuranosyl)-2(lH)-pyridone (31), is obtained under similar reaction conditions as the 3-isomer, using tetraethylammonium fluoride, yield 63%. Rf 0.47 (CH2Cl2-MeOH 9:1), 1HNMR ~300 MHz, DMSQ-d6) ~8.4 (s, 1, H6), 7.6 - 7.2 (m, 10, axomatic), 6.44 (s, 1, H3), 6.24 (dxd, J = 6.3Hz, 1, ~ , 5.24 (d, J = 4.41 Hz, 1, 3'-OH), 5.14 (t, 1, 5'-OH), 4.23 (m, 1, H3'), 3.85 (m, 1, H4'), 3.59 (m, 2, ~5' x 5"), 2.49 (m, 1, H2'), 2.02 (m, 1, H2")i FAB mass spectrum (m/z) 555 (M+Li), 439 (M+Li-sugar).
A mixture of 4-O-diphenylcarbamoyl-3-iodo-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-2(1~)-pyri-done (30), (0.22g, 0.0006 mol), dimethoxytritylchloride (O.27g, 0.0008 mol), dimethylaminopyridine (DMAP) (O.025g) and diisopropylethylamine (O.14 mL, 0.0008 mol), in dry pyridine (5 mL) is stirred at room temperature for 6 hours and left overnight at 0C.
Pyridine is distilled under reduc~d pressure and the residue purified by silica gel flash chromatography using EtOAc-Hexane containing 0.5~ Et3N. The appro-priate fractions (visualized under W lamp) are combined, concentrated under reduced pressure and the -61- 07-21(2~1)A
resulting residue dried under high vacuum to give O.26g (76%) of ~-O-diphenylcarbamoyl-3-iodo-1-[5-O-[bis(4-methoxyphenyl)phenylmethyl]-2-deoxy-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone ~32), as an S amorphous material (Homogenous on TLC). Rf = 0.76 [CH2Cl2-MeOH (10%)]. IHNMR (CDCl3), ~:7.8 (d, J =
7.7Hz, 1, H6), 7.4 - 6.82 (m, 23, aromatic), 6.35 (d, J = 7.7Hz, H5), 6.2 (dxd, 1, H') 4.5 (m, l, H3'), 4.05 (m, l, H4'), 3.9 ~m, 2, H5'x 5") 3.8 (s, 6, 2X-OCH3), 2.50 (m, 1, H2'), 2.35 (m, 1, H2"); FAB mass spectrum `
(m/z): 857 ~M+Li), 533 (M+Li-DMT); HRFABMS observed 857.1904, calculated for C44~39IN2O8Li 857.1911.
4-O-~iphenylcarbamoyl-5-iodo-1-[5-O-[bis (4-methoxyphenyl)phenylmethyl]-2-deoxy-beta-D-erythro-pentofuranosyl]-2(1H)-pyridone (33), is prepared under similar reaction conditions. Yield 65%. R- 0.73 [CH2Cl~-MeOH (10%)]; 1HNMR (DMSO-d6) ~: 8.07 (s, l, H6), 7.46 - 6.8 (m, 23 aromatic), 6.51 (s, 1, ~3), 6.25 (dxd, l, J = 6.8Hz, 1, Hl'), 5.31 (d, J = 4.35 Hz, 1, 3'-OH), 4.19 (m, 1, H3'), 3.98 (m, 1, H4'), 3.22 (m, 2, H5' x 5"), 2.3 (m, l, E2~), 2.1 (m, l, H2"); FAB mass spectrum (m/z) 857(M+Li), 553 (M~Li-DMT).
To a chilled (ice bath) solution, 4-O-diphe-nylcarbamoyl-3-iodo-1-[5-O-[bis(4-methoxyphenyl)phe-nylmethyl]-2-deoxy-beta-D-erythro-pentofuranosyl]-2(1~) -pyridone ~32), (0.24g, 0.00028 mol) in dichloromethane (5 mL) containing diisopropylethylamine (0.1 mL, 0.00057 mol), is added dropwise a solution of ~-cyano-ethoxy-N,N-diisopropylamine chlorophosphine (0.lg, O.00042 mol), in dichloromethane (1 mL). The reaction mixture is stirred at room temperature for 2 hours.
TLC (EtOAc) reveals completion of ~he reaction. The solution is concentrated to dryness, the residue dissolved in EtOAc (10 mL), and washed with 10%
-62- 07-21(281)A
NaHCO3 (3 x 10 mL), followed by water (3 x 10 mL).
The organlc layer ls dried over anhydrous Na2 S04, concentrated under reduced pressure and the resulting syrupy residue purified by silica gel flash chroma-tography using EtOAc-Hexane (1:1) as the eluent to give 0.2g (63%) of 4-O-diphenylcarbamoyl-3-iodo-1-[5 -O-[bis(4-methoxyphenyl)phenylmethyl]-[3-O-[bis(l-methylethyl)amino](2-cyanQethoxy)phosphino]-2-deoxy-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone (34), as a white amorphous solid. IH, NMR (300 MHz, CDCl3) ~:8.20 (d, J = 7.6 Hz, H6), 8.05 (d, J = 7.6 Hz, H6), 7.4 - 6.8 (m, 23, aromatic), 6.35 (m, 1, H1'~, 6.2 x 6.02 (d, J = 7.6 Hz, 1, a5), 4.65 x 4.55 (m, 1, H3'), (4.15 (m, 1, H4'), 3.77 (2s, 6~ 2x -OCH3), 3.85 - 3.3 (m, 4, H5' x 5", -CH2-0-P-), 2.75 x 2.25 (m, 2, H2' x H2"), 2.6 x 2.4 (2t, 2, -CH2CN), 1.4 - 1.05 [(m, 14, N(i-Pr)2]; FAB mass spectrum (m/z) 1057 (M~Li), 753 (M+Li-DMT); HRFABMS: observed 1057.2999, calculated for Cs3H56IN~Og PLi 1057.2990.
4-O-diphenylcarbamoyl-5-iodo~ 5-O-[bis (4-methoxyphenyl)phenylmethyl]-[3-O-[bis(l-methyl-ethyl)amino](2-cyanoethoxy) phosphino]-2l-deoxy]-beta-D-erythro-pentofuranQsyl)-2(1H)-pyridone (35) is prepared as described for the 3-iodo derivative. Yield 74%; lENMR
(CDCl3) ~: 8.19 x 8.15 ~2s, 1, H6~, 7.5 - 6.8 (m, 23, aromatic), 6.51 (s, 1, 3H), 6.37 (d x d, 1, ~1'), 4.5 (m, 1, ~3') 4.19 (m, 1, H~'), 3.78 (2s, 6, 2 x -OCH3), 3.7 - 3.2 (m, 4, ~5' x 5" x -C~2-0-P), 2.75 x 2.15 (m, 2, ~2' x H2'l), 2.6 x 2.4 (2t, 2, -CH2CN) 1.35 - 1.05 lm, 14-N (i~Pr)2]. FAB mass spectrum (m/z) 1057 (M+Li), 753 (M+Li~DMT), ERFABMS observed 1057.
3035, calculated for Cs3~50lN4Og P 1057.2990.
. .
~2~
-63- 07-21(281)A
A mixture of 4-O-dlphenylcarbamoyl-3-io-do-1-~2-deoxy 3,5 O-[1,1,3,3-tetrakls(1-methyleth-yl)-1,3-dislloxanediyl]-beta-D-erythro-pentofuranosyl]-2(1H)-pyridone (28), (1.2g, 0.0015 mol), vinyl tri-n-butyltin (O.8 mL, 0.0027 mol) and palladium chloride bistriphenylphosphlne (0.lg) in dry aceto-nitrile (20 mL) ls heated at 60~C for 16 hours, under argon atmosphere. The solutlon is concentrated under reduced pressure and excess vinyl tri-n-butyltin ls removed by repeated (3 times) distillation with DMF.
The resulting viscous oil is dissolved in ethylace~ate (15 mL) filtered through celite and the flltrate concentrated to give a syrupy residue. This residue is purified by flash chromatography using EtOAc Hexane (1:4) as the eluent. The fractlons containing the desired product (visualized under W ) are combined, concentrated under reduced pressure and the resulting residue dried under high vacuum in a desicator to give 0.85g (82%) of 4-O-diphenylcarbamoyl-3-ethenyl-1 [2-deoxy-3,5-0-[1,1,3, 3-tetrakis(l-methyl ethyl)-1,3-disiloxanediyl]-beta-D-ery~hro-pentofuranosyl]-2(1H)-pyridone (36), as a pale yellow amorphous material.
Rf = 0.63 (EtOAc-Hexane 1~ HNMR 1300 MHz, CDCl3) ~:7.79 (d, J = 7.69Hz, 1, ~6), 7.42 - 7.2 (m, 10, aromatic), 6.45 (dxd, J = 17.7~z & 11.85~z, 1, CH=CH2) 6.35 (d, J = 7.69Hz, 1, H5), 6.2 (m, 2, Hl' x CH=CH2), 5.38 (dxd, J = 2.5 x 11.8~2)i 4.39 (m, 1, H3'), 4.2 (m, 2, ~5' x 5"), 3.8 (m, 1, ~4'), 2.6 (m, 1, ~2'), 2.26 (m, 1, H2"), 1.15 o 0.85 (m, 28, O~Si-(i-Pr)2)2;
FAB mass spectrum (m/z) 697 (M+Li), 339 (~+Li-sugar);
,. H~FABMS observed (N~Li) 697.3321, calculated for C3,H50N2O7 Si2Li 697.3317.
2 ~ 2 ~
-64- 07-21(281)A
To a chllled (ice bath) solution of 4-O-di-phenylcarbamoyl-3-ethenyl-1[2-deoxy-3,5-O-[1,1,3,3-tetrakls (l-methylethyl)-1,3-disiloxanediyl]-beta-D-erythro~ pentofuranosyl]-2(1H)-pyridone (36) (0.39, 0.00063 mol) in acetonitrile (5 mL) is added tetraethyl-ammonium fluoride (O.035g, 0.0002 mol). The reaction mixture is stirred at 0C for 30 minutes and at room temperature for 1 hour. During this period, desily-ation is complete as revealed by TLC of the reaction mixture. The solution is concentrated under reduced pressure and the residue purified by flash chroma-tography using CH2Cl2-MeOH (5%) containing 0.5% Et3N
to give 0.18g (94%) of 4 0-diphenylcarbamoyl-3-ethenyl-1(2-deoxy-~eta-D-erythro-pentofuranosyl)-2(lH)-pyridone (37). Rf = 0.26 (CH2Cl2-MeOH, 5%); lHNMR (300MHz, DMSO-d6) ~: 7.98 (d, J = 7.70Hz, H6), 7.55 - 7.2 ~m, 10, aromatic), 6.5 (m, 2, H5 & CH-C~2), 6.49 (d, J =
7.7Hz, 1, H5), 6.39 (dxd, J = 6.4Hz, 1, Hl'), 5.35 (dxd, 1, J = 3.01Hz, ll.9Hz), 5.24 (d, 1, J = 4.25 Hz, 2'-OH), 5.04 (t, 1, J = 5.14 Hz, 5'-OH), 4.23 (m, 1, H3'), 3.86 ~m, 1, H4'), 3.62 (m, 2, H5' x 5"), 2.35 (m, 1, H2'), 2.00 (m, 1, H2"), FAB mass spectrum (m/z) 4.49 (M~H), 333; HRF~BMS observed (M+Li) 455.1795 calculated for C2sH24N~o6Li 455.1794.
A mlxture of 4-_-diphenylcarbamoyl-3-ethe-nyl-1~2-deoxy-beta-D-erythro-pentofuranosyl)-2~lH)-pyri-done (37), (0.lg, 0.00022 mol), dimethoxytritylchloride (O.llg, 0.00032 mol) and diisopropylethylamine (O.08 mL, 0.00046 mol) in anhydrous pyridine ~3 mL) is stirred for 24 hours at xoo~ temperature under argon atmosphere. Pyridine is distilled off under reduced pressure and this residue purified by flach chroma-` tography using EtOAc-~exane (1:1) containing 0.5%
Et3N. Fractions containing the desired product (visualized under W lamp) are combined, concentrated 2~2~2 -65- 07-21(281)A
and this residue dried in a dessicator under high vacuum to afford 4-O-diphenylcarbamoyl-3-ethenyl-1-[5-O ~bis(4-methoxyphenyl)phenylmethyl]-2-deoxy-beta-D-ery~hro-pentofuranosyl]-2(lH)-pyridone (38), (0.13g, 78%) as white amorphous material. Rf 0.6 [CH2Cl2-MeOH (5%)]. 1HNMR (DMSO-d6) 7.8? (d, J =
7.7Hz, 1, H6), 7.46 - 6.8 (m, 23, aromatic) 6.5 (dxd, 1, -CH=CH2), 6.3 (m, 3, Hl', H5' x CH=CH2), 5.35 (m, 2, 2'-OH x -CH=CH2), 4.27 (m, 1, H3'), 3.99 (m, 1, H4'), 3.72 Is, 6, 2x-OCH3), 3.2~ (m, 2, H5' x H5"), 2.4 (m, 1, H2'), 2.05 ~m, 1, H2"); FAB mass spectrum (m/z): 757 (M+Li), 453.
To an ice cooled solution of 4-O-diphenyl-carbamoyl-3-ethenyl-1-[5-O-[bis(4-methoxyphenyl)phenyl-methyl]-2-deoxy-be~a-D-erythro-pentofuranosyl]-2(1H)-pyridone (38), (O.llg, 0.00015 mol) in dry dichloro-methane (5 mL) containing diisopropylamine (O.05 mL, 0.00029 mol), is added dropwise a solution of N,N,-diisopropylamine-~-cyanoethoxychlorophosphine (0.052g, 0.0002 mol) in dichloromethane (1 mL). The resulting mixture is stirred at 0C for 1 hour and at room temperature for 3 hours. TLC (EtOAC) revealed complete conversion to the product. The reaction mixture is concentrated under reduced pressure, the residue is dissolved in ethyl acetate (10 mL) and washed with 10%
NaHCO~ (2 x 10 mL) followed by water (2 x 10 mL).
The organic layer is dried (Na~S04) and concentrated under reduced pressure to give a syrupy material.
This syrupy material is purified by flash chromato-graphy using EtOAc containing 0.5% Et3N. The frac-tions containing 4-O-diphenylcarbamoyl-3-e~henyl-1-[5-O-[bis(4-methoxyphenyl~phenylmethyl]-3-O-~bis(l-methylethyl) amino] ~2~cyanoethoxy~phosphino]-2-de-oxy-beta-D-erythro-pentofuranosyl]-2(1~)-pyridone 2~2~
-66- 07-21(281)A
(39) are pooled, concentrated ~nd dried in a desicator under vacuum to afford (0.13g, 87%) as a white amorphous material. lHNMR (CDCl3) ~:7.93 x 7.8 (2d, J = 7.7Hz, 1, H6), 17.43 - 6.8 (m, 23, aromatic~ 6.55 - 6.3 (m, S 2, H1' x CH=CH2), 6.25 (m, 2, HS' x CH=CH2), 5.35 (m, 1, -CH=CH~), 4.50 (m, 1, H3'), 4.20 ~m, 1, H4'), 3.76 (s, 6, 2x-OCH3), 3.45 [m, 4, HS' x 5" & -N-lCH(CH3)2]2, 2.60 x 2.40 (2 (-5, -CH2-CH2CN x H2'), 2.25 (m, 1, H2"), 1.35 - 1.0 lm, 12,-N-[CH(CH3)2]2; FAB mass spectrum (m/z): 957 (M+Li), 733 & 653.
EXAMpr~ g To 5.0 ~m (7.3 x 10- 3 mole) 4-0-diphenylcar-bamoyl-l-[3,5-0-[1,1,3,3-tetrakis(l-methyl-ethyl)-1,3-disiloxanediyl]-beta-D-erythro-pentofurano-syl]-2(1H)-pyridone (4), in 30 mLs dry DMF can be added 0.24 gm (1 x 10- 2 mole) NaH while stirring.
To this is added 1.26 gm (1 x 10-2 mole) dimethyl sulfate. The reaction mixture is kept under argon until complete and then volatiles are removed under reduced pressure. The residue is taken up in EtOAc and the organic layer washed with saturated aqueous NaHCO3 and distilled water, then dried (MgSO4), filtered and solvent removed. Purification by silica gel chromatography can be used to give the desired product, 4-O-diphenylcar~amoyl~ [3,5-0-[1,1,3,3-tetra-kis(l-methylethyl)-1,3-disiloxanediyl]-2'-O-methyl]-~e~a-D-ery~hro-pentofuranosyl ]-2~lH)-pyridone (40).
To 3.1 gm (4.5 x 10-3 mole) 4-O-diphenylcarbamoyl-1-[~3, 5-0-[1,1,3,3-tetrakis(l-methylethyl)-1,3-disiloxanediyl]
-2-0-methyl ] -~eta-D-erythro-pentofuranosyl ] -2 ( lH) -pyridone (40) in 30 mL CH3CN at 0C is added 9.65- gm ( 2 . O x 10- 3 mole) tetraethylammonium fluoride trihydrate (Aldrich, Milwaukee, WI) while stirring. The reaction -67- 07-21(281)A
mixture is kept at OC for 30 minutes, warmed to room temperature and kept at room temperature for 30 mlnutes. Solvent is removed under reduced pressure and the desired product can be isolated by silica gel flash chromatography to give 4-O-diphenylcarbamoyl-l-(2-O-methyl -be ta-D-erythro-pentofuranosyl) 2(lH)-pyridone (41). To 1.4 gm (3.2 x 10-~ mole) 4-O-diphenylcarbamoyl-l-(2'-O-methyl-~eta-D-erythro-pentofuranosyl)-2(1~)-pyridone (41), in 15 mL dry pyridine is added 1.3 gm (3.95 x 10- 3 mole) diiso-propylethylamine and 30 mg 4-N,N-dimethylamino-pyridine (2.5 x 10-4 mole) and 1.2 gm (3.5 x 10-3 mole) dimethoxy tritylchloride. The reaction mixture is stirred at room temperature for 4 hours or until the reaction is substantially complete. Vola-tiles are removed and excess pyridine is removed by repeated (3 x 20mL) distillation of toluene under reduced pressure. The residue is purified by silica gel chromatography and 4-O-diphenylcarbamoyl-1-[5-O-~bis-(4-methoxyphenyl)phenylmethyl]-2'-O-methyl-beta-D-erytAro-pentofuranosyl~-2(lH)-pyridone (42), can be obtained. To 0.55 gm (7.3 x 10- 4 mole) 4-O-diphenyl-carbamoyl-1-[5'-O- fbis- (4-methoxyphenyl)phenylmethyl]-2'-O-methyl-beta-D-erythro pentofuranosyl]-2(lH~-pyridone (42), in C~2C12 is added 0.45 mL (2.5 x 10-3 mole) N, N-diisopropylethylamine. To this is added, dropwise over about 15 minutes 0.19 gm (8.0 x 10-~ mole~ ~-cyanoethyloxy-N,N-diisopropyl chloro-phosphine (American Bio~etics, Inc., ~ayward, CA) in 5 mL CR2Cl2. The reaction mixture is stirred and kept under an argon atmosphere at room temperature for an additional hour. The solution is concentrated to .~ dryness under vacuum usi~g a ro~ary evaporator and the residue taken up in EtOAc, washed with saturated aqueous NaHCO3 (3 x 20 mL) and distilled water, then dried (MgSO4~. The solution is concentrated to dryness and dried under vacuum to give a product 2 ~ 8 2 -6~- 07-21(281)A
which can be isolated by taklng lt up ln EtOAc and precipitating with hexane to give substantially pure 4-O-diphenylcarbamoyl-l-(5-O-~bis (4-methoxy phenyl) phenylmethyl]-3-O-[bis~l-methylethyl)amino (2-cyano-ethoxy)phosphino~-2-O-methyl-beta-D-eryt~ro~pento-furanosyl]-2(lH)-pyridone (43).
A mixture of 4-O-diphenylcarbamoyl-1-[3,5-O-[1,1,3,3-tetrakls(1-methylethyl)-1,3-disiloxanediyl]-beta-D-erythro-pentofuranosyl]-2(lH)-pyridone (4), (6.8 gm, 1 x 10-2 mole), N-iodosuccinimide (2.7 gm, 1.2 x 10-2 mole) in dry DMF containing dichloroacetic acid (0.36 gm, 2.8 x 10- 3 mole) is heated at 60C
under argon atmosphere until the reaction is complete.
The resulting mixture is concentrated in vacuo and the residue can be purified by silica gel flash chroma-tography to obtain as a major product 4-O diphenylcar-bamoyl-3 iodo-l-[3,5-O-[1,1,3,3-tetrakis (l-methyl-ethyl)-1,3-disiloxanediyl]-beta-D-erythro-pento-furanosyl ]-2(1H)-pyridone (44) and as a minor product 4-O-diphenylcarbamoyl-5-iodo-1~[3,5-O-[1,1,3, 3-tetrakis~1-methylethyl)-1,3-disiloxanediyl]-~eta-D-erythro-pentofuranosyl]-2(lH)-pyridone (45).
A mixture of 3.7 gm ~4.6 x 10-3 mole) of 4-O-diphenylcarbamoyl-3-iodo-1-[3,5-O-[1,1,3,3-tetra-kis(l-methylethyl)-1,3-disiloxanediyl] -beta-D-erythro-pentofuranosyl]-2(lH)-pyridone (44), and 0.88 gms (2.8 x 10-3 mole) tetrabutylammonium fluoride tri-hydrate in acetoni~rile is stirred a~ 0C for 30 minutes followed by stirring at room temperature for 30 minutes. The solution is concentrated in vacuo and 2~2~2 -69- 07-21(281)A
the residue can be purified by silica gel flash chromatography to obtain 4-O-diphenylcarbamoyl-3-iodo-l-beta-D-erythro-pentofuranosyl-2(1H)-pyridone (12).
In a similar fashion, the 5-iodo isomer, 4-O-diphenylcarbamoyl-5-iodo-1-beta-D-erythro-pento-furanosyl-2 (lH)-pyridone (13) can be obtained from 4~0-diphenylcarbamoyl-5-iodo 1-[3,5-0-[1,1,3,3-tetrakis ~l-methylethyl)-1,3-dlsiloxanediyl]-beta-D-erythro-pentofuranosyl]-2(lH~-pyridone (45).
EXAMPLE l?
All DNA oligonucleotides of the invention are synthesized on an ABI 380B synthesizer (Applied Biosystems, Inc., Foster City, CA) on a 1 micromole scale. Purification is achieved by polyacrylamide gel electrophoresis (PAGE). Oligonucleotides are labelled at their S' end by transfer of 32p phosphate from g~mma-32P-ATP using T4 polynucleotide kinase as described in T. Maniatis, E. F. Fritsch, and J.
Sambrook, "Molecular Cloning", Cold Spring Harbor Laboratory, (1982). Radiolabelled probe is separated from the reaction mixture by Sephadex G-50 chromato-graph (eluted with water). About 1 x 106 cpm/filter of labelled probe is included in the hybridization solution. After overnight hybridization, blots are washed in 6 x SSC (Standard Saline Citrate) at the appropriate temperature for 5 minutes. Autoradiograms are obtained using Kodak XAR-5 film with one intensi-fying screen (24 hours).
The 0.8 Kb DNA fragment encoding the small subunit of soybean ribulose-l, 5-bisphosphate (RuBP) carboxylase (S.C. Berry-Low et al., J. Mol. and_A~lied 2~2~
-70- 07-21(281)A
Gen., 1:483 (1982)) is obtained from plasmid pSRS 0.8 using the following conditlons. 6.8 uL (400 ug) of pSRS o.8 plasmid solution ls combined with 8 ~L of lOX
restriction buffer [SO mM NaCl, 10 mM tris Cl(pH=
7.5), 10 mM MgCl~, 1 mM dithiothreitol], 63 ~L deionized water, and 2 ~L EcoRI (20 units/~L) restriction enzyme. To obtain the homologous wheat RuBP fragment (R. Broglie, Biotechnology, 1:55 (1983)) 3 uL pw9 (1.3 mg/mL) plasmid is combined with 8 ~L of lOX restriction buffer, 67 ~L deionized water and 2 ~L PST (20 units/~L) restriction enzyme. Both reactions are incubated at 37C for l hour and subsequently heated to 70C for 5 minutes to denature the enzymes. The DNA in 20 ~L of each reaction mixture is separated by electrophoresis on a 1.2% agarose gel using lX TBE (Tris, Borate, EDTA) as buffer. After electrophoresis at 30V, overnight, the gel is washed 2X with 25PmM HC1 for 15 minutes, then washed (2X water) and the DNA denatured by soaking the gel in 1.5M NaCl, 0.5M NaOH (2 X 15 minutes). The denatured gel is washed twice with water and neutralized with 3.OM sodium chloride (NaCl), 0.5M tris HCl pH = 7.4 (2 X 20 mins). The DNA
fragments are transferred onto nitrocellulose paper according to Southern (E.M. Southern, J. Mol. Bio., 98:503 (1975)). Panels are prehybridized in 6 X SSC, lO X Denhardt's solution (0.2% BSA. 0.2% Ficell, and 0.2% polyvinylpyrolide) containing 100 ~g/mL yea~t tRNA at room temperature for 1-2 hours. Hybridization is carried out at 52-54C for 16-18 hours.
, A nitrocellulose filter is prepared with both soy and wheat RuBP carboxylase DNA in substantial accordance with the teaching of Example 13 and a single -71- 07-21(281)A
stranded oligonucleotide probe complementary to a target sequence on the 0.8 kb EcoRI fragment of soy RuBP is synthesi7ed. The target portion of the approxlmate 800bp DNA fragment has the sequence:
*
3'-CCGTCGAAGGTGTACCAGGT-5' The 20-base probe has the se~uence:
5'-GGCAGCTTCCACATGGTCCA-3' The sequence for wheat RuBP carboxylase DNA is the same except that an A is substltuted for C in the starred position. The effects of the G-A mismatch pairing when soy probe is hybridized with wheat target is determined by comparing the melting temperatures (Tm), the temperature at which 50% of the probe dissociates. The melting temperature of soy probe without 2'-deoxy-3-deazauridine hybridized to soy RuBP
DNA and soy probe hybridized to wheat RuBP DNA where there is a single base mismatch present is measured.
The hybridization panel is pretreated with about 15 mLs of 6 x SSC buffer (0.9 M NaCl, 0.09 M
sodium citrate), lOX Denhardt's solution and 110 ~g/mL yeast tRNA for about 3 hours at 5~C.
Normal oligonucleotide probes containing native bases and probes in which 2'-deoxy-3-deazauri-dine replaces cytidine in ~he nonmal seguence axe labelled at the 5' end with y_32p to a specific activity of about 3.9 x 106 cpm/pmole. About 0.15 pmole of labelled soy probe i added to fre~h hybrid~
ization ~uffer (10 mL~ and the panel is heated in the buffer at about 54C for 12 hour~. The panel is washed at 58C in 6 x SSC for 5 minutes and a~ auto-,'i:
~2~ 8~
72- 07-21(281)A
radiogram exposure (24 hours) obtained using a Kodak XAR-5 film with one intensifying screen. Soy RuBP DNA
is stron~ly exposed on the autoradiogram while wheat RuBP DNA is only weakly detectable. The panel is then washed at 60C for S mlnutes. The soy DNA signal remains strong and no wheat DNA signal is detected.
The panel is then washed at 63C and the soy signal lS
still visible but only to the extent of about half that visible at 60C. Thus, the melting temperature (Tm) of the soy RuBP probe and soy target DNA is about 63C and the Tm for the soy RuBP complement and wheat target DNA is estimated to be about 58C. The Tm for the probes listed in Example 18, Table III, were determined in a similar manner.
A soy RuBP probe is constructed in substan-tial accordance with the teaching of Example 14, in-cluding the same sequence, however, the cytidine at position 9 from the 5' end is replaced with 2'-deoxy-3-deazauridine. This probe is 5' labelled in substan-tial accordance with the teaching of Example 12 and has the following sequence:
5'-GGCAGCTTYCACATGGTCCA-3' `!' Y = 2'-deoxy-3-deazauridine ~ freshly prepared nitrocellulose panel is employed and the 2'-deo~y3-deazauridine containing probe is allowed to hybridiæe, and then washed in sub-stantial accordance with the teaching of Example 14.
.. Washes are conducted at 60, 70, 80 and 90C. The soy specific probes hybridize only to the soy RuBP DNA
and are distinctly visible throughout the wash cycle 2 ~
-73- 07-21(281)A
with wheat DNA weakly vlsible. At 90C the intensity of the 3NA band in the autoradiogram was about 25% of the intensity observed at 70C. (Intensity at 70C
is about equal to 60C).
In a separate experiment the soy probe contalning 2'-deoxy-3-deazauridine is resynthesized, labelled with 32p and a freshly prepared nitrocellu-lose panel is probed. Washes are conducted at temp-eratures of 64~, 66 and 70C. Again excellent selectivity and detectability are maintained. A 5 minute wash at 100C in 0.1 x SSC is accomplished with about one-half of the original (64C) concentration of probe remaining hybridized to the soy RuBP DNA.
The oligonucleotide probe complemen~ary to wheat RuBP DNA is synthesized in substantial accor-dance with the teaching of Example 12, with 2'-deoxy-3-deazauridine replacing cytidine at position 9 from the 5' end. The 20-base probe has the following sequence:
5'-GGC AGC TTY CAC ATA GTC CA-3' Y = 2'-deoxy-3-deazauridine A freshly prepared nitrocellulose panel is treated with the 2'-deoxy-3-deazauridine containing wheat probe in substan~ial accordance with the teach-ing o~ E~ample 13. Washes are conducted at 70, 80 and 90C. In this instance, the wheat probe detects only the wheat target D~A. At 90C about 50% of the original signal remains on ~he autoradiogram.
.
EXAMPLE l?
Three 17-base pair probes to the gene for Bacillus thuringiensis toxin (B.t.t.) are synthesized ~ ~ 2 ~
_74_ 07-21(281)A
ln substantial accordance with the teaching of Example 12. One wlth 2'-deoxy-3-deazauridine replacing cytidine at pOSltion 8 from the 5' end (Btt-8), another with 2'-deoxy-3-deazauridlne replacing cyti-dine at position 12 from the 5' end (Btt-12) and the normal probe without 2'-deoxy-3-deazauridine (Btt-N).
The probes have the following sequences:
5'-ATG M T CCG AAC AAT CG-3' (Btt-N) 5'-ATG AAT CYG AAC M T CG-3' Y = 2'-deoxy-3-deazauridine ~Btt-8) 5'- ATG AAT CCG AAY AAT CG-3' Y = 2'-deoxy-3-deazauridine (Btt-12) These probes are 32P-labeled in substantial accordance with the teaching of Example 12 and used to identify E. coli colonies containing the Btt gene.
Seventeen colonies are screened. Three of ~he ~. coli colonies contain the correct Btt gene, the remainder contain only a portion of the Btt gene and do not contain the portion to which the probe is designed to hybridize. Three duplicate colony filters are prepared in which the DNA from the E. coli colonies growing in a culture dish is transferred to the filter (ICN Bio-chemicals, "Protocols for DN~ and RNA Transfer, DNA
Electrotransfer and Protein Transfer to Biotrans~
Nylon Membran~s," 1985, Pall Corporation). One filter is probed with the Btt-N probe; ~he ~econd filter is probed with Btt-8 and the third probed with Btt-12.
The hybridization is carried out at about 40-42C for 20 hours. Each filter is subsequently washed in 1 X
SSC 1% SDS (sodium dodecyl sulfate) for 3 minutes at ~Ja~4l8~, 75- 07-21(281)A
45C. This wash is repeated and the filters are exposed to x-ray film for 4 hours. All three probes hybridize to the same colonles, all of which contain the correct Btt gene.
The filters are then washed at 65C for 5 minutes ln 1 X SSC 1% SDS. The normal probe, Btt-N, lS completely removed at this temperature, however, both Btt-8 and Btt-12 containing 2'-deoxy-3-deazauri-dine remain hybridized to the colonles containlng the Btt gene. The two 2'-deoxy-3-deazauridine containing probe filters are washed for 1 hour in 50% formamide containing about 10 mM sodium phosphate (Na~PO4), pH
6.5 at 65C. Both Btt-8 and Btt-12 are still hybri-dized after treatment and easily detected by auto-radiography.
Single-stranded oligonucleotide probes are synthesized in accordance with Example 12, without 2'-deoxy-3-deazauridine and with 2'-deoxy-3-deazauri-dine (indicated by "Y") substltuted for a cytidine.
The probes are hybrldized with complementary target DNA, and the corresponding melting temperatures are compared as ~hown in Table III. The hybrid molecules generated with probes containing 2'-deo~y-3-deazauri-dine have considerably higher apparent melting tempera-tures than hybrid molecules containing probes without 2'-deoxy-3-deazauridine, Table III lists probes of the invention complementary to soybean ribulose bis-phosphate (RuBP) carbo~ylase small subunit DNA (soy), wheat RuBP carboxylase small subuni~ DNA (wheat), the gene for Bacillus ~hurin~iensis toxin ~ ~t) and human ,. cytomegalovirus gene (HCV). Control probes (not shown) have the same sequences as the probes shown in Table III, however, control probes have a cytidine in place of "Y".
2~2~2 -76~ 07-21(281~A
_able III
Probe Sequence Tm*
Tm* Probes Probe Control of the Target Probe Sequence (5'-3') Length HT~ Probes Invention soy AGCT TCC AYA TGG TCC 16 Mer 40 56 >90C
soy AGCT TCC AYA TGG TCC A 17 mer 45 ~58 >90C
soy GCA GCT TYC ACA TGG TCC l8 mer 45 60 >90C
soy GGCA GCT TYC ACA TGG TCC A 20 mer 52 63 >90C
wheat GGC AGC TTY CAC ATA GTC CA 20 mer 52 ~65 >90C
Bt~ ATG M T CYG AAC AAT CG 17 mer 4l ~65 >90C
Btt ATG AAT CCG M Y AAT CG 17 mer 4l <65 >90C
HCV TAG CGG CGA CGY ACG TAC AC 20 mer 52 ND >90C
HC~ CTC TAG GYT GTC GGC CAG CC 20 mer 52 ND >90C .
soy TAG GCA GCT TCC AYA TGG TCC , AGT AGC 27 mer 40 76 >gOC
Y = 2'-deoxy-3-deazauridine * = Temperature at which approximately l/2 the signal of labeled probe remains *~ = Hybridization temperature C
2~32~
-77- 07-21(281)A
.
A soy RuBP probe is constructed in substan-tial accordance with the teaching of Example 14, including the same probe sequence, however, the cytidine at position 19 ~from the 5'-end) is replaced with 2'-deoxy~3-deazauridine:
5'-GGC AGC TTC CAC ATG GTC YA - 3' Y = 2'-deoxy~3-deazauridine This probe is 32-P labelled in substantial accordance with the teaching of Example 12 and employed in a Southern experiment using a freshly prepared nitro-cellulose blot in substantial accordance with Example 14. The extent of hybridizatlon of the probe is about equal to both soy and wheat DNA at 60C. Other washes are conducted at 63C, 70C and 83C. After the 83C
wash, about 15% of the concentration distinguishable at 63C still remains distinguishable for both soy and wheat RuBP DNA seguences.
A soy RuBP probe is constructed in substan-tial accordance wi~h the teaching of Example 14, including the same seguence, however, the cytidine at position 9 (from the 5' end) is replaced with 4-O-di-phenylcarbamoyl-3-iodo-1-(2-deoxy-beta-D-erythro-pento-furanosyl)-2(1~)-pyridone (30), which becomes 3-iodo-4-hydroxy-l-(2-deoxy-~eta-D-ery~hro-pentofuranosyl)-2(1~) pyridone, after depxotection. This probe is 32-P
labelled in substantial accordance with the teaching of Example 12 and has the following sequence:
.
S ' -GGC AGC T~X CAC ATG GTC CA - 3 ' X = 3-iodo-4-hydroxy-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-2(lH)-pyridone.
2 ~ 2 -78 07~ ~(2~ ~A) ' A freshly prepared nitrocellulose panel is employed and the 3-iodo-4-hydroxy-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-2(lH)-pyridone (12) containing probe allowed to hybridize in substantial accordance with the teaching of Example 12, however, ln this case 6 picomole of 32-P radlolabelled probe is employed.
Washes are accomplished in substantial accordance with the teaching of Example 21. At 80C about 50% of the concentration visible at 60C stlll remains visible.
Therefore, the apparent Tm (at which 50% of the signal remains) is about 80C.
A soy RuBP probe is constructed in substan-tial accordance with Example 20, however, X = 5-iodo-4-hydroxy-1-(2-deoxy-beta-D-eryt~ro-pentofuranosyl)-2 (lH)-pyridone, which is 4-0-diphenylcarbamoyl-5-iodo-1-(2-deoxy-beta~D-erythro-pentofuranosyl)-2(lH)-pyridone (31) prior to deprotection. A freshly prepared nitro-cellulose panel is employed and hybridization carried out at 52C wi~h 3 picomoles of 32-P labelled probe.
Washes are conducted at 58, 63, 70, 80 and 100C
in 6 x SSC for 5 minutes. At 100C approximately 50%
of the signal visible at 63C (the Tm of the probe without 5-iodo-4-hydroxy-1-(2-deoxy-~eta D~e~y~hro-pentofuranosyl)-2 (lH)-pyridone (13)) still remains visible. Therefore, the apparent Tm (at which 50% of the signal remains) is about 100C. Both soy and wheat RuBP DN~ are visible to an equal extent. When the hybridization is carried out at 37C, and the panel washed at 80C about 25% of ~he concentration visible at 60C still remains visible.
. .
A soy RuBP probe is constructed in substan-tial accordance with the teaching of Example 20, -79- 07-21(281)A
however, X = 4-O-dlphenylcarbamoyl-3-ethenyl~ 2-deoxy-beta-D-erythro-pentofuranosyl)-2(lH)-pyridone (37).
A freshly prepared nitrocellulose panel is employed and hybridizatlon carried out at 53C with 32-P
labelled probe. In this case, the autoradlographlc signal for both soy and wheat sequences were equally intense after washing at 63C for 5 minutes. Further washes were conducted at 69, 80 and 100C. About 75% of the signal visible at 63C (the Tm of the probe without 4-O-diphenylcarbamoyl-3-ethenyl-1 (2-deoxy-beta-D-erythro-pentofuranosyl)-2(1H)-pyridone (37)) remains at 80C for both soy and wheat D~A.
About 10% of the signal visible at 63C remains after the 100C wash. Therefore, the apparent Tm (at which 50% of the signal remains) is about 80C.
A soy RuBP probe is constructed in substan-tial accordance with the teaching of Example 14, however, the probe (Soy 27) is extended to include 27 bases of soy sequence:
5' TAG GCA GCT TCC ACA TGG TCC AGT AGC 3' (Soy 27) and the cytidine at position 14 from the 5' end is replaced with 2'-deoxy -3-deazauridine:
5' TAG GCA GCT TCC AYA TGG TCC AGT AGC 3' Y = 2'-deoxy-3-deazauridine.
The probe is 32P-labelled and hybridized to a freshly prepared panel in substantial accord~nce with Example ,. 14, except the hybridization temperature is 4aC.
.
'~ ~s~ 2 -80- 07-21(281)A
Washes are carried out at 66, 75, 79, 90 and 100C. Both soy and wheat DNA are visualized to an equal extent throughout the wash cycle. At 90C about 25% of the DNA visible at 79C still remains visible.
S At 100C the slgnal is weak but still easily discern-ible.
Although the invention has been described with respect to specific modifications, the details thereof are not to be construed as limitations, for it will be apparent that various equivalents, changes and modifications may be resorted to without departing from the spirit and scope thereof and it is understood that such equivalent embodiments are to be included herein.
Claims (41)
THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A single-stranded nucleic acid probe which comprises a nucleoside residue of the formula:
in which R1, and R2 independently are C1-C5 alkyl, C2-C5 alkenyl, halo or hydrogen, R3 is hydrogen, hydroxy, C6-C14 aryloxy or C1-C5 alkoxy.
in which R1, and R2 independently are C1-C5 alkyl, C2-C5 alkenyl, halo or hydrogen, R3 is hydrogen, hydroxy, C6-C14 aryloxy or C1-C5 alkoxy.
2. The probe of Claim 1 in which R1, R2 and R3 are hydrogen.
3. The probe of Claim 1 in which R1 and R2 are hydrogen and R3 is hydroxy.
4. The probe of Claim 1 in which R1 is iodine and R2 and R3 are hydrogen.
5. The probe of Claim 1 in which R1 is iodine, R2 is hydrogen and R3 is hydroxy.
6. The probe of Claim 1 in which R1 is ethenyl and R2 and R3 are hydrogen.
7. The probe of Claim 1 in which R1 is ethenyl, R2 is hydrogen and R3 is hydroxy.
8. The probe of Claim 1 in which R1 and R3 are hydrogen and R2 is iodine.
9. The probe of Claim 1 in which R1 is hydrogen, R2 is iodine and R3 is hydroxy.
-82- 07-21(281)A
-82- 07-21(281)A
10. The probe of Claim 1 in which the probe is 15 to 50 nucleotides in length.
11. The probe of Claim 1 in which the probe is is to 25 nucleotides in length.
12. The probe of Claim 1 in which the single-stranded nucleic acid is DNA.
13. The probe of Claim 1 in which the single-stranded nucleic acid is RNA.
14. The probe which is selected from the group consisting of in which Y = 4-hydroxy-1(2-deoxy-beta-D-erythro-pentofuranosyl-2(H)-pyridone.
15. The probe having the following sequence:
GGCAGCTTXCACATGGTCCA, in which X = 3-iodo-4-hydroxy-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-2(1H)-pyridone
GGCAGCTTXCACATGGTCCA, in which X = 3-iodo-4-hydroxy-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-2(1H)-pyridone
16. The probe having the following sequence:
GGCAGCTTXCACATGGTCCA, in which X = 5-iodo-4-hydroxy-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-2(1H)-pyri-done.
GGCAGCTTXCACATGGTCCA, in which X = 5-iodo-4-hydroxy-1-(2-deoxy-beta-D-erythro-pentofuranosyl)-2(1H)-pyri-done.
17. The probe having the following sequence: GGCAGCTTTXCACATGGTCCA, in which X = 4-O-di-phenylcarbamoyi-3-ethenyl-1-(2-deoxy-beta-D-erythro-pentofuranosyl))-2(1H)-pyridone.
18. A method for preparing hybrid DNA or RNA molecules which comprises contacting a single-stranded nucleic acid probe which comprises a nucleo-side residue of the formula:
-83- 07-21(281)A
in which R1 and R2 independently are C1-C5 alkyl, C2-C5 alkenyl, halo or hydrogen, R5 is hydrogen, hydroxy, C6-C14 aryloxy or C1-C5 alkoxy, with target nucleic acid sequences under conditions suitable for hybridization, then recovering the hybrid molecule under conditions that remove a hybrid molecule not containing a probe which comprises the nucleoside residue of the above described formula.
-83- 07-21(281)A
in which R1 and R2 independently are C1-C5 alkyl, C2-C5 alkenyl, halo or hydrogen, R5 is hydrogen, hydroxy, C6-C14 aryloxy or C1-C5 alkoxy, with target nucleic acid sequences under conditions suitable for hybridization, then recovering the hybrid molecule under conditions that remove a hybrid molecule not containing a probe which comprises the nucleoside residue of the above described formula.
19. The method of Claim 18 in which the hybrid molecule is recovered at temperatures of 50 -100°C.
20. The method of Claim 18 in which the hybrid molecule is recovered at temperatures of 65 85°C.
21. The method of Claim 18 in which R1 and R2 are hydrogen and R3 is hydroxy.
22. The method of Claim 18 in which R1, R2 and R3 are hydrogen.
23. A hybridized molecule comprising a probe of Claim 1.
-84- 07-21(281)A
-84- 07-21(281)A
24. The hybridized molecule of Claim 23 in which R1, R2 and R3 are hydrogen.
25. The hybridized molecule of Claim 23 in which R1 and R2 are hydrogen and R3 is hydroxy.
26. The nucleoside residue of Claim 1 in which R1 is iodine and R2 and R3 are hydrogen.
27. The nucleoside residue of Claim 1 in which R1 is iodine, R2 is hydrogen and R3 is hydroxy.
28. The nucleoside residue of Claim 1 in which R1 and R3 are hydrogen and R2 is iodine.
29. The nucleoside residue of Claim 1 in which R1 is hydrogen, R2 is iodine and R3 is hydroxy.
30. The nucleoside residue of Claim 1 in which R1, is ethenyl, and R2 and R3 are hydrogen.
31. The nucleoside residue of Claim 1 in which R1 is ethenyl, R2 is hydrogen and R3 is hydroxy.
32. A compound of the formula:
in which R1 and R2 independently are C1-C5 alkyl, C2-C5 alkenyl, halo or hydrogen, R3 is hydrogen, halo, C1-C5 alkoxy or tri(C1-C5 alkyl)substituted silyloxy, R4 is triphenylmethyl or mono-, di- or tri -85- 07-21(281)A
(C1-C5 alkoxy)substituted triphenylmethyl, R5 is hydrogen phosphonate or R'-?-R" in which R' is .beta.-cyanoethyl, C1-C5 alkoxy, or C1-C5 alkyl, and R"
is morpholino, or mono- or di(C1-C1 alkyl)substituted amino, R6 is tri(C1-C5 alkyl or C1-C5 alkoxy) substi-tuted silyloxy, benzoyl, methylcarbonyl, carbamoyl, or carbamoyl substituted with C1-C5 alkyl, phenyl, acetyl or isobutyryl.
in which R1 and R2 independently are C1-C5 alkyl, C2-C5 alkenyl, halo or hydrogen, R3 is hydrogen, halo, C1-C5 alkoxy or tri(C1-C5 alkyl)substituted silyloxy, R4 is triphenylmethyl or mono-, di- or tri -85- 07-21(281)A
(C1-C5 alkoxy)substituted triphenylmethyl, R5 is hydrogen phosphonate or R'-?-R" in which R' is .beta.-cyanoethyl, C1-C5 alkoxy, or C1-C5 alkyl, and R"
is morpholino, or mono- or di(C1-C1 alkyl)substituted amino, R6 is tri(C1-C5 alkyl or C1-C5 alkoxy) substi-tuted silyloxy, benzoyl, methylcarbonyl, carbamoyl, or carbamoyl substituted with C1-C5 alkyl, phenyl, acetyl or isobutyryl.
33. The compound of Claim 32 in which R1, R2 and R3 are hydrogen, R4 is dimethoxytriphenyl-methyl, R' is .beta.-cyanoethyl, R" is N,N-diisopropyl amino and R6 is diphenylcarbamoyl.
34. The compound of Claim 32 in which R1 and R2 are hydrogen, R3 is triisopropylsilyloxy, R4 is dimethoxytriphenylmethyl, R' is .beta.-cyanoethyl, R"
is N,N-diisopropyl amino and R6 is diphenylcarbamoyl.
is N,N-diisopropyl amino and R6 is diphenylcarbamoyl.
35. The compound of Claim 32 in which R1 and R2 are hydrogen, R3 is tert butyl-dimethyl-silyloxy, R4 is dimethoxytriphenylmethyl, R' is .beta.-cyanoethyl, R" is N,N-diisopropyl amino, and R6 is diphenylcarbamoyl.
36. The compound of Claim 32 in which R1 is iodine, R2 is hydrogen, R3 is hydrogen, R4 is dimethoxytriphenylmethyl, R' is .beta.-cyanoethyl, R" is N,N-diisopropyl amino and R6 is diphenylcarbamoyl.
37. The compound of Claim 32 in which R1 is iodine, R2 is hydrogen, R3 is triisopropylsilyl-oxy, R4 is dimethoxytriphenylmethyl, R' is .beta.-cyanoethyl, R" is N,N-diisopropyl amino and R6 is diphenylcarbamoyl.
38. The compound of Claim 32 in which R1 is hydrogen, R2 is iodine, R3 is triisopropylsilyl-oxy, R4 is dimethoxytrityl, R' is .beta.-cyanoethyl, R" is N,N-diisopropyl amino and R6 is diphenylcarbamoyl.
39. The compound of Claim 32 in which R1 is hydrogen, R2 is iodine, R3 is hydrogen, R4 is dimeth--86- 07-21(281)A
oxytriphenylmethyl, R' is .beta.-cyanoethyl, R" is N,N-di-isopropyl amino and R6 is diphenylcarbamoyl.
oxytriphenylmethyl, R' is .beta.-cyanoethyl, R" is N,N-di-isopropyl amino and R6 is diphenylcarbamoyl.
40. The compound of Claim 32 in which R1 is ethenyl, R2 is hydrogen, R3 is triisopropylsilyl-oxy, R4 is dimethoxytriphenylmethyl, R' is .beta.-cyanoethyl, R" is N,N-diisopropyl amino and R6 is diphenylcar-bamoyl.
41. The compound of Claim 32 in which R1 is ethenyl, R2 is hydrogen, R3 is hydrogen, R4 is dimeth-oxytriphenylmethyl, R' is .beta.-cyanoethyl, R" is N,N-di-isopropyl amino and R6 is diphenylcarbamoyl.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US07/400,205 US5134066A (en) | 1989-08-29 | 1989-08-29 | Improved probes using nucleosides containing 3-dezauracil analogs |
| US400,205 | 1989-08-29 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2024182A1 true CA2024182A1 (en) | 1991-03-01 |
Family
ID=23582650
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002024182A Abandoned CA2024182A1 (en) | 1989-08-29 | 1990-08-28 | Probes |
Country Status (4)
| Country | Link |
|---|---|
| US (2) | US5134066A (en) |
| EP (1) | EP0415901A3 (en) |
| JP (1) | JPH06102676B2 (en) |
| CA (1) | CA2024182A1 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CH541566A (en) * | 1969-04-11 | 1973-09-15 | Schering Ag | Process for the preparation of nucleosides |
| US3705147A (en) * | 1969-08-22 | 1972-12-05 | Univ Utah | 3-deazapyrimidine nucleosides and method of preparation thereof |
| JPS5136758B2 (en) * | 1971-12-14 | 1976-10-12 | ||
| US3873516A (en) * | 1973-05-29 | 1975-03-25 | Miles Lab | Process for the preparation of 1-{8 {62 -D-2-substituted-2-deoxyribofuranosyl{9 -pyrimidines |
| US4321366A (en) * | 1978-05-12 | 1982-03-23 | Research Corporation | Process of preparing 2-azido-2-deoxyarabinofuranosyl halides |
| US4211773A (en) * | 1978-10-02 | 1980-07-08 | Sloan Kettering Institute For Cancer Research | 5-Substituted 1-(2'-Deoxy-2'-substituted-β-D-arabinofuranosyl)pyrimidine nucleosides |
| EP0015584A3 (en) * | 1979-03-12 | 1980-12-10 | Kailash Kumar Dr. Prof. Gauri | Nucleotides, methods for their preparation and medicaments |
| SU937459A1 (en) * | 1980-09-12 | 1982-06-23 | Всесоюзный Научно-Исследовательский Институт Молекулярной Биологии | Process for producing 2,3-didesoxynucleosides |
| US4334059A (en) * | 1980-10-20 | 1982-06-08 | Bio Logicals Inc. | Silylated arabino-base compounds |
| US4711955A (en) * | 1981-04-17 | 1987-12-08 | Yale University | Modified nucleotides and methods of preparing and using same |
| US4556643A (en) * | 1982-07-26 | 1985-12-03 | Agracetus | Assay method and probe for polynucleotide sequences |
| JPS5993095A (en) * | 1982-11-17 | 1984-05-29 | Sankyo Co Ltd | 3-deazauridine derivative |
| US4605735A (en) * | 1983-02-14 | 1986-08-12 | Wakunaga Seiyaku Kabushiki Kaisha | Oligonucleotide derivatives |
| US4599303A (en) * | 1983-12-12 | 1986-07-08 | Hri Associates, Inc. | Nucleic acid hybridization assay employing probes crosslinkable to target sequences |
| US5015733A (en) * | 1983-12-20 | 1991-05-14 | California Institute Of Technology | Nucleosides possessing blocked aliphatic amino groups |
| US4563417A (en) * | 1984-08-31 | 1986-01-07 | Miles Laboratories, Inc. | Nucleic acid hybridization assay employing antibodies to intercalation complexes |
| US4681873A (en) * | 1985-07-29 | 1987-07-21 | Warner-Lambert Company | 4-amino-3-halo-2-pyridinone nucleoside and nucleotide compounds |
| US5093232A (en) * | 1985-12-11 | 1992-03-03 | Chiron Corporation | Nucleic acid probes |
| EP0266099A3 (en) * | 1986-10-28 | 1990-09-19 | The Johns Hopkins University | Oligonucleoside alkyl or arylphosphonate derivatives capable of crosslinking with or cleaving nucleic acids |
| US5030557A (en) * | 1987-11-24 | 1991-07-09 | Ml Technology Venture | Means and method for enhancing nucleic acid hybridization |
| US5006646A (en) * | 1989-02-22 | 1991-04-09 | Yuki Gosei Kogyo Co., Ltd. | Process for preparing 2'-deoxy-5-trifluoromethyl-beta-uridine |
-
1989
- 1989-08-29 US US07/400,205 patent/US5134066A/en not_active Expired - Lifetime
-
1990
- 1990-08-24 EP EP19900870134 patent/EP0415901A3/en not_active Ceased
- 1990-08-28 CA CA002024182A patent/CA2024182A1/en not_active Abandoned
- 1990-08-28 JP JP2226438A patent/JPH06102676B2/en not_active Expired - Lifetime
-
1992
- 1992-05-06 US US07/879,648 patent/US5359053A/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| US5359053A (en) | 1994-10-25 |
| EP0415901A3 (en) | 1992-10-28 |
| JPH06102676B2 (en) | 1994-12-14 |
| US5134066A (en) | 1992-07-28 |
| JPH0398599A (en) | 1991-04-24 |
| EP0415901A2 (en) | 1991-03-06 |
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Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| FZDE | Discontinued | ||
| FZDE | Discontinued |
Effective date: 19970828 |