NZ622268B2 - 5-methoxy, 3'-oh unblocked, fast photocleavable terminating nucleotides and methods for nucleic acid sequencing - Google Patents
5-methoxy, 3'-oh unblocked, fast photocleavable terminating nucleotides and methods for nucleic acid sequencing Download PDFInfo
- Publication number
- NZ622268B2 NZ622268B2 NZ622268A NZ62226812A NZ622268B2 NZ 622268 B2 NZ622268 B2 NZ 622268B2 NZ 622268 A NZ622268 A NZ 622268A NZ 62226812 A NZ62226812 A NZ 62226812A NZ 622268 B2 NZ622268 B2 NZ 622268B2
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- New Zealand
- Prior art keywords
- compound
- ome
- alkanediyl
- group
- nucleic acid
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- 125000003729 nucleotide group Chemical group 0.000 title claims abstract description 115
- 239000002773 nucleotide Substances 0.000 title claims abstract description 100
- 108020004707 nucleic acids Proteins 0.000 title claims description 72
- 150000007523 nucleic acids Chemical class 0.000 title claims description 71
- 150000001875 compounds Chemical class 0.000 claims abstract description 284
- -1 5-methoxy-substituted nitrobenzyl Chemical group 0.000 claims abstract description 92
- 238000010348 incorporation Methods 0.000 claims abstract description 77
- 239000002777 nucleoside Substances 0.000 claims abstract description 38
- 229910052739 hydrogen Inorganic materials 0.000 claims description 394
- 125000000956 methoxy group Chemical group [H]C([H])([H])O* 0.000 claims description 222
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 149
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 147
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 claims description 145
- 238000006243 chemical reaction Methods 0.000 claims description 92
- 229920003013 deoxyribonucleic acid Polymers 0.000 claims description 83
- 238000003786 synthesis reaction Methods 0.000 claims description 69
- 230000015572 biosynthetic process Effects 0.000 claims description 67
- 230000002194 synthesizing Effects 0.000 claims description 66
- 229910052799 carbon Inorganic materials 0.000 claims description 61
- 238000007792 addition Methods 0.000 claims description 56
- 239000001257 hydrogen Substances 0.000 claims description 54
- 239000001226 triphosphate Chemical group 0.000 claims description 50
- 125000004435 hydrogen atoms Chemical group [H]* 0.000 claims description 48
- 235000011178 triphosphate Nutrition 0.000 claims description 46
- 125000000217 alkyl group Chemical group 0.000 claims description 44
- 101700011961 DPOM Proteins 0.000 claims description 38
- 101710029649 MDV043 Proteins 0.000 claims description 38
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- 101700054624 RF1 Proteins 0.000 claims description 38
- 238000003384 imaging method Methods 0.000 claims description 38
- 125000003118 aryl group Chemical group 0.000 claims description 35
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 claims description 35
- UNXRWKVEANCORM-UHFFFAOYSA-I triphosphate(5-) Chemical group [O-]P([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O UNXRWKVEANCORM-UHFFFAOYSA-I 0.000 claims description 34
- 150000003839 salts Chemical class 0.000 claims description 33
- 239000011780 sodium chloride Substances 0.000 claims description 33
- 239000007787 solid Substances 0.000 claims description 31
- 125000005647 linker group Chemical group 0.000 claims description 28
- 125000004432 carbon atoms Chemical group C* 0.000 claims description 27
- VHJLVAABSRFDPM-UHFFFAOYSA-N 1,4-dimercaptobutane-2,3-diol Chemical compound SCC(O)C(O)CS VHJLVAABSRFDPM-UHFFFAOYSA-N 0.000 claims description 25
- 239000002253 acid Substances 0.000 claims description 23
- 150000003833 nucleoside derivatives Chemical class 0.000 claims description 23
- 239000011324 bead Substances 0.000 claims description 20
- 125000004093 cyano group Chemical group *C#N 0.000 claims description 20
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 claims description 17
- 125000000732 arylene group Chemical group 0.000 claims description 16
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 claims description 16
- 125000002252 acyl group Chemical group 0.000 claims description 12
- 229910052760 oxygen Inorganic materials 0.000 claims description 12
- 230000000295 complement Effects 0.000 claims description 11
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 claims description 11
- 229920000388 Polyphosphate Polymers 0.000 claims description 10
- 125000003710 aryl alkyl group Chemical group 0.000 claims description 10
- 239000001205 polyphosphate Substances 0.000 claims description 10
- 235000011176 polyphosphates Nutrition 0.000 claims description 10
- 125000000547 substituted alkyl group Chemical group 0.000 claims description 10
- 239000000758 substrate Substances 0.000 claims description 10
- 125000002264 triphosphate group Chemical group [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 9
- UMGDCJDMYOKAJW-UHFFFAOYSA-N Thiourea Chemical compound NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 0.000 claims description 9
- 125000003342 alkenyl group Chemical group 0.000 claims description 9
- 125000003368 amide group Chemical group 0.000 claims description 9
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 claims description 9
- 125000001072 heteroaryl group Chemical group 0.000 claims description 9
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N Coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 claims description 8
- XPPKVPWEQAFLFU-UHFFFAOYSA-J Pyrophosphate Chemical group [O-]P([O-])(=O)OP([O-])([O-])=O XPPKVPWEQAFLFU-UHFFFAOYSA-J 0.000 claims description 8
- 229910006069 SO3H Inorganic materials 0.000 claims description 8
- 125000003282 alkyl amino group Chemical group 0.000 claims description 8
- 125000000304 alkynyl group Chemical group 0.000 claims description 8
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- 125000005843 halogen group Chemical group 0.000 claims description 8
- 238000007480 sanger sequencing Methods 0.000 claims description 8
- 238000005406 washing Methods 0.000 claims description 8
- 125000004423 acyloxy group Chemical group 0.000 claims description 7
- 125000003396 thiol group Chemical group [H]S* 0.000 claims description 7
- IEQIEDJGQAUEQZ-UHFFFAOYSA-N Phthalocyanine Chemical compound N1C(N=C2C3=CC=CC=C3C(N=C3C4=CC=CC=C4C(=N4)N3)=N2)=C(C=CC=C2)C2=C1N=C1C2=CC=CC=C2C4=N1 IEQIEDJGQAUEQZ-UHFFFAOYSA-N 0.000 claims description 6
- 150000007513 acids Chemical class 0.000 claims description 6
- 125000003545 alkoxy group Chemical group 0.000 claims description 6
- 230000003595 spectral Effects 0.000 claims description 6
- 125000004663 dialkyl amino group Chemical group 0.000 claims description 5
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- ANRHNWWPFJCPAZ-UHFFFAOYSA-M CHEMBL593252 Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 claims description 4
- 235000001671 coumarin Nutrition 0.000 claims description 4
- 229960000956 coumarin Drugs 0.000 claims description 4
- 239000001177 diphosphate Chemical group 0.000 claims description 4
- VMHLLURERBWHNL-UHFFFAOYSA-M sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 4
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- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 3
- 108060006184 phycobiliprotein family Proteins 0.000 claims description 3
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- 125000000896 monocarboxylic acid group Chemical group 0.000 claims 1
- 238000001668 nucleic acid synthesis Methods 0.000 claims 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 claims 1
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- 238000003776 cleavage reaction Methods 0.000 abstract description 56
- 230000002441 reversible Effects 0.000 abstract description 47
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- 125000003835 nucleoside group Chemical group 0.000 abstract description 8
- 238000004458 analytical method Methods 0.000 abstract description 7
- 238000003559 rna-seq method Methods 0.000 abstract 1
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 180
- UHOVQNZJYSORNB-UHFFFAOYSA-N benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 170
- 239000000203 mixture Substances 0.000 description 165
- WYURNTSHIVDZCO-UHFFFAOYSA-N tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 148
- 239000000243 solution Substances 0.000 description 145
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 142
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- FPGGTKZVZWFYPV-UHFFFAOYSA-M tetrabutylammonium fluoride Substances [F-].CCCC[N+](CCCC)(CCCC)CCCC FPGGTKZVZWFYPV-UHFFFAOYSA-M 0.000 description 38
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- 239000011975 tartaric acid Substances 0.000 description 1
- 229960001367 tartaric acid Drugs 0.000 description 1
- 125000004213 tert-butoxy group Chemical group [H]C([H])([H])C(O*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 125000003718 tetrahydrofuranyl group Chemical group 0.000 description 1
- 125000001412 tetrahydropyranyl group Chemical group 0.000 description 1
- 125000003831 tetrazolyl group Chemical group 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 150000004897 thiazines Chemical class 0.000 description 1
- 125000000335 thiazolyl group Chemical group 0.000 description 1
- 125000001544 thienyl group Chemical group 0.000 description 1
- 125000002813 thiocarbonyl group Chemical group *C(*)=S 0.000 description 1
- 125000004568 thiomorpholinyl group Chemical group 0.000 description 1
- RYYWUUFWQRZTIU-UHFFFAOYSA-K thiophosphate Chemical group [O-]P([O-])([O-])=S RYYWUUFWQRZTIU-UHFFFAOYSA-K 0.000 description 1
- 150000003585 thioureas Chemical class 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 125000003944 tolyl group Chemical group 0.000 description 1
- 230000002588 toxic Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000027 toxicology Toxicity 0.000 description 1
- 230000001131 transforming Effects 0.000 description 1
- 125000005627 triarylcarbonium group Chemical group 0.000 description 1
- 125000004306 triazinyl group Chemical group 0.000 description 1
- 125000001425 triazolyl group Chemical group 0.000 description 1
- IMFACGCPASFAPR-UHFFFAOYSA-O tributylazanium Chemical compound CCCC[NH+](CCCC)CCCC IMFACGCPASFAPR-UHFFFAOYSA-O 0.000 description 1
- 125000002306 tributylsilyl group Chemical group C(CCC)[Si](CCCC)(CCCC)* 0.000 description 1
- 229910052722 tritium Inorganic materials 0.000 description 1
- YZCKVEUIGOORGS-NJFSPNSNSA-N tritium Chemical compound [3H] YZCKVEUIGOORGS-NJFSPNSNSA-N 0.000 description 1
- 229960000281 trometamol Drugs 0.000 description 1
- 239000005436 troposphere Substances 0.000 description 1
- 238000004450 types of analysis Methods 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
- 229910052724 xenon Inorganic materials 0.000 description 1
- FHNFHKCVQCLJFQ-UHFFFAOYSA-N xenon(0) Chemical compound [Xe] FHNFHKCVQCLJFQ-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/06—Auxiliary integrated devices, integrated components
- B01L2300/0627—Sensor or part of a sensor is integrated
- B01L2300/0654—Lenses; Optical fibres
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2300/00—Additional constructional details
- B01L2300/16—Surface properties and coatings
- B01L2300/168—Specific optical properties, e.g. reflective coatings
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
- C07H19/073—Pyrimidine radicals with 2-deoxyribosyl as the saccharide radical
-
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
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- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
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- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/16—Purine radicals
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- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
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Abstract
The disclosure relates to 3'-OH unblocked nucleotides and nucleosides labeled and unlabeled with 5-methoxy-substituted nitrobenzyl-based photocleavable terminating groups (compounds I – VII) for use in methods and systems related to DNA and RNA sequencing and analysis. These compounds may be used as reversible terminators as they exhibit fast nucleotide incorporation kinetics, single-base termination, high nucleotide selectivity, and rapid terminating group cleavage that results in a naturally occurring nucleotide. reversible terminators as they exhibit fast nucleotide incorporation kinetics, single-base termination, high nucleotide selectivity, and rapid terminating group cleavage that results in a naturally occurring nucleotide.
Description
DESCRIPTION
—METHOXY, 3'—OH UNBLOCKED, FAST PHOTOCLEAVABLE TERMINATING
NUCLEOTIDES AND S FOR NUCLEIC ACID SEQUENCING
BACKGROUND OF THE INVENTION
The present application claims the benefit of priority to US. ional Application
61/627,211, filed October 7, 2011, and US. Provisional Application 61/534,347, filed
September 13, 2011, the contents of both applications are incorporated herein by reference.
1. Field of the Invention
The present invention relates generally to compositions and methods for DNA
sequencing and other types of DNA analysis. More particularly, the invention relates in part
to fast 3’—OH unblocked nucleotides and nucleosides with photochemically cleavable groups
and methods for their use in a number of DNA sequencing methods, including applications in
biomedical research.
11. Description of Related Art
Methods for rapidly sequencing DNA are needed for analyzing diseases and
mutations in the population and developing therapies (Metzker, 2010, which is incorporated
herein by reference). Commonly observed forms of human sequence variation are single
nucleotide polymorphisms (SNPs), which occur in approximately l-in—300 to 000 base
pairs of c sequence and structural variants (SVs) including block substitutions,
ion/deletions, inversions, segmental duplications, and copy number variants. Structural
variants can account for 22% of all variable events and more variant bases than those
contributed by SNPs (Levy et al., 2007, which is incorporated herein by nce). This
finding is consistent with that of Scherer, , and gues who analyzed 270
individuals using microarray based s (Redon et al., 2006, which is incorporated herein
by nce). Building upon the complete sequence of the human genome, efforts are
underway to identify the underlying genetic link to common diseases and cancer by SNP and
SV mapping or direct ation. Technology developments focused on rapid, high—
throughput, and low cost DNA cing would facilitate the understanding and use of
genetic information, such as SNPs and SVs, in applied medicine.
In general, 10%-to-15% of SNPs will affect protein function by altering specific
amino acid residues, will affect the proper processing of genes by changing splicing
mechanisms, or will affect the normal level of expression of the gene or protein by varying
regulatory mechanisms. SVs may also play an important role in human biology and disease
(Iafrate et al., 2004; Sebat et al., 2004; Tuzun et al., 2005; Stranger et al., 2007, which are
incorporated herein by reference). It is envisioned that the identification of informative SNPs
and SVs will lead to more accurate diagnosis of ted disease, better prognosis of risk
susceptibilities, or identity of sporadic mutations in tissue. One application of an individual’s
SNP and SV profile would be to significantly delay the onset or progression of disease with
prophylactic drug therapies. Moreover, an SNP and SV e of drug metabolizing genes
could be used to prescribe a specific drug regimen to provide safer and more cious
results. To accomplish these ous goals, genome sequencing will move into the
resequencing phase with the potential of partial sequencing of a large ty of the
population, which would involve sequencing specific regions in parallel, which are
buted throughout the human genome to obtain the SNP and SV profile for a given
complex disease.
Sequence variations underlying most common diseases are likely to involve multiple
SNPs, SVs, and a number of combinations thereof, which are dispersed throughout associated
genes and exist in low frequency. Thus, DNA sequencing technologies that employ
strategies for de novo cing are more likely to detect and/or discover these rare, widely
dispersed variants than technologies targeting only known SNPs.
One example how NGS logies can be applied in the detection of SNPs, SVs,
single nucleotide variants (SNVs) and a number of combinations thereof is cancer
diagnostics. These assays have ionally been a single-marker, -assay ch that
has ly progressed to assaying multiple markers with a single experimental approach.
However, each cancer is genetically complex often with many mutations occurring
simultaneously in numerous genes. Therefore, traditional methods lead to ive and
onsuming testing, while providing information only on a select few of known sequence
variants. Recent advances in NGS technologies have allowed targeted approaches that center
on many medically actionable gene targets associated with various cancer types (See Su et
al., 2011; Beadling et a]. 2012). Due to recent successes of sequencing efforts, such as The
Cancer Genome Atlas (TCGA) project, the International Cancer Genome Consortium (ICGC)
project, and the Catalogue of Somatic Mutations in Cancer (COSMIC) database, there is a
large dium of knowledge regarding these gene targets in many cancer types and the
result of therapeutics on cancers containing those mutations (See Futreal et al., 2004).
Additional work, in part as a result of the Pediatric Cancer Genome Project, has shown that
2012/055231
pediatric cancers have ct genetic profiles marked by a fewer number of ons and a
prevalence of mutations in alternative molecular pathways (See, Wu et al., 2012; Meldrum et
a]. 2011). The largest current unmet need in cancer diagnostics is a fast, high-throughput
technology with the needed cy and sensitivity for early-stage detection to identify rare
sequence ts that belong to a limited subpopulation of cells undergoing a cancerous
ormation.
Traditionally, DNA sequencing has been accomplished by the “Sanger” or “dideoxy”
method, which involves the chain termination of DNA synthesis by the incorporation of 2’,3’-
ynucleotides (ddNTPs) using DNA polymerase (Metzker et al., 2005, which is
incorporated herein by reference). Since 2005, there has been a fundamental shift away from
the application of automated Sanger sequencing for genome analysis. Advantages of next-
generation cing (NGS) technologies include the ability to produce an enormous
volume of data cheaply, in some cases in excess of a hundred million short sequence reads
per instrument run. Many of these approaches are commonly referred to as sequencing—by—
synthesis (SBS), which does not clearly delineate the different mechanics of sequencing
DNA (Metzker, 2010; Metzker 2005, which are incorporated herein by reference). DNA
polymerase—dependent strategies have been classified as cyclic reversible termination (CRT),
single nucleotide addition (SNA, e.g., pyrosequencing), and real—time sequencing. An
approach whereby DNA polymerase is replaced by DNA ligase is referred to as sequencing—
by—ligation (SBL). These approaches have been described in Metzker (2010), which is
incorporated herein by reference.
Sequencing technologies include a number of methods that are grouped broadly as (a)
te preparation, (b) sequencing and imaging, and (c) data analysis. The unique
combination of specific protocols distinguishes one technology from r and determines
the type of data produced from each platform. These differences in data output present
challenges when comparing platforms based on data y and cost. Although quality
scores and accuracy estimates are provided by each manufacturer, there is no consensus that a
‘quality base’ from one platform is equivalent to that from another platform.
Two methods used in preparing templates for NGS reactions include: clonally
amplified templates ating from single DNA molecules and single DNA molecule
templates. cing methods that use DNA polymerases are classified as cyclic reversible
termination (CRT), single-nucleotide addition (SNA) and real-time sequencing, (See Metzker
2010). Sequencing by ligation (SBL), an approach in which DNA rase is ed by
DNA ligase, has also been used in the NGS technologies, (see, e.g., Shendure et al., 2005;
v et al., 2008). Imaging methods coupled with these sequencing strategies range from
measuring inescent signals to four-color imaging of single molecular events. The
voluminous data produced by these NGS platforms place substantial demands on ation
technology in terms of data storage, tracking and quality control (see Pop & rg, 2008).
The need for robust methods that produce a representative, non—biased source of
nucleic acid material from the genome under investigation remains an important goal.
Current methods generally involve randomly ng genomic DNA into smaller sizes from
which either fragment templates or mate-pair templates are created. A common theme
among NGS technologies is that the template is ed or immobilized to a solid e or
support. The immobilization of spatially separated template sites allows thousands to billions
of sequencing reactions to be performed simultaneously.
Although clonally amplified methods offer certain advantages over bacterial g,
some of the protocols are typically cumbersome to implement and require a large amount of
genomic DNA material (3—20 ug). The preparation of single-molecule templates is more
straightforward and requires less starting al (<1 ug). Moreover, these methods do not
require PCR, which s mutations in clonally amplified templates that masquerade as
ce variants. AT-rich and h target sequences may also show ication bias
in product yield, which results in their underrepresentation in genome alignments and
assemblies. Quantitative applications, such as RNA—seq (See Wang et al., 2009), perform
more effectively with non-amplified te sources, which do not alter the representational
nce of mRNA molecules.
An important aspect of the CRT method is the reversible terminator, of which there
are two main types: 3’—0—blocked and 3’—OH unblocked (Metzker, 2010). The use of a
ddNTP, which acts as a chain terminator in Sanger sequencing, provided the basis for the
initial development of reversible blocking groups attached to the 3’—end of nucleotides
(Metzker et a]. 1994; Canard & i, 1994). Blocking groups such as 3’allyl-dNTPs
(Metzker et al., 1994; US. Patent 6,664,079; Ju et al., 2006; US. Patent 7,057,026; US.
Patent 7,345,159; US. Patent 7,635,578; US. Patent 7,713,698) and 3’—0—azidomethyl—
dNTPs (US. Patent 7,057,026; Guo et al., 2008; Bentley et al., 2008; US. Patent 7,414,116;
US. Patent 7,541,444; US. Patent 7,592,435; US. Patent 7,556,537; US. Patent 7,771,973)
have been used in CRT. 3’—0—Blocked terminators require the cleavage of two chemical
bonds to remove the fluorophore from the nucleobase and restore the 3’—OH group. A
drawback in using these reversible terminators is that the blocking group attached to the 3’-
end typically causes a bias against incorporation with DNA polymerase. Mutagenesis of
DNA polymerase is often required to facilitate oration of 3’blocked terminators.
Large numbers of genetically ered DNA polymerases have to be created by either site—
directed or random nesis containing one or more amino acid substitutions, insertions,
and/or deletions and then identified by high-throughput screening with the goal of
incorporating 3’—blocked tides more efficiently.
The difficulty in identifying a modified enzyme that efficiently incorporates 3’-0—
blocked terminators by screening large ies of mutant DNA polymerases has led to the
development of 3’—unblocked reversible terminators. It was trated that a small
photocleavable group attached to the base of a 3’—OH unblocked nucleotide can act as an
effective reversible terminator and be ently incorporated by wild-type DNA
polymerases (Wu et al., 2007; Metzker, 2010; Litosh et al., 2011, Gardner et al., 2012; US.
Patents 7,897,737, 7,964,352; and 8,148,503, US. Patent Appl. Publication 2011/0287427).
For example, 5-hydroxymethyl-2’-deoxyuridine (HOMedU) is found naturally in the
genomes of numerous bacteriophages and lower eukaryotes (Gommers—Ampt, 1995, which is
incorporated herein by reference). Its ymethyl group can serve as molecular handle to
attach a small photocleavable terminating group. Other naturally occurring hypermodif1ed
bases that can be further modified to function as reversible terminators include
-hydroxymethyl-2’-deoxycytidine (HOMedC), which is found naturally in the s of
T2, T4, and T6 bacteriophages (Wyatt & Cohen, 1953; Gommers—Ampt, 1995) and of
s (Kriaucionis & Heintz, 2009; Tahiliani et al., 2009; Ito et al., 2010). The
pyrrolopyrimidine ring structure (7-deazapurine) is also found naturally in side
antibiotics (Carrasco & Vazquez, 1984, which is incorporated herein by reference) and tRNA
bases (Limbach, et al., 1994, which is incorporated herein by reference), and the compounds
7—deaza—7—hydroxymethyl—2’—deoxyadenosine (C7—HOMedA) (Rockhill et al., 1997) and 7—
deaza—7—hydroxymethyl-2’—deoxyguanosine (C7—HOMedG) (McDougall et al., 2001) have
been reported.
One aspect of the present ion is the use of a modified 2-nitrobenzyl group
ed to the nucleobase of hydroxymethyl nucleoside and nucleotides. Described over a
half century ago, solutions of 2-nitrotoluene (Wettermark, 1962) and its derivatives
(Wettermark, 1962; Hardwick et al., 1960; Mosher et al., 1960; Sousa & Weinstein, 1962;
Weinstein et al., 1966) were reported to exhibit the ty of photochromism, a
phenomenon considered to be the result of transient formation of an aci-nitro anion
intermediate (Weinstein et al., 1966; Morrison, 1969). Without being bound by theory, it is
generally accepted that absorption of a photon by the nitro group results in hydrogen
abstraction from the bon (Mosher et al., 1960; Berson & Brown, 1955; De Mayo, 1960),
formation of the aci-nitro anion intermediate, and then release of the ‘Caged’ effector
molecule and creation of a nitrosocarbonyl by—product (Corrie, 2005). These early studies
suggested that (x—substitution of the benzylic carbon (Wettermark, 1962) or substitution of the
4-position of the benzene ring with an electron-donating group (Sousa & ein, 1962;
Weinstein et a], 1966) increased the rate of the photochromic effect. These findings led to
the development of photosensitive 2—nitrobenzyl protecting groups (Barltrop et al., 1966;
Patchornik, 1968; Patchornik et al., 1970). The degree to which the rate of photochemical
cleavage is altered, however, typically depends on numerous factors that are reported to
include substitution of the ic carbon (Walker et al., 1986; Hasan et al., 1997; Giegrich
et al., 1998), functional group(s) attached to the benzyl ring (Wootton & am, 1989;
Hasan et al., 1997; Giegrich et al., 1998), and the leaving group (Walker et al., 1986) as well
as pH (McCray et al., 1980; Walker et al., 1986; Wootton & Trentham, 1989), solvent (Sousa
& Weinstein, 1962; McGall et al., 1997; Giegrich et al., 1998), and light intensity (McCray et
al., 1980; McGall et al., 1997). One property, however, that has not been studied is
stereochemistry, whereby, substitution of 2—nitrobenzyl’s benzylic or (x—carbon results in a
chiral . For the case of tide synthesis, coupling of a c (x—substituted 2—
nitrobenzyl alcohol would result in two diastereomers, which differ only by the absolute
configuration (R or S) at the benzylic carbon.
Another class of 3’—OH unblocked nucleotides has been described by Mitra et a].
(2003) and ti et a1. (2008), which rely on steric hindrance of the bulky dye group to
stop incorporation after the addition of the first nucleotide. It is noted that the substituted 2-
nitrobenzyl nucleotide s bed by Wu et a]. (2007), Litosh et a]. (2011), and
Gardner et al., 2012 cause termination of DNA synthesis t the requirement of bulky
substituents such as fluorescent dyes. A further class of 3’—unblocked nucleotides has been
described by Helicos Biosciences. These nucleotides use a second nucleoside or nucleotide
analog that acts as an tor of DNA synthesis (Bowers et al., 2009; US. Patent
7,476,734). A significant difference in termination properties is observed when comparing
compounds of the t invention with those described by Bowers. For e, Bowers
et al. described eady—state kinetics employing two—base homopolymer templates, for
which kp01(+2) rates were measured for all of their 3’-OH unblocked ‘virtual’ terminators.
Bowers et a1. conducted their termination experiments at submicromolar nucleotide
concentrations (i.e., from 100 to 250 nM), termination assays. In contrast, l
compounds of the present invention were performed at 10 uM over the time course of 0.5 to
min. Both compounds dU.V and dU.VI were rapidly incorporated at the first base
position (100% by 2 min) and then terminated DNA synthesis at that position. No
appreciable signal could be detected at the expected second—base position up to tion
times of 20 min. See Gardner et al., 2012 for more details.
3’—OH unblocked reversible terminators typically have several advantages over 3—0—
blocked nucleotides. For example, for many 3’—OH unblocked reversible terminators the
cleavage of only a single bond removes both the terminating and fiuorophore groups from the
nucleobase. This in turn results in a more efficient strategy for restoring the tide for
the next CRT cycle. A second advantage of 3’—OH unblocked reversible terminators is that
many of these compounds show more favorable enzymatic incorporation and, in some cases,
can be incorporated as well as a natural nucleotide with wild—type DNA polymerases (Wu et
al., 2007; Litosh et al., 2011; Gardner et al., 2012; U.S. Patent 7,897,737; U.S. Patent
352; U.S. Patent 8,148,503; U.S. Patent Appl. ation 2011/0287427), although in
other cases this efficiency has not been observed (Bowers et al., 2009; U.S. Patent
734). One challenge for 3’—OH unblocked terminators is creating the appropriate
modifications to the base that lead to termination of DNA synthesis after a single base
addition. This is important because an unblocked 3’-OH group is the natural substrate for
incorporating the next incoming nucleotide.
eneration sequencing (NGS) technologies have facilitated important
ical discoveries, yet chemistry improvements are still needed for a number of reasons,
including reduction of error rates, ion of slow cycle times. To be effective in NGS
, it is typically desirable for ible terminators to exhibit a number of ideal
properties including, for example, fast kinetics of nucleotide incorporation, single-base
termination, high nucleotide selectivity, and/or rapid cleavage of the terminating group.
Thus, there is a need for developing new nucleosides and nucleotides that meet these
challenges.
SUMMARY OF THE INVENTION
In some aspects, the present disclosure provides novel compounds and compositions
that are useful in efficient cing of genomic information in high throughput sequencing
reactions. In r aspect, reagents and combinations of reagents that can efficiently and
affordably provide genomic information are provided. In further aspects, the present
invention provides ies and arrays of reagents for stic methods and for developing
targeted therapeutics for individuals.
In some aspects, the present disclosure provides new compounds that may be used in
DNA sequencing. For e, the present disclosure provides compounds of the formula:
R5 R5
R6 OMe R5 R6
R5 OMe
02N R4 O2N R4
H OZN R4 NH2 H
0 NH
R3 0 I NH H R3 2
0 \N
R3 I / | UN
R N O /
R1 R1 N
OH R2 OH
OH R R2
(1), 2 (11),
OMe R6 OMe
OZN O2N R4
H H
0 R3 NH
N \
' ANH <N N
, l a
N NH2 R1 N
FO:1 PO;
(111), R2 0“
(IV), R2 (V),
R5 R6 OMe
R5 OMe
02N R4
OZNH R4 *
R3 NH
N 0
R1 05%</ AN H2 R1
0” R2 0”
(VI), or R2 (VII),
wherein:
R1 is hydroxy, monophosphate, phate, sphate, oc-thiotriphosphate or
polyphosphate;
R2 is hydrogen or hydroxy;
R3 is alkyl(cgg) or substituted alkyl(cgg);
R4 is
en, hydroxy, halo, amino, nitro, cyano, azido or mercapto;
alkyl(cg6), acyl(cg6), alkOXy(Cg6), acyloxy(cg6), alkylamino(cg6), lamino(cg6),
amido(cg6), or a substituted version of any of these groups;
R5 and R6 are each independently:
hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto;
alkyl(cg6), alkenyl(cg6), alkynyl(cg6), aI'yl(Cg6), aralkyl(cgg), heteroaryl(cg6),
acyl(cg6), alkOXy(Cg6), acyloxy(cg6), alkylamino(cg6), dialkylamino(cg6),
amido(cg6), or a substituted version of any of these ;
a group of formula:
HzNMXE‘
H N2 VbomH‘e’i
HZNwam/VYVWONWX?HO
m n
wherein
X is
—O—, —S—, or —NH—; or
alkanediyl(cg12), diyl(cg12), alkynediyl(cg12), or a
substituted version of any of these groups;
Y is —O—, —NH—, diyl(cg12) or substituted alkane-
diy1(C:12);
n is an integer from 0—6; and
m is an integer from 0—6; or
a —linker—reporter;
or a salt, er, or optical isomer thereof.
In some embodiments, the compounds are further defined as a compound of formulas
I, II, III, IV, V, VI or VII. In some embodiments, R1 is hydroxy, monophosphate,
diphosphate, triphosphate, 0t-thiotriphosphate, or polyphosphate.
In some embodiments, R2 is hydrogen, hydroxy. In some embodiments, R3 is
alkyl(cgg), for example, alkyl(c3_4), including isopropyl or utyl. In some embodiments, R4
is hydrogen, nitro. In some embodiments, R5 is hydrogen, iodo, or alkOXy(Cg6), including, for
example, y. In some embodiments, R5 is a group of formula:
HZNMXE';
wherein
X is
—O—, —S—, or —NH—; or
alkanediyl(cg12), alkenediyl(cg12), alkynediyl(cg12), arenediyl(cg12),
heteroarenediyl(cg12), or a substituted n of any of these groups;
and
n is an integer from 0—6.
In some embodiments, X is alkynediyl(cz_g), for example, —CEC—. In some
embodiments, n is zero. In some embodiments, R5 is a group of formula:
H2N\AY/\)i<”/\/YV\IONWX}€HO
m n
X is
—O—, —S—, or —NH—; or
alkanediyl(cg12), diyl(cg12), alkynediyl(cg12), arenediyl(cg12),
heteroarenediyl(cg12), or a tuted version of any of these groups;
Y is —O—, —NH—, diyl(cg12) or substituted alkanediyl(cg12);
n is an integer from 0—6; and
m is an integer from 0—6.
In some embodiments, X is alkynediyl(cz_g), for example, —CEC—. In some
embodiments, Y is —CH2—. In some embodiments, n is zero. In some embodiments, m is
zero. In some embodiments, R5 is a —linker—reporter. In some embodiments, the linker is:
Wherein
X is
—O—, —S—, or —NH—; or
alkanediyl(cg12), alkenediyl(cg12), alkynediyl(cg12), arenediyl(cg12),
heteroarenediyl(cg12), or a substituted version of any of these groups;
n is an integer from 0—6.
In some embodiments, X is alkynediyl(cz_g), for example, —CEC—. In some
embodiments, n is zero. In some ments, the linker is:
’l‘inY/QICQ/VYVWONWX?‘H H
m n
Wherein
X is
—O—, —S—, or —NH—; or
alkanediyl(cg12), alkenediyl(cg12), alkynediyl(cg12), arenediyl(cg12),
heteroarenediyl(cg12), or a substituted version of any of these groups;
Y is —O—, —NH—, alkanediyl(cg12) or substituted alkanediyl(cg12);
n is an integer from 0—6; and
m is an integer from 0—6.
In some embodiments, X is alkynediyl(c2_g), for example, —CEC—. In some
embodiments, Y is —CH2—. In some embodiments, n is zero. In some embodiments, m is
zero. In some embodiments, the reporter is based on a dye, wherein the dye is zanthene,
cein, ine, BODIPY, cyanine, coumarin, pyrene, phthalocyanine,
phycobiliprotein, or a squaraine dye. In some embodiments, the reporter is:
CI CHZSO3H
SCHz—fi—NH(CH2)5—§—§‘o ,or
038 so;
O W / // / o
"‘I’W
In some embodiments, R6 is hydrogen. In some embodiments, the starred carbon
atom is in the S ration. In some embodiments, the starred carbon atom is in the R
configuration. In some embodiments, the compound is further defined as:
OMe OMe
02N OZN
t-BU t-BU
O NH2 O NH2
\ \
/ I N / I N
A A
O /R\ /\\ O
_ /P\\ _ _
O O O O O O
OH OH
OMe OMe
OZN 02N
PBu O O
O O
z\ 10%;!“ NH / NH
HO 2 NH2 O\/O\P/0 H2
W /F<\_ /P\\ IF<\
o o o o o 0
OMe OMe
OZN i OZN iOH
t—Bu t—Bu
O O
NH / | NH
HO N NH2 HO\P/O\ /O\P/O N N NH2
0 /P\\ ,F’\\ /P\\ O
o o o o o 0
OH OH
, ,
OMe OMe
OMe OMe
OZN 02N
bBu bBu
O O
/ | NH / | NH
/ /
o N N NH2 HO\F)/o\ /o\P/o N N NH2
0 /\\ o
_ A\ R\
o o o o o 0
OH OH
, ,
OMe OMe
ZN’ E o oZN’ E o
t—Bu OAfiiH t—Bu 0%1H
N o N o
o A\ R\ ,F’\\ o
_ o o _o o o 0
OH OH
2012/055231
02N NH2
t-BuHOO:(:NO E/O
_ /F<\ /P\\ IF<\
O _
O O O O O
,01'
or a salt and/or protonated form of any of these formulas.
In some embodiments, the compound is further defined as:
OH OH
7 ,
H2N H2N
OMe OMe
02N O 02N NH2
t—Bu OAELNH t-Bu o \N
N O ”A0
HO 0 o o HO 0 o o
\P/ \P/
/\\ /\\ \IP/ \P/ \P/ \P/ O
OOOOOR\\
O /\\
_ _ /\\
_ /\\
OO'OO‘OR
OH ,Or OH
wherein R is :0 or =S, or a salt and/or protonated form of any of these formulas.
In some embodiments, the compound is further as:
WO 40257
t—Bu O
HO\ ,0\ /o\ o N N NH2
OZN o
t—BU O NH
/F<\ /R\ R\ O
O O _ _ O O _ O R
OZN NH2
t—Bu \ N
0 | NAG
HO\ /0\ /o\ /o
P P P
\ \\ \\ O 9
_ o’\0‘o’o-o/s k
wherein R is :0 or =S, or a salt and/or ated form of any of these formulas.
In some embodiments, the compound is further defined as:
WO 40257
OMe OMe
OZN 02N
t-BU
O NHZ t-Bu
O NH2
/ I UN / I
N N/\JN HO\P/O\P/O\P/ N HO\P/O F)/o\P/o N
_O/\O_ “0—0/0\ \ \\ o
:- 3 _O/\O_/\O_/\O\ \ \ I:0:
OH OH
7 7
NH(CH2CH20)8CH2CH2— IcI
SO3HO o:3:ON 0 ||
H2NO OMe
COOH 02N
t—BU
O NH2
/ \N
I A
HO P/O\ /0\P/ N N
/F{\ /\\ O
_ \\
o _
o o _ o o
CI CI
0 0 OH
H300 E
OCH3
HOOC
I H
OMe OMe
02N O OZN o
t-Bu o | j: t-Bu II
N 0 N O
Ho\ /0\ /o\ /o Ho\ /0\ /o\ /o
IF<\ /R\ O /F<\ /R\ /R\ O
‘0000'00_
/F{\ _oooooo_
OH OH
OZN o
luau oAfL/NQ
N O
HO\P/O\P/O\P/O
/\\ /\\ O
_ /\\
_ O O O O _ O O
WO 40257
t—Bu O
/ NH
HO\ /0\ /o\ ,o N N NH2
_ /F{\ R\ R\
O _
O O "O O
701'
02N NH2
t-Bu O I \1
N 0
HO\ /0\ /o\ /o
_ /F{\ /R\ /R\
O _
O O _O O
or a salt and/or protonated form of any of these formulas.
In another aspect of the invention there are provided methods of sequencing a target
nucleic acid comprising the following steps:
(i) ing the 5’-end of a primer to a solid surface;
(ii) hybridizing a target c acid to the primer attached to the solid surface to
form a ized primer/target c acid complex;
(iii) obtaining a polymerase and one or more compounds described herein, with the
proviso that compound of different formulas I-VII have different fluorophores;
(iv) reacting the hybridized primer/target nucleic acid complex with a polymerase
and one or more of the compounds of step (iii) to form a growing primer
strand via a polymerase reaction;
(V) imaging the growing primer strand to identify the incorporated compound of
step (iv) via its fluorophore;
(vi) exposing the solid surface with the growing primer strand to a light source to
remove a leavable terminating moiety of the formula:
with the les as defined herein referenced in step (iii), ing in an
extended primer with naturally-occurring components; and
(vii) repeating steps (iv) through (vi) one or more times to identify a plurality of
bases in the target nucleic acid, where the extended primer of step (vi) of the
previous cycle reacts in place of the hybridized primer/target nucleic acid
complex in step (iv) of the subsequent cycle.
In some embodiments, step (vi) is conducted in the presence of sodium azide. In
some embodiments, the sodium azide concentration is from 0.1 mM to 10 mM, for examples,
about 1 mM. In some ments, step (vi) is conducted in the presence of sodium acetate.
In some embodiments, the sodium acetate concentration is from 0.1 mM to 10 mM, for
example, about 1 mM.
In some embodiments, steps (V) or (vi) is conducted in the ce of thiourea. In
some ments, the thiourea concentration is from 10 mM to 500 mM, for example,
about 100 mM.
In some embodiments, step (vi) is conducted in the presence of dithiothreitol (DTT).
In another aspect, there are provided methods of sequencing a target nucleic acid
comprising the following steps:
(i) attaching the 5’—end of a nucleic acid to a solid surface;
(ii) hybridizing a primer to the nucleic acid attached to the solid surface to form a
hybridized primer/target c acid complex;
(iii) obtaining a rase and one or more compounds described herein, with the
proviso that compound of different formulas I-VII have different fluorophores
(iv) reacting the ized primer/target nucleic acid complex with a polymerase
and one or more of the nds of step (iii) to form a growing primer
strand via a polymerase reaction;
(v) imaging the growing primer strand to identify the incorporated compound of
step (iv) via its fluorophore;
(vi) exposing the solid surface with the growing primer strand to a light source to
remove a photocleavable terminating moiety of the a:
with the variables as defined herein, resulting in an extended primer with
naturally-occurring components; and
(vii) repeating steps (iv) through (vi) one or more times to identify a plurality of
bases in the target nucleic acid, where the extended primer of step (vi) of the
previous cycle reacts in place of the ized primer/target nucleic acid
complex in step (iv) of the subsequent cycle.
In some embodiments, step (vi) is conducted in the presence of sodium azide. In
some embodiments, step (vi) is conducted in the presence of threitol (DTT).
In some embodiments, the incorporation of at least one compound according to step
(iv) occurs at about 70% to about 100% of the efficiency of incorporation of its natural
nucleotide counterpart. In some ments, the incorporation efficiency occurs at about
85% to about 100%.
In some embodiments, the polymerase is selected from the group consisting of reverse
transcriptase, terminal erase, and DNA polymerase. In some embodiments, about 85%
to about 100% of the photocleavable terminating moieties are removed by exposure to a light
source in step (vi). In some embodiments, incorporation of at least one nd according
to step (iv) is followed by termination of strand growth at an efficiency of from about 90% to
about 100%.
In some embodiments, a pulsed multiline excitation detector is used for imaging in
step (v).
In some embodiments, the method further comprises washing the growing primer
strand prior after step (iv) or step (vi).
In some embodiments, the method further ses, prior to step (iv), capping any
s or growing primer strands that did not react in step (iv).
In some embodiments, the method further comprises sequencing multiple target
nucleic acids synchronistically.
In another aspect of the invention, there are provided methods of converting a non—
naturally occurring component in a nucleic acid molecule into a naturally-occurring
component comprising:
(i) incorporating a compound described herein;
(ii) exposing the resulting nucleic acid to a light source to remove a
leavable terminating moiety of the formula:
with the variables as defined , from the c acid.
In some embodiments, the method r comprises converting non—naturally
occurring components in multiple c acid molecules into naturally—occurring
components synchronistically. In some embodiments, the method further comprises
ating multiple nucleic acid syntheses synchronistically.
In another aspect, the invention es methods of terminating a nucleic acid
sis comprising the step of placing a 3’—OH unblocked nucleotide or nucleoside
described above in the environment of a polymerase and allowing incorporation of the 3’—OH
unblocked nucleotide or nucleoside into a nucleic acid molecule. In some embodiments,
efficiency of termination of DNA synthesis upon incorporation of the 3’—OH unblocked
nucleotide or nucleoside ranges from about 90% to about 100%. In some embodiments, the
efficiency of incorporation of the 3’—OH ked nucleotide or nucleoside ranges from
about 70% to about 100% compared to the efficiency of incorporation of a naturally—
ing nucleotide or nucleoside with the same base as the 3’—OH unblocked nucleotide or
nucleoside.
In another aspect, the invention provides methods of performing Sanger or Sanger-
type sequencing comprising using a compound described herein as a terminating nucleotide
analog.
In another aspect, there are provided methods of determining the sequence of a target
nucleic acid comprising
(i) adding a target nucleic acid to a Sanger or Sanger-type sequencing apparatus,
(ii) adding one or more compounds described herein to the cing apparatus,
with the proviso that where more than one type of base is present, each base is
attached to a ent fluorophore;
(iii) adding a complementary primer and a polymerase enzyme,
(iv) performing a polymerase on to orate at least one of the nds
of step (ii) into a growing nucleic acid strand, and
(V) analyzing the result of the Sanger sequencing reaction with fluorescence
sequencing instrumentation or by pulsed multiline excitation fluorescence,
wherein steps (i)-(iii) can be performed in any order.
In some embodiments, incorporation of at least one compound according to step (iv)
is followed by termination of strand growth at an efficiency of from about 90% to about
100%. In some embodiments, the oration of at least one compound according to step
(iv) occurs at about 70% to about 100% of the efficiency of incorporation of a native
substrate with the same base in the polymerase reaction. In some ments, the
incorporation efficiency occurs at about 85% to about 100%. In some embodiments, the
polymerase is ed from the group ting of e transcriptase, terminal
transferase, and DNA polymerase.
In r aspect, the invention provides methods of incorporating a non-naturally
occurring component into a nucleic acid comprising:
(i) adding a target nucleic acid to a sequencing apparatus;
(ii) adding one or more compounds described herein to the sequencing apparatus,
with the proviso that where more than one type of base is present, each base is
attached to a different fluorophore;
(iii) adding a polymerase enzyme; and
(iv) performing a polymerase reaction to incorporate at least one of the compounds
of step (ii) into a growing nucleic acid strand,
wherein steps (i)-(iii) can be performed in any order.
In some embodiments, the method further comprises:
(v) analyzing the result of the polymerase chain reaction for incorporation of at
least one compound from step (ii).
In some embodiments, incorporation of at least one compound according to step (iv)
is ed by termination of strand growth at an efficiency of from about 90% to about
100%. In some embodiments, the incorporation of at least one compound according to step
(iv) occurs at about 70% to about 100% of the efficiency of incorporation of native substrate
with the same base in the polymerase reaction a native substrate with the same base in the
rase reaction.
In another aspect, the invention es methods of performing mini-sequencing or
minisequencing-type sequencing comprising addition of a compound described herein to a
equencing or minisequencing—type sequencing method.
In some embodiments of any of the methods described above, the nd is further
defined as a compound of formula I, II, III, IV, V, VI, or VII.
In another aspect, the invention provides a system sing:
a flowcell comprising a plurality of beads, wherein:
each bead attached to a DNA molecule, wherein a compound described
herein has been incorporated into using a polymerase; and
the flowcell is at least partially transparent to visible and UV light;
an imaging device configured to capture images of the flowcell;
a filter wheel comprising at least four spectral filters, wherein the filter wheel
is configured to cycle between each filter;
a lamp configured to create a light path from the flowcell through a filter in
the filter wheel to the imaging device; and
an ultraviolet light source configured to provide ultraviolet light to the DNA
molecules on the flowcell.
In some embodiments, the flowcell is a luidic flowcell. In some embodiments,
the system further ses an objective lens between the filter wheel and the flowcell. In
some embodiments, the system further ses a mirror configured to direct the light path
to the imaging device.
In some aspects, the present disclosure provides for cancer diagnostics that are fast,
hroughput, accurate and sensitive for early—stage detection to identify rare sequence
variants that belong to a limited subpopulation of cells undergoing a cancerous
transformation.
Other s, es and advantages of the present disclosure will become apparent
from the following detailed description. It should be understood, however, that the detailed
description and the specific examples, while indicating specific embodiments of the
invention, are given by way of illustration only, since various changes and modifications
within the spirit and scope of the invention will become apparent to those d in the art
from this ed description. Note that simply because a particular compound is ascribed to
one particular generic formula does not mean that it cannot also belong to another generic
formula.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to
further demonstrate certain aspects of the present disclosure. The invention may be better
understood by reference to one of these gs in combination with the detailed description
of specific embodiments presented herein.
— Structures of 2-Nitr0benzyl Alkylated HOMedNTP Analogs. “R” is H,
iso—propyl, or utyl. “R’” is H, 4—OMe, 5—OMe, 4,5—di—OMe, or 6—NOz. See keys for
specific examples. “*” s two different stereochemical configurations at this carbon
atom. The portion of the formulas within the dashed ellipsoids ghts the terminating
functional groups that are d upon exposure to UV light.
— Elimination of Transient Product (TP) with DTT. Fluorescent gel
image of UV photochemical cleavage time series of dU.VI incorporated by Therminator
polymerase in the presence of (A) 1 mM NaN3 and (B) 1 mM NaN3, 50 mM DTT. Lanes:
“P” (primer) contains Therminator bound to oligoTemplate-4 hybridized with BODIPY-FL
labeled primer-1 in 1x ThermoPol buffer (Wu et al., 2007; Litosh et al., 2011), “I”
poration) contains that found in lane “P” plus 100 nM dU.VI, and time point lanes
contain that found in lane “1” plus listed times s were exposed to 0.70 W/cm2 365 nm
light. “1P” denotes incorporated product and “CP” denotes cleaved product.
— X-ray Crystal Structure of (S)(5-methoxy—Z-nitrophenyl)—2,2-
dimethyl—l-propyl (1S)-camphanate. Crystallographic measurements were made on a
crystal of (S)(5-methoxynitrophenyl)-2,2-dimethyl-l-propyl (1S)-camphanate with
dimensions of 0.50 mm X 0.05 mm X 0.05 mm as described in Litosh et al. , which is
incorporated herein by reference. Data collection: CuKoc ion, A = 1.54178 A, T = 110
:: 20K, 26max = 120.00, 32,513 reflections collected, 2,913 unique (Rim = 0.0517). Final GooF
= 1.091, R] = 0.0681, wR2 = 0.1695, R indices based on 2,913 reflections with I>2sigma(I)
ement on F2), 290 parameters, 43 restraints. Lp and absorption corrections applied, ,a =
0.819 mm”. Absolute structure parameter: 0.05 :: 0.09. X-Ray llography data:
C22H29N07, M: 419.46. Orthorhombic, a = 6.29, b = 15.00, c = 22.27 A (a, [3, y = 90°), V=
2,099.29 A3, space group P212121, Z= 4, Dc = 1.327 g/cm'3, F(000) = 896.
— DTT Eliminates the Nitroso Intermediate (TP). scent gel image
of UV photochemical cleavage experiment of dU.VI incorporated by TherminatorTM
polymerase. Lanes: “P” (primer) ns TherminatorTM bound to oligoTemplate-4
hybridized with BODIPY-FL labeled primer-l in l>< ThermoPol buffer, “I” (incorporation)
contains that found in lane “P” plus 100 nM dU.VI. Reagents A-G listed as final
concentrations in the key were added, and samples were exposed to 0.70 W/cm2 365 nm light
for 10 sec. “IP” s incorporated product, “CP” denotes cleaved product, and “TP”
denotes transient product.
FIGS. 5A & B — Optical Set-Up for UV Photochemical Cleavage Measurements.
shows a schematic of the modified 0.5 mL Eppendorf tube cut in half, PM100 power
meter, a 1,000 um pinhole cassette using a 3-axis manual translation stage to align the arc
beam. shows a sample holder and modified 0.5 mL Eppendorf tube with an internal
alignment card to align the arc beam to the center of a 10 uL or 20 uL reaction .
— Example of photochemical cleavage on. Upon uced
photochemical cleavage, the terminating 2-nitrobenzyl derivative is released to yield a natural
hydroxymethyl nucleotide. The combination of a stereospecific (S)—tert—butyl group attached
at the benzylic carbon coupled with a 5-OMe group d on the 2 enzyl ring
substantially increased the rate of the photochemical cleavage reaction. For the case of C7—
HOMedG, the rate increased by more than one order of magnitude over its corresponding
parent analog.
— tert—Butyl tution at the a-Carbon and Methoxy Substitution at
the 5 on Correlates with Improved Photochemical Cleavage Rates. This figure
compares photochemical cleavage rates of the parent, (S)—0t—tert—butyl, and (S)—(x—tert—butyl—5—
OMe 2-nitrobenzyl groups alkylated on C7—HOMedA, HOMedC, C7—HOMedG, and
HOMedU nucleosides. Lower DT50 values indicate faster hemical cleavage rates.
A schematic representation of a system for imaging fluorescent beads on a
flowcell.
— e of Bead Preparation and Immobilization Method. Schematic
illustration of the steps of an exemplary mpreparing a bead sample on a l.
. Schematic illustration of the steps of incorporation, fluorescence imaging,
and photochemical cleavage in a CRT cycle.
. Illustration of tile images from three CRT cycles and subsequent base—
calling from individual beads.
WO 40257
— Chemical Formulas of 3'-OH Reversible Terminators Attached to
Generic Dyes (“Fluor”). The portion of the formulas assed by the dashed ellipsoid
denotes the dye-labeled terminating onal groups and that are cleaved upon exposure to
UV light.
— Diagram of the Random Nick Sequencing (RNS) method. Lightning
TerminatorsTM denoted in the figure are comprised of the reversible terminators of the present
invention.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
I. Reversible Terminators and Methods of Synthesis Thereof
In one aspect, the present disclosure provides new nds that may be used to
function as reversible terminators in a y of different DNA sequencing applications. The
compounds provided by the present disclosure are also referred to as reversible terminators,
3’—OH unblocked reversible terminators, and as Lightning TerminatorsTM. In some
embodiments, compounds of the following as are provided:
R N O
1 N
0 R1 R1 N
OH FO:1
R2 OH
0“ R
(I), R2 (11), 2
R5 R5
R6 OMe R6 0M6
02N R4 OZN R4
H H
R3 0 0 R3 NH
Hi“ N
/ 0N/
R1 N N NH2 R1 N N
FO:1 O
(111), R2 0“
(IV), R2 (V),
R5 R6 OMe
R5 OMe
02N R4
OZNH R4 *
R3 NH
</ fl
N o
R1H205% AN R1
0” R2 0”
(VI), or R2 (VII),
wherein:
R1 is hydroxy, monophosphate, diphosphate, triphosphate, oc-thiotriphosphate or
polyphosphate;
R2 is hydrogen or y;
R3 is cgg) or substituted cgg);
R4 is
hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto;
alkyl(cg6), acyl(cg6), alkOXy(Cg6), acyloxy(cg6), alkylamino(cg6), lamino(cg6),
amido(cg6), or a substituted version of any of these groups;
R5 and R6 are each independently:
hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto;
alkyl(cg6), alkenyl(cg6), alkynyl(cg6), aI'yl(Cg6), aralkyl(cgg), aryl(cg6),
acyl(cg6), alkOXy(Cg6), acyloxy(cg6), alkylamino(cg6), dialkylamino(cg6),
amido(cg6), or a substituted version of any of these groups;
a group of formula:
HzNMXE‘
H N2 VbomH‘e’i
HN Y N X
Om 1’1
WO 40257
wherein
X is
—O—, —S—, or —NH—; or
alkanediyl(cg12), alkenediyl(cg12), alkynediyl(cg12), or a
S substituted version of any of these groups;
Y is —O—, —NH—, alkanediyl(cg12) or substituted alkane-
diy1(C:12);
n is an integer from 0—6; and
m is an r from 0—6; or
a —linker—reporter;
or a salt, tautomer, or optical isomer thereof.
Dye—labeled 0t-tBuOMenitrobenzyl alkylated hydroxymethyl nucleotides may
be synthesized according to the following schemes and procedures.
A. Synthesis of dye labeled 7-[(S)(5-methoxy-Z-nitrophenyl)-2,2-dimethyl—
propyloxy]methyldeaza-2 '-deoxyadenosine-5'-triphosphates
OMe OMe
OZN 02N
CI t—BU CI t—BU
CI 0 O NH2
/ I )N / I j“ / I )N
TBSO N N/ HO N N/ HO N N/
O (i). (ii) 0 (iii) 0
—> —>
OTBS OH OH
N H2N
OMe OMe
OZN OZN
t—Bu t-BU
o NH2 o NH2
/ \N
I / ‘N
a I
. g
('V) HO N N (V) HO\ /O\ /O\P/ N N
’ O ) /F<\ /F{\ /\\ O
_ O _ _
o o o o o
OH OH
Scheme 221. Synthesis of dye labeled 7-[(S)(5-methoxy-Z-nitrophenyl)-2,2-dimethyl—
propyloxy]methyldeaza-2'-deoxyaden0sine-5'-triph0sphate. Reagents and conditions:
(i) (S)(5-meth0xynitr0pheny1)-2,2-dimethy1—1-pr0pan01, 110°C; (ii) F, THF,
room temperature; (iii) NH3, 1,4—di0xane/MeOH, 100°C; (iv) N—
propargyltrifluoroacetamide, 3)4(0), Cul, Et3N, DMF; (v) POCl3, (MeO)3PO, 0°C; (n—
BU3NH)2H2P207; I’Z-BU3N; DMF; l M HNEt3HCO3.
+ so; 803
H2N o O\ O NH2
HOOC
(vi)
t—Bu
O NH2
/ \N
I A
HO\ /O\ /O\ / N N
/F{\ IR
_ /F{\
0 _
o o _ o 0
(vii)
tB' u 0 NH2
/ \N
I A
\P/O\P/ N N
'o’\o‘o’ 0'0] 0\ \\ \\ k O 9
Scheme 2c. (vii) 6—FAM NHS, 0.1 M NazCO3/NaHCO3 buffer (pH 9.2).
lI\JH(CH2CH20)8CH2CH2—fi
SO3H o=s=o 0 ||
H2N 0 NH
0 e/
COOH 02N
g t—Bu
0 NH2
(viii)
HO\P/O\P/O\P/O N N
_O/\\ \ \
_ /\O _O/\O T:0:
Scheme 2d. (viii) CF488A NHS, 0.1 M NazCO3/NaHCO3 buffer (pH 9.2).
B. Synthesis of dye labeled (5-methoxynitrophenyl)-2,2-dimethyl-
propyloxy]methyl—2'-deoxyuridine-5'-triph0sphate
50>—
Br NBoc OZUIeNBEEOOMI: NHt- .3 NH
TBSO HO HO
(i) (ii)
0 (iii)
I:OZN _> _>
OTBS OH OH
OZN O
t—Bu O I 1:4
N O
(iv)
’ HO\P/O\P/O\P/O/\\ /\\ /\\ O
_ O O _ O O _ O O
Scheme 321. Synthesis of dye labeled 5-[(S)(5-methoxynitrophenyl)-2,2-dimethyl-
propyloxy]methyl-2'-deoxyuridine-5'-triph0sphate. Reagents and conditions: (i) (S)-1—(4—
i0d0meth0xynitr0phenyl)-2,2-dimethy1—1-pr0pan01, 110°C; (ii) NH4F, MeOH, 50°C;
(iii) N—propargyltrifluoroacetamide, Pd(PPh3)4(0), CuI, Et3N, DMF; (iv) POC13, proton
, (MeO)3PO, 0°C; (n—Bu3NH)2H2PzO7, n—Bu3N, DMF; 1 M HNEt3HCO3;
OZN o
t—Bu o NH
| MAO
, /\\ O
_ /\\ /\\
_ O O O O _O O
Scheme 3b. (v) Alexa Fluor 532 NHS, 0.1 M NazCO3/NaHCO3 buffer (pH 9.2).
CI CI
0 0 OH
HsCO j \ E
OCH3
HOOC
02N o
t—Bu o/fLNH
N O
(vi) ’ HO\P/O\P/O\P/O /\\ /\\ O
_ /\\
_ O O O O _O O
Scheme 3c. (v) 6—JOE NHS, 0.1 M NazCO3/NaHCO3 buffer (pH 9.2).
2012/055231
, / O
OZN O
t-Bu 0%NH
N O
(Vii)
’ HO\P/O\P/O\P/O
/\\ /\\ O
_ /\\
_ O O O O _O O
Scheme 3d. (v) Cy3 NHS, 0.1 M NazCO3/NaHCO3 buffer (pH 9.2).
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C. Synthesis of dye labeled 7-[(S)(5-methoxynitrophenyl)-2,2-dimethyl-
propyloxy]methyldeaza-2 '-de0xyguan0sine—5 '-triph0sphate
CI CI
HO CI
/ '1 / '1
TBSO N N’ NHTBS TBSO N N/ NHTBS
j (')|
0 j (ii), (iii)
OTBS OTBS
OMe OMe
OZN OZN
t—Bu CI t—Bu O
0 O
/ \N
I / NH
HO N NANHZ HO N N/)\NH2
(iv) (V)
:0: O
, ,
OH OH
>_NH H2N
OMe OMe
OZN OZN
t—Bu O t—Bu O
O O
/ NH NH
| A / ' A
HO N NH2 N NH2 N
(V' ) HO\P/O\P/O\P,O 0 /\\ /\\ /\ O
—> _ O _
O 0 0‘0 0
OH OH
Scheme 43. Synthesis of dye labeled 7-[(S)(5-methoxy-Z-nitrophenyl)-2,2-dimethyl-
oxy]methyldeaza-2'-deoxyguan0sine-5'-triph0sphate. Reagents and conditions:
(i) MsCl, DMAP, CHzClz, 0°C; (ii) (S)-l-(4-iodomethoxynitrophenyl)—2,2-dimethyl-lpropanol
, 115°C; (iii) n-Bu4NF, THF, room temperature; (iv) syn-pyridine-Z-aldoxime,
l,l,3,3-tetramethyl ine, 1,4-dioxane/DMF, 70°C; (v) N—propargyltrifluoroacetamide,
Pd(PPh3)4(0), Cul, Et3N, DMF; (vi) POCl3, proton sponge, (MeO)3PO, 0°C; (n—
BU3NH)2H2P207; I’Z-BU3N; DMF; 1 M HNEt3HCO3.
WO 40257
(Vii) t—Bu O
/ NH
_ R\ /\\ /F{\
O _ O ‘O O
(viii) t—Bu O
HO\ /O\ /O\ /O
P P P
\ \
_ o’\o‘o’\o ‘0’ 0\\ :0:
Scheme 4c. (vii) 6—ROX NHS, 0.1 M NazCO3/NaHCO3 buffer (pH 9.2).
D. Synthesis of dye labeled 5-[(S)(5-methoxynitrophenyl)-2,2-dimethyl-
propyloxy]methyl—2'-deoxycytidine-5'-triph0sphate
' ' '
i-Pr
OMe OMe OMe O
o\\//
/S i-Pr
o N O O o
2 OZN OZN
I._Pr
t—Bu o NH t-Bu o NH t—Bu o \N
I I I
HO N 0
(i) TBSO N 0 N 0
.. TBSO
O (H)
—> O —> O
OH OTBS OTBS
I I
OMe OMe
OZN NH2 OZN NH2
t—Bu O \N t—Bu O \N
l x Af/k
N 0 N O
(iii) TBSO (iv) HO (V)
O ’
—> O
OTBS OH
N H2N
OMe OMe
OZN NH2 02N NH2
t—Bu o \N t-Bu o \N
l n ' A
N 0 \P/O\P/O N O
HO (Vi)
O —> \ \\ \\ k O _ 0’ \0‘0’ 0 ‘0’ o 9
OH OH
Scheme 521. Synthesis of dye labeled 5-[(S)(5-methoxynitrophenyl)-2,2-dimethylpropyloxy
l-2'-deoxycytidine-5'-triph0sphate. Reagents and conditions: (i) TBSCl,
imidazole, DMF, room temperature; (ii) 2,4,6-triisopropylbenzenesulfonyl chloride, DMAP,
Et3N, CHzClz, room temperature; (iii) NH3, 1,4—dioxane, 90°C; (iv) n—Bu4NF, THF, room
temperature, 82%; (v) N—propargyltrifluoroacetamide, Pd(PPh3)4(0), Cul, Et3N, DMF; (vi)
POCl3, proton sponge, (MeO)3PO, 0°C; (n—Bu3NH)2H2PZO7, n-Bu3N, DMF; l M
HNEt3HCO3.
033 803
O / / o
N+ N
OZN NH2
(vi)
t—Bu 0A6,“NJso
HO\ /o\ /o\ /o
P P P
_ GAO\_ \\ K 0 o’\0'o/o 9
Scheme 5b. (vi) Cy5 NHS, 0.1 M NazCO3/NaHCO3 buffer (pH 9.2).
Alexa FIuor647—N
02N NH2
(vi) t-Bu 0%,“
N O
HO\ /O\ /O\ /O
P P P
\ \ \ O
‘0’ \0‘o’\o -o’\o K 9
Scheme 5c. (vi) Alexa Fluor 647 NHS, 0.1 M NazCO3/NaHCO3 buffer (pH 9.2).
In some embodiments, it was observed that the stereochemistry of the alkyl
substituted at the (x-carbon can improve the hemical cleavage properties of a 2-
nitrobenyl group. In some embodiments, it was observed that faster photochemical cleavage
rates result by combining the stereospecific group at the oc-carbon with another chemical
group attached to the 2-nitrobenzyl ring. See, for example,
Compounds of the present disclosure may be made using the methods bed
above and in the Example section below. For example, a summary of a sis for making
(x-tBu-S-OMenitrobenzyl alcohol, including an opure form thereof, is provided in
Example 8. These methods can be further modified and optimized using the principles and
techniques of organic try as applied by a person skilled in the art. Such principles and
techniques are taught, for example, in March ’s Advanced c Chemistry: ons,
Mechanisms, and Structure (2007), which is incorporated by reference herein.
Compounds employed in methods of the invention may contain one or more
asymmetrically-substituted carbon or nitrogen atoms, and may be isolated in optically active
or racemic form. Thus, all chiral, diastereomeric, racemic form, ic form, and all
geometric isomeric forms of a structure are intended, unless the specific stereochemistry or
ic form is specifically ted. Compounds may occur as racemates and racemic
mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. In some
embodiments, a single diastereomer is obtained. The chiral centers of the compounds of the
present invention can have the S or the R configuration. For e, in some aspects of the
present disclosure, tution and its stereochemistry of the oc—carbon of the benzyl ether of
modified 5-hydroxymethyl pyrimidine or 7-hydroxymethyldeazapurine bases affects
biological function and ge rates of reaction of 3’—OH unblocked, base—modified dNTPs.
Compounds of the invention may also have the advantage that they may be more
efficacious than, be less toxic than, be longer acting than, be more potent than, produce fewer
side effects than, be more easily absorbed than, and/or have a better cokinetic profile
(e.g., higher oral bioavailability and/or lower clearance) than, and/or have other useful
pharmacological, physical, or chemical properties over, compounds known in the prior art,
whether for use in the indications stated herein or otherwise.
Chemical as used to represent compounds of the invention will typically only
show one of possibly several ent tautomers. For example, many types of ketone groups
are known to exist in equilibrium with corresponding enol groups. Similarly, many types of
imine groups exist in equilibrium with enamine groups. less of which tautomer is
depicted for a given compound, and regardless of which one is most prevalent, all tautomers
of a given chemical formula are intended.
In addition, atoms making up the compounds of the present invention are intended to
include all ic forms of such atoms. Isotopes, as used herein, include those atoms
having the same atomic number but different mass numbers. By way of l example and
without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of
carbon include 13C and 14C. Similarly, it is contemplated that one or more carbon atom(s) of
a compound of the present invention may be replaced by a silicon atom(s). Furthermore, it is
contemplated that one or more oxygen atom(s) of a compound of the present invention may
be replaced by a sulfur or selenium atom(s).
In some ments, the 3’—OH unblocked reversible terminators provided herein
have an thiophosphate group, preferably an alpha-thiotriphosphate group. See, for
e, compounds 54b, 55b, 59b, 60b, 63b, 64b, 68b, and 69b, in Example 8 below. It is
well known in the art that DNA polymerases exhibit 3’-5’ exonuclease activity. The function
of the 3’—5’ exonuclease ty is to remove the just incorporated nucleotide from the primer
strand. Many commercially available DNA polymerases delete or mutate the 3’—5’
exonuclease domain to reduce this activity below able levels. Nonetheless, even low
level activity of some DNA polymerases can result in poor sequence data quality due to
dephasing of the primary signal. In some embodiments, the 3’—OH unblocked reversible
terminators having alpha-thiotriphosphate groups may be used to , minimize and/or
eliminate the residual 3’-5’ exonuclease activity. Without being bound by theory, it is well
known in the art that alpha-thiotriphosphates are resistant to exonuclease activity. See, for
example, an Patent EP 0 640 146 to Rosenthal and Brenner, which is incorporated
herein by reference.
Compounds of the present invention may also exist in prodrug form. Since prodrugs
are known to enhance numerous ble qualities of pharmaceuticals (e.g., solubility,
bioavailability, manufacturing, etc), the nds employed in some methods of the
invention may, if desired, be delivered in g form. Thus, the invention contemplates
prodrugs of compounds of the present invention as well as methods of delivering prodrugs.
Prodrugs of the compounds ed in the invention may be prepared by modifying
functional groups present in the compound in such a way that the modifications are cleaved,
either in e manipulation or in vivo, to the parent compound. Accordingly, gs
include, for example, compounds described herein in which a hydroxy, amino, or carboxy
group is bonded to any group that, when the prodrug is administered to a subject, cleaves to
form a hydroxy, amino, or carboxylic acid, respectively.
The present disclosure further provides nucleotide and nucleoside compounds as well
as salts, esters and phosphates thereof, that can be used in rapid DNA sequencing technology.
However, it should be recognized that the particular anion or cation forming a part of any salt
of this invention is not critical, so long as the salt, as a whole, is pharmacologically
acceptable. onal examples of ceutically acceptable salts and their methods of
preparation and use are presented in Handbook ofPharmaceutical Salts: Properties, and Use
(2002), which is incorporated herein by reference. The compounds are optionally in the form
of ribonucleoside triphosphates (NTPs) and deoxyribonucleoside triphosphates (dNTP). The
nucleotide and nucleoside compounds in some cases include a chemically or enzymatically
cleavable group labeled with a reporter group such as a cent dye. The nucleotide and
nucleoside compounds include chemically or enzymatically removable protecting groups that
are designed to ate DNA synthesis as well as cleave rapidly, so that these monomers
can be used for rapid sequencing in a parallel format. The presence of such rapidly cleavable
groups d with fluorescent dyes on the nucleotide and nucleoside nds can
enhance the speed and accuracy of sequencing of large oligomers of DNA in parallel, to
allow, for example, rapid whole genome cing, and the identification of polymorphisms
and other valuable genetic information.
These 3’—OH ked terminators are well-tolerated by a number of commercially
available DNA polymerases, representing a key advantage over 3’-0—blocked terminators.
The benzyl group of the nds disclosed herein can also can be derivatized to include a
selected fluorescent dye or other reporter group.
11. Properties of ible Terminators
As discussed above, in one aspect, there are provided novel alkylated 2—nitrobenzyl
nucleotides with fast photochemical cleavage properties that may be used as improved
reversible terminators for cyclic reversible terminator (CRT) sequencing ations. Such
applications are bed in Metzker (2005, 2010), which are both incorporated by reference
herein. In some embodiments, there are provided modifled a—7-hydroxymethyl—2’-
denosine (C7—HOMedA) (Rockhill et al., 1997) and 7—deaza—7—hydroxymethyl—2’-
deoxyguanosine (C7—HOMedG) (McDougall et al., 2001) along with HOMedC and HOMedU
with a variety of tuted 2—nitrobenzyl groups. See In some embodiments, the
reversible terminators disclosed herein exhibit a number of suitable properties, including fast
kinetics of nucleotide incorporation, single-base termination, high nucleotide selectivity,
and/or rapid cleavage of the terminating group.
tographic conditions were identified to separate C7—HOMedA analogs into
single diastereomeric nucleotides, with the first eluting isomer denoted as dsl and the second
as ds2. To evaluate the photochemical cleavage effect of the stereochemistry of an 0t—
pyl group substitution with the 2-nitrobenzyl ring modifications of 4-methoxy (4-OMe)
and 6-nitro (6—N02), three C7—HOMedA analogs dA.III.a — dA.III.c, were synthesized, as
well as the parent dA.I (see Examples section below). Incorporation assays were performed
with these obenzyl ted C7-HOMedATP analogs and then subjected to UV
photochemical ge experiments in sodium azide solution (Table 1).
Table 1. Photochemical Cleavage Rates of C7-HOMedA Analogs
DT50 ill 1 HIM NaN3
C -HOMedA analog
N0 DTT 50 mM DTT
dA.I 3.6 :: 0.1 3.5 :: 0.1
dA.III.a dsl 4.5 :: 0.2 4.4 :: 0.2
dA.III.a (182 2.2 :: 0.1 2.1 :: 0.1
dA.III.b dSl 7.0 :: 0.3 6.1 :: 0.4
dA.III.b (182 1.1 :: 0.1 1.0 :: 0.1
dA.III.c dsl 3.4 :: 0.2 3.0 :: 0.2
dA.III.c dSZ 2.8 :: 0.2 2.5 :: 0.1
In all cases, the ds2 isomers of dA.III.a — dA.III.c showed faster photochemical
cleavage rates (i.e., lower DT50 values) by factors of 2.0><, 6.4><, and l.2><, respectively,
compared with those of their dsl counterparts. stingly, the dsl isomers exhibited
similar I.c) or higher (dA.III.a or dA.III.b) DT50 values compared with the parent
dA.I analog. These data provide evidence that stereochemistry of the substituted 0t—isopropyl
group is an important determinant, and coupled with a 4-OMe substitution, the dA.III.b ds2
analog produced the lowest DT50 value for the propyl C7—HOMedA set.
Previous work demonstrated that the Ot—tert—butyl analog dU.V exhibited excellent
CRT properties such as single-base termination and high nucleotide selectivity (Litosh et al.,
2011). This allowed to further examination of the stereospecific effect using a different 0tsubstitution
group coupled with various OMe ring tutions by synthesizing four (Hert-
butyl C7—HOMedG analogs, dG.V.a — dG.V.d, along with the parent dG.I (.
Consistent with the propyl-C7-HOMedATP analogs, UV photochemical cleavage
experiments ed that ds2 isomers of dG.V.a — dG.V.d showed faster rates by factors of
3.l><, 4.5><, 4.4><, and 3.0><, respectively, compared with those of their dsl counterparts (Table
WO 40257
Table 2. hemical Cleavage Rates of edG Analogs.
DT50 in 1 mM NaN3
C -HOMedG analog
N0 DTT 50 mM DTT
dG.I 9.2 :: 0.3 8.1 :: 0.2
dG.V.a dsl 11.0 :: 0.4 10.7 :: 0.2
dG.V.a ds2 3.6 :: 0.3 3.5 :: 0.3
dG.V.b dsl 4.9 :: 0.3 4.6 :: 0.3
dG.V.b ds2 1.1 :: 0.1 1.3 :: 0.2
dG.V.c dsl 3.5 :: 0.3 3.0 :: 0.1
dG.V.c ds2 0.8 :: 0.1 0.8 :: 0.1
dG.V.d dsl 2.4 :: 0.1 2.3 :: 0.2
dG.V.d ds2 0.8 :: 0.1 0.8 :: 0.1
Both 5—OMe ds1 and ds2 isomers exhibited faster photochemical cleavage rates of
1.4>< fold each compared with the corresponding 4—OMe isomers. The bis—substituted 4,5—di—
OMe ds1 isomer showed faster cleavage rates compared with mono—substituted 4—OMe (2.0><)
or 5—OMe (1.5 X) isomers. Conversely, the 5—OMe ds2 and 4,5—di—OMe ds2 s exhibited
identical DT50 values of just 0.8 sec. In the absence of an (x—substitution group, Hasan et a].
(1997) reported a rate increase of only 1.2>< for a 5-OMenitrobenzyl analog over its
corresponding parent. Comparison of ds1 and ds2 isomers of dG.V.c with dG.V.a revealed
higher rate increases of 3.6>< and 4.4><, tively, suggesting that the stereospecific tert—
butyl group enhances the effect of the 5-OMe group. With four—color CRT applications, this
combination provides good flexibility in ring system utility, as a linker ure can also be
attached to the 4—position to create dye—labeled analogs (US. Patents 7,897,737 7,964,352,
and 8,148,503; US. Patent Appl. Publication 287427; Metzker, 2010).
To determine the stereochemistry of these (x—tert—butyl C7—HOMedG analogs, the (IS)-
camphanate of (R/S)—1-(5-methoxynitrophenyl)-2,2-dimethylpropanol was resolved into
its enantiopure (5) alcohol by fractional crystallization (Corrie et al., 1992) (. This (5)
alcohol and (S)-(x—tert—butyl—2—nitrobenzyl alcohol (US. Patent 8,148,503; Litosh et al.,
2011)were each coupled to C7—HOMedG (. RP—HPLC analysis of their corresponding
triphosphates revealed that both ds2 isomers of dG.V.a and dG.V.c had identical peak
retention times as that for dG.V and dG.VI, respectively, thus allowing us to ine that
both ds2 isomers have the same (S) configuration at the bon. By inference, the
corresponding ds1 isomers of dG.V.a and dG.V.c have been assigned the (R) configuration.
These (S) alcohols were then coupled to the remaining nucleosides to examine the
effect of the leaving group on the hemical cleavage rate. For example, UV
photochemical ge experiments revealed that DT50 values for the parent 2-nitrobenzyl
analogs varied from 2.0 sec for dC.I to 9.2 sec for dG.I (and Table 3).
Table 3. Photochemical Cleavage Rates of Reversible Terminators
DT50 ill 1 HIM NaN3
Nucleotide analog
N0 DTT 50 mM DTT
dA.I 3.6 :: 0.1 3.5 :: 0.1
dA.V 2.1 :: 0.1 2.0 :: 0.2
dA.VI 0.8 0.1”] 0.8 0.1
dC.I 2.0 :: 0.3 1.6 :: 0.2
dC.V 1.2 :: 0.1 1.0 :: 0.2
dC.VI 0.6 0.1”] 0.6 0.1
dG.I 9.2 :: 0.3 8.1 :: 0.2
dG.V 3.0 :: 0.1 2.9 :: 0.2
dG.VI 0.8 :: 0.1 0.8 :: 0.1
dU.I 2.1 :: 0.1 1.7 :: 0.1
dU.V 1.4:: 0.1 1.3 :: 0.1
dU.VI 0.7 0.1”] 0.7 0.1
[alTransient product (TP) observed by gel electrophoresis; considered as cleaved t in
DT50 value.
Substitution of the benzylic carbon with (S)—tert—butyl resulted in increased cleavage
rates by factors of l.5>< — 3.l>< and the additional tution with a 5-OMe group further
increased rates by factors of 3.0>< — 11.5>< compared with the parent analogs. The greatest
rate improvement was observed when comparing C7—HOMedG analogs, reducing DTso
values from 9.2 to 0.8 sec ( black bars). The complete set of (S)—5—OMe—(x—tert—butyl
reversible terminators showed a more narrow range of DT50 values from 0.6 to 0.8 sec.
These data suggest that the combined effects of the (S)—0t—tert—butyl and 5—OMe groups play
an important role in diminishing ge rate variation ed with particular nucleotide
g groups, having the cal application of providing normalized and faster cleavage
conditions for the CRT cycle. Unexpectedly, transient products were observed from
incorporation assays for (S)—5—OMe—0t—tert—butyl—C7—HOMedA, —HOMedC and —HOMedU,
but not —C7—HOMedG, following brief exposure to UV light (HOMedU only shown in FIG 2,
left side). As the only difference being the just incorporated nucleotide, we hypothesize that
the faster cleaving (S)—5-OMe—0t-tert—butyl—2—nitrobenzyl group produces a more ve 2—
nitrosoketone by-product that attacks the minal nucleotide of the growing primer strand.
To investigate conditions to quench the nitroso intermediate, a number of amino and
thiol agents were tested during UV photochemical cleavage experiments (. Of these,
only dithiothreitol (DTT) nd, 1964) eliminated the transient product ( right side).
In some embodiments, the effective DTT concentration is from 1 mM to l M. In some
embodiments, the effective DTT tration is from 5 mM to 100 mM. In some
embodiments, the effective DTT concentration is from 10 mM to 50 mM. In some
embodiments, the effective DTT concentration is about 50 mM. In some embodiments, the
photochemical cleavage step takes place in the presence of sodium azide. In some
embodiments, the effective sodium azide tration is from 0.1 mM to l M. In some
embodiments, the effective sodium azide concentration is from 1 mM to 100 mM. In some
embodiments, the effective sodium azide concentration is from 1 mM to 50 mM. In some
embodiments, the effective sodium azide concentration is about 1 mM.
To test rate effects, UV photochemical cleavage experiments were repeated for all
compounds in the presence of DTT, of which DT50 values for several parent and dsl s
were reduced (Tables 1, 2, and 3). Barth et a]. (2005) proposed that DTT s the nitroso
group by nucleophilic addition, although a later review by Corrie (2005) describes protective
thiols as unnecessary as the evidence for biological interference from of the nitrosoketone by-
product remains minimal. In our examples described in this invention, DTT plays an
important tive role against such undesired ons.
The stereospecific (S) configuration of the (x—substituted group combined with a
—methoxy group were found to be determinants in creating fast-cleaving reversible
ators. The reactive nitrosoketone by-product can be effectively eliminated during
photochemical cleavage in the presence of DTT, providing appropriate conditions in
maintaining the biological integrity of the CRT reaction.
III. Nucleotide and side Compounds and Their Use in DNA cing
The reversible terminators of the present ion may be used in DNA sequencing
methods based on a variety of approaches, including:
0 “Sanger” or “dideoxy” methods, which involve the chain termination of DNA
synthesis by the incorporation of 2’,3’—dideoxynucleotides (ddNTPs) using
DNA polymerase. See Metzker et al., 2005, which is incorporated herein by
reference.
Sequencing—by—synthesis (SBS), which typically does not clearly delineate the
different mechanics of cing DNA. See Metzker, 2010; Metzker 2005,
which are incorporated herein by nce.
DNA polymerase—dependent gies, which are also classified as cyclic
ible termination (CRT), single nucleotide addition (SNA, e.g.,
pyrosequencing), and real—time sequencing. See Metzker, 2010, which is
incorporated herein by reference.
Single molecules sequencing using the Random Nick Sequencing (RNS)
approach.
In some embodiments, the ion provides methods of sequencing a target nucleic
acid comprising the following steps:
(0 attaching the 5’-end of a primer to a solid surface;
(ii) hybridizing a target nucleic acid to the primer attached to the solid surface;
(iii) adding a compound according to any of structures described herein, with the
proviso that where more than one type of base is present, each base is attached
to a different reporter group;
0V) adding a nucleic acid replicating enzyme to the ized primer/target
nucleic acid complex to incorporate the composition of step (iii) into the
growing primer strand, wherein the incorporated composition of step (iii)
terminates the polymerase reaction at an efficiency of n about 70% to
about 100%;
(V) washing the solid surface to remove unincorporated components;
(vi) detecting the incorporated reporter group to identify the incorporated
composition of step (iii);
(vii) optionally adding one or more chemical compounds to permanently cap
unextended primers;
(viii) removing the terminating moiety comprising photochemically cleaving off the
terminating moiety, ing in an extended primer with 5-hydroxymethyl
pyrimidine or oxymethyldeazapurine bases;
(iX) washing the solid surface to remove the d terminating group; and
(x) repeating steps (iii) through (viii) one or more times to fy the plurality of
bases in the target nucleic acid.
In some variations, the order of steps (iii) and (iv) is reversed. In further variations, the
polymerase and the compound are added at the same time. In embodiments, they are in the
same solution.
In another aspect the invention provides a method of sequencing a target nucleic acid
comprising the ing steps:
(0 attaching the 5’—end of a target nucleic acid to a solid surface;
(ii) hybridizing a primer to the target c acid attached to the solid
e;
(iii) adding a compound according to any of structures described herein,
with the o that where more than one type of base is present, each
base is attached to a different reporter group;
0V) adding a nucleic acid replicating enzyme to the hybridized
primer/target nucleic acid complex to incorporate the composition of
step (iii) into the growing primer strand, wherein the incorporated
composition of step (iii) terminates the polymerase reaction at an
efficiency of between about 70% to about 100%;
(V) washing the solid surface to remove unincorporated components;
(V0 detecting the incorporated er group to identify the incorporated
composition of step (iii);
(vii) optionally adding one or more chemical compounds to permanently
cap unextended primers;
(viii) removing the ating moiety comprising photochemically cleaving
off the terminating moiety, resulting in an extended primer with a 5-
hydroxymethyl pyrimidine or 7—hydroxymethyl—7-deazapurine bases;
(iX) washing the solid surface to remove the cleaved terminating group; and
(X) repeating steps (iii) through (ix) one or more times to fy the
plurality of bases in the target nucleic acid.
In some variations, the order of steps (iii) and (iv) is reversed.
In some ments the compound is incorporated by a nucleic acid replicating
enzyme that is a DNA rase. In some embodiments the DNA polymerase is selected
from the group consisting of Taq DNA polymerase, Klenow(exo—) DNA polymerase, Bst
DNA polymerase, VENT® (exo-) DNA polymerase (DNA polymerase A cloned from
Thermococcus Zitoralis and containing the Dl4lA and E143A mutations), Pfu(exo-) DNA
polymerase, and DEEPVENTTM (exo-) DNA polymerase (DNA polymerase A, cloned from
the Pyrococcus species GB-D, and containing the Dl4lA and E143A mutations). In some
embodiments the DNA polymerase is selected from the group consisting of AMPLITAQ®
DNA polymerase, FS (Taq DNA polymerase that contains the G46D and F667Y mutations),
SEQUENASETM DNA polymerase (Taq DNA polymerase that contains the F667Y
mutation), THERMOSEQUENASETM II DNA polymerase (blend of
SEQUENASETM DNA polymerase and T. acidophilum osphatase),
THERMINATORTM DNA polymerase (DNA polymerase A, cloned from the Thermococcus
species 9°N—7 and containing the Dl4lA, E143A and A485L mutations),
THERMINATORTM II DNA polymerase (THERMINATORTM DNA polymerase that
contains the additional Y409V mutation), and VENT® (exo-) A488L DNA polymerase
(VENT® (exo-) DNA polymerase that contains the A488L mutation).
Compounds of the present disclosure may be made using the methods ed in the
Examples section. These methods can be further d and optimized using the principles
and ques of organic chemistry as d by a person skilled in the art. Such principles
and techniques are taught, for example, in Wu et a]. (2007; Litosh et a]. (2011); Stupi et al.
(2012); Gardner et al., 2012; US. Patent 7,897,737, 352, and 8,148,503; US. Patent
Appl. Publ. 2011/0287427, which is incorporated herein by reference.
In some embodiments, sample components enable the determination of SNPs. The
method may be for the high-throughput identification of informative SNPs. The SNPs may
be obtained directly from c DNA material, from PCR amplified material, or from
cloned DNA material and may be assayed using a single nucleotide primer extension method.
The single tide primer extension method may se using single unlabeled dNTPs,
single labeled dNTPs, single 3’—0—modified dNTPs, single base—modified 2’—dNTPs, single
alpha—thio-dNTPs or single labeled 2’,3’-dideoxynucleotides. The mini—sequencing method
may comprise using single unlabeled dNTPs, single labeled dNTPs, single 3’—0—modified
dNTPs, single base—modified 2’—dNTPs, single alpha-thio-dNTPs or single labeled 2’,3’—
dideoxynucleotides. The SNPs may be ed ly from c DNA material, from
PCR amplified material, or from cloned DNA materials.
A. tide and side Compounds and Their Use in CRT
In some aspects of the present invention, nucleotide and nucleoside compounds
provided herein (reversible terminators) may be used in DNA cing technology based
on cyclic reversible termination (CRT). CRT is a cyclic method of detecting the
synchronistic, single base additions of multiple templates. This approach differentiates itself
from the Sanger method (Metzker, 2005, which is incorporated herein by reference) in that it
can be performed t the need for gel ophoresis, a major bottleneck in advancing
this field. Like Sanger sequencing, however, longer read-lengths translates into fewer
sequencing assays needed to cover the entire genome. The CRT cycle typically comprises
three steps, incorporation, imaging, and deprotection. The term “deprotection” may be used
synonymously with “cleavage”,
so that the three steps could also be described as
incorporation, imaging, and cleavage. For this procedure, cycle efficiency, cycle time, and
sensitivity are important s. The cycle efficiency is the product of deprotection and
incorporation efficiencies and determines the CRT read-length. The CRT cycle time is the
sum of incorporation, imaging, and deprotection times. For rapid CRT for whole genome
sequencing, the nucleotide and nucleoside nds as disclosed herein may be used,
which can exhibit fast and efficient deprotection properties. These compounds can be labeled
with reporter groups such as cent dyes, attached directly to the benzyl group having an
azido substitution on the alpha carbon, providing, e.g., cent, ible terminators
with similar deprotection properties. It has remained difficult to accomplish the goal of long
CRT reads because reversible terminators typically act as poor substrates with commercially
available DNA polymerases. Modified nucleotide analogs of the present invention may be
used to improve this technology by providing substrates that incorporate as well or better than
a natural nucleotide with commercially available DNA polymerases.
Photocleavable groups attached to the base of a 3’—OH unblocked nucleotide, such as
the groups described herein, can act as an ive reversible terminator and be efficiently
incorporated by wild—type DNA polymerases. See Wu et al., 2007; Metzker, 2010; Litosh et
al., 2011; Gardner et al., 2012; US. Patents 7,897,737, 7,964,352, and 8,148,503; US. Patent
Appl. Publ. 2011/0287427, which are incorporated herein by nce. For example,
-hydroxymethyl-2’-deoxyuridine (HOMedU) is found naturally in the genomes of numerous
bacteriophages and lower eukaryotes (Gommers—Ampt, 1995, which is incorporated herein by
reference). Its ymethyl group can serve as molecular handle to attach a small
photocleavable terminating group. Other naturally occurring hypermodif1ed bases that can be
r modified in the manner described herein to function as reversible terminators include
-hydroxymethyl-2’-deoxycytidine (HOMedC), which is found naturally in the genomes of
T2, T4, and T6 bacteriophages (Wyatt & Cohen, 1953; Gommers—Ampt, 1995) and of
s (Kriaucionis & Heintz, 2009; Tahiliani et al., 2009; Ito et al., 2010). The
pyrrolopyrimidine ring structure (7-deazapurine) is also found lly in nucleoside
antibiotics sco & Vazquez, 1984, which is incorporated herein by reference) and tRNA
bases (Limbach, et al., 1994, which is incorporated herein by reference), and the compounds
7—deaza—7—hydroxymethyl—2’—deoxyadenosine (C7—HOMedA) (Rockhill et al., 1997) and 7—
deazahydroxymethyl-2’-deoxyguanosine (C7—HOMedG) gall et al., 2001) may
also be further modified in the manner described herein to function as reversible terminators.
In some embodiments described herein, the photocleavable group is a substituted 2—
enzyl nucleotide, which may be efficiently hemically cleaved, for example, with
365 nm UV light. See US. Patent Appl. Publ. 2010/0041041, which is incorporated herein
by reference. It is generally understood the wavelengths >300 nm are used to ze
damage to DNA and proteins (Corrie, 2005) with several specific wavelengths other than 365
nm being 340 nm (Kaplan et al., 1978) and 355 nm (Seo, 2005).
In some embodiments, the 3’—OH unblocked reversible terminators described herein
typically have several advantages, including, for e, that photocleavage of only a single
bond removes both the terminating and fluorophore groups from the nucleobase. This in turn
may be used to more efficiently restore the nucleotide for a subsequent CRT cycle. A second
advantage of 3’—OH unblocked reversible terminators provided herein is that many of these
compounds show more favorable enzymatic incorporation and, in some embodiments, can be
incorporated as readily as a natural nucleotide with wild-type DNA polymerases.
One challenge for 3’-OH unblocked terminators is creating the appropriate
modifications to the base that lead to termination of DNA synthesis after a single base
addition. This is typically ant because an unblocked 3’-OH group is the natural
substrate for incorporating the next incoming nucleotide. The compounds bed herein
address this challenge. For example, in some embodiments, there the ible terminators
provided herein lead to ation of DNA synthesis after a single base addition.
In some embodiments, the compounds disclosed herein may be used in CRT to read
directly from genomic DNA. Fragmented genomic DNA can be hybridized to a high-density
oligonucleotide chip containing priming sites that span selected chromosomes. Each priming
sequence is separated by the estimated read—length of the CRT method. Between base
additions, a fluorescent imager can simultaneously image the entire high-density chip,
marking significant improvements in speed and sensitivity. In specific embodiments, a
fluorophore, which is attached to the benzyl group having an azido tution on the alpha
carbon or its derivatives described herein, is removed by a specific chemical or enzymatic
reaction releasing the benzyl group for the next round of base addition. In another specific
embodiments, a fluorophore, which is attached to the benzyl group having an amide
substitution on the alpha carbon or its tives described herein, is d by a specific
enzymatic or al reaction releasing the benzyl group for the next round of base
addition. After approximately 500 CRT cycles, the complete and contiguous genome
sequence information can then be compared to the reference human genome to determine the
extent and type of sequence variation in an individual’s sample. Reversible terminators that
exhibit higher incorporation and deprotection efficiencies will typically achieve higher cycle
encies, and thus longer read-lengths.
CRT Efficiency is defined by the formula: fl = 0.5, where RL is the ength
in bases and Cefl is the overall cycle efficiency. In other words, a read—length of 7 bases
could be achieved with an overall cycle efficiency of 90%, 70 bases could be achieved with a
cycle ency of 99% and 700 bases with a cycle efficiency of 99.9%. The efficiency of
incorporation of compounds according to the invention may range from about 70% to about
100% of the oration of the analogous native nucleoside. Preferably, the efficiency of
oration will range from about 85% to about 100%. Photochemical cleavage
efficiencies will preferably range from about 85% to about 100%. Further, termination of
nucleic acid ion will range from about 90% to about 100% upon incorporation of
compounds according to the ion. Nucleotide and nucleoside compounds in one
embodiment have a cycle efficiency of at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9%.
Another aspect of the present invention is directed towards the use of quencing,
which is a non—electrophoretic, bioluminescence method that measures the release of
inorganic pyrophosphate (PPi) by proportionally converting it into visible light by a series of
enzymatic reactions (Ronaghi et al., 1998, which is incorporated herein by reference).
Unlike other sequencing approaches that use modified nucleotides to terminate DNA
synthesis, the pyrosequencing assay manipulates DNA polymerase by the single addition of a
dNTP in limiting amounts. DNA polymerase then s the primer upon incorporation of
the complementary dNTP and pauses. DNA synthesis is reinitiated following the addition of
the next complementary dNTP in the dispensing cycle. The order and intensity of the light
peaks are recorded as flowgrams, revealing the underlying DNA ce. For
homopolymer repeats up to six nucleotides, the number of dNTPs added is directly
proportional to the light signal. Homopolymer repeats greater than six nucleotide can result
in insertion , which are the most common error type for pyrosequencing. Modified
nucleotide s of the present invention may improve this technology by accurate
sequencing through homopolymer repeats, particularly those r than six nucleotides in
length.
Another aspect of the present invention is directed towards the use of Sanger
sequencing, for example, as applied to zygote detection. Despite much advancement,
improvements in the dideoxy-BigDye terminator cing chemistry for accurate
heterozygote detection are needed. It is generally believed that a uniform peak height
distribution in the primary data makes alling and heterozygote detection more reliable
and accurate. The termination pattern in Sanger sequencing is primarily due to sequence—
dependent bias incorporation by DNA polymerase, which can selectively incorporate natural
nucleotides over modified nucleotides (Metzker et al., 1998, which is incorporated herein by
reference). These bias incorporation effects are more nced with the dye—terminator
chemistry than with the dye-primer try. This can be uted to s of the large
fluorescent dye ures attached to the terminating nucleotide, lowering enzyme activity at
least 10-fold to that of the natural substrate. Thus, the reduction of bias incorporation effects
by DNA polymerase towards dye—labeled terminators could lead to improved heterozygote
detection. Modified nucleotide s of the present invention may improve this technology
by incorporating as well or better than a natural nucleotide, thus eliminating incorporation
bias in Sanger sequencing.
r aspect of the present invention is directed towards the use of clonally
amplified templates and single DNA molecule templates. The front—end ofNGS logies
can be partitioned into two camps: clonally amplified templates from single DNA molecules
and single DNA molecule templates. It is well recognized in the art that DNA can be
lized to a solid surface by either attaching a primer to said surface and hybridizing a
target nucleic acid to said primer (US. Patent No. 5,770,367; Harris et al., 2008, which are
incorporated herein by reference) or by attaching a target nucleic acid to said surface by
clonally amplification and hybridizing a primer to said target nucleic acid (Dressman et al.,
2003; Margulies et al., 2005, which are incorporated herein by reference). Either
immobilization ration can be used in the present invention for then binding a DNA
polymerase to initiate either the CRT method or the pyrosequencing method.
An aspect, the present invention is directed towards the use of single template
molecules, that t of large DNA fragments (i.e., 0.1 — 0.5 megabase). In some
embodiments, an adaptor—free strategy called Random Nick Sequencing (RNS) can be
employed. It has l advantages including (a) no requirement for PCR or adaptor
on, (b) ant sequencing of the same template to improve accuracy, and (c) e
sequencing reaction sites across the single molecule template providing localized de novo
assemblies. For example, the PCR process creates mutations in clonally amplified tes
that masquerade as sequence variants. AT—rich and GC—rich target sequences may also show
amplification bias in product yield, resulting in their underrepresentation in genome
alignments and assemblies. Knowing the location of sequencing reactions for a given single
molecule template will simplify the location assignment and zation of complex
genomic and structural regions in the assembly of s.
In RNS approach, sugar—nonspecific ses such as that isolated from Vibrio
vulnificus (vvn) create random single—stranded nicks in dsDNA and digest both ssDNA and
RNA (See Hsia et al., 2005). VW binding occurs in the minor groove of DNA, avoiding
sequence—dependent recognition of nucleobases. This represents an advantage in nicking
along a DNA molecule in a random fashion without sequence—dependent bias. The upstream
strand of the nick becomes a priming site for polymerase to begin the RNS reaction. For this
approach to work in some embodiments, the polymerase must be able to displace the
downstream strand while extending the upstream strand. Several well-known DNA
polymerases that possess this property include (p29 (See Soengas et al., 1995, which is
incorporated herein by reference) and Bst (See Aliotta et al., 1996, which is incorporated
herein by reference) polymerases. xo—) (see Gardner et al., 1999, which is
incorporated herein by reference) and TherminatorTM polymerases also show strand
displacement ties, albeit maybe limited to 50 bases before stalling. Following UV
cleavage, hydroxymethyl tides are created which serve as excellent template bases for
subsequent rounds of RNS (). The strand cement during RNS creates a
bifurcated dsDNA (flap) structure similar to that created with the Invader assay (See
hev et al., 1999). Flap endonucleases (FENl) are known to cleave these bifurcated
structures as shown in , generating a downstream 5’—PO4 end strand t a
nucleotide gap (Kaiser et al., 1999). This creates a ligatable substrate to repair the dsDNA
molecule for subsequent rounds of RNS.
B. Polymerase Assays
Another aspect of the present invention is directed s the use of polymerase
assays. Natural and modified nucleotides were tested for incorporation efficiency using the
“polymerase end point assay” (Wu et al., 2007, which is incorporated herein by reference).
This assay examines oration efficiency on matched and mismatched template bases.
Incorporation efficiency is measured by determining the concentration at which the
nd orates on half the primer-template complexes (leo). Titrations of
increasing compound tration were performed to te curves from which the lC50
can be determined.
The sequence of the template DNA is selected depending on which compound will be
tested. For example, the first interrogation base after the primer in the template sequence is
the complement base of the compound when measuring incorporation efficiency, and one of
three mismatched bases when measuring mismatch discrimination properties.
To the annealed reaction, a DNA polymerase (e. g., THERMINATORTM DNA
polymerase, 0.25 units per reaction, New England Biolabs), 1>< Thermopol Buffer, and a
known concentration of either natural or modified tide are added to each 10 uL
on and incubated at 75°C for 10 minutes, cooled on ice, and quenched with 10 uL of
stop solution (98% formamide: 10 mM NazEDTA, pH = 8.0, 25 mg/ml Blue Dextran).
Stopped reactions are heated to 75°C for 30 seconds to denature the DNA, and then placed on
ice. The extension products are analyzed on a 10% Long Ranger (Lonza) polyacrylamide gel
using an ABI model 377 DNA sequencer. Additional details are provided in Example 1,
below.
C. Mismatch Discrimination and Termination
Another aspect of the present invention is directed towards improved discrimination
against ch incorporation, for example, through the use of the reversible terminators
bed herein. It has been reported that substitution at the oc—carbon of the 2—nitrobenzy1
group can increase the rate of the cleavage reaction (Reichmanis et al., 1985; Cameron and
Frechet, 1991; Hasan et al., 1997, all three are incorporated herein by reference). Without
being bound by theory, the results presented herein suggest that substitution at the oc—carbon
of the obenzy1 group can also affect the termination of DNA synthesis for 3’-OH
unblocked nucleotide triphosphates and e discrimination against mismatch
incorporation. Furthermore, and based on the results discussed in greater detail below, it was
found that the stereochemistry of the substitution of oc-carbon of the 2-nitrobenzyl group can
have a significant impact on the extent of mismatch discrimination and the rate of the
cleavage reaction. t being bound by theory, at least two factors were found to
typically influence termination of DNA sis after a single incorporation: a) substitution
at the a-carbon of the 2-nitrobenzyl group, and b) substitution at the 2-position of the benzyl
ring.
D. UV-Cleavage Rates
Another aspect of the present invention is directed s ing reversible
ators with improved UV-cleavage rates. Cleavage of the terminating substituted
2-nitrobenyl group when analogs are incorporated into the primer strand with 365 nm UV
light allows for the next cycle of incorporation to resume. Without being bound by theory, at
least two s were found typically influence UV—cleavage rates of incorporated tide
analogs: a) stereo-chemistry of the oc-carbon substitution of the 2-nitrobenzyl group, and b)
substitution on the benzyl ring.
E. Next-generation cing (NGS) technologies
Another aspect of the present ion is directed towards applying the reversible
terminators and methods provided herein to next-generation sequencing methods.
Sequencing technologies include a number of s that are grouped broadly as (a)
template preparation, (b) sequencing and imaging, and (c) data analysis. The unique
combination of ic protocols distinguishes one technology from r and determines
the type of data produced from each platform. These differences in data output present
nges when comparing platforms based on data quality and cost. Although quality
scores and accuracy estimates are provided by each cturer, there is no consensus that a
‘quality base’ from one platform is equivalent to that from another platform. The compounds
and s described herein may be used in combination with and/or applied to one or more
of the template formats described below.
Methods used in preparing templates for NGS reactions include: clonally amplified
templates originating from single DNA molecules, and single DNA molecule templates.
Sequencing methods that use DNA polymerases are classified as cyclic reversible termination
(CRT), single-nucleotide addition (SNA) and real-time sequencing. Sequencing by ligation
(SBL), an approach in which DNA polymerase is replaced by DNA ligase, has also been used
in the NGS technologies. See, e.g., Shendure et al., 2005 and Valouev et al., 2008, which are
WO 40257
incorporated herein by reference. Imaging methods coupled with these sequencing strategies
range from measuring inescent signals to olor imaging of single molecular
events. In some embodiments, such combined methods are r combined with suitable
information technology systems capable of ng the voluminous data produced by NGS
platforms, including aspects related to data storage, tracking and quality control. See Pop &
Salzberg, 2008, which is incorporated herein by reference.
a) Template Preparation
In some ments, the present invention is directed towards ng and/or
ing the reversible terminators and sequencing methods with one or more templates or
template preparation methods. For example, in some embodiments, robust template
preparation methods are used. These produce representative, non—biased sources of nucleic
acid material from the genome under investigation. In some embodiments, the method
involves randomly ng c DNA into smaller sizes from which either fragment
templates or mate-pair templates are created. In some embodiments, for example those
associated with NGS technologies, the template is attached or immobilized to a solid surface
or support. The lization of spatially separated template sites may be used to allow for
thousands to billions of sequencing reactions to be performed simultaneously.
Clonally amplified templates. In some embodiments, the present invention comprises
the use of clonally amplified templates or clonally amplified template preparation s.
For e, such templates may be used with imaging systems that have not been designed
to detect single fluorescent events. For example, two common amplification methods are
emulsion PCR (also called emPCR) and solid—phase amplification. See Dressman et al., 2003
and Fedurco et al., 2006, which are both incorporated herein by reference. In some
embodiments, emPCR may be used to prepare sequencing templates in a cell—free system,
which has the advantage of ng the arbitrary loss of genomic sequences — a problem
that is typically nt in bacterial cloning s. In some embodiments, a library of
fragment or mate-pair targets is created, and adaptors containing universal priming sites are
d to the target ends, allowing complex genomes to be amplified with common PCR
primers. For example, after ligation, the DNA is separated into single strands and captured
onto beads under conditions that favor one DNA molecule per bead. See Metzker 2010, Fig.
la, which is incorporated herein by reference. For example, after the successful amplification
and enrichment of emPCR beads, millions can be immobilized in a polyacrylamide gel on a
standard microscope slide (used with the Polonator instrument; See, e.g., Shendure et al.,
2005, which is incorporated herein by reference), chemically crosslinked to an aminocoated
glass surface (used with the Life/APG SOLiD and Polonator instruments; see, e.g., Kim et
al., 2007, which is incorporated herein by reference) or deposited into either individual
PicoTiterPlate (PTP) wells (used with the Roche/454 instrument; Margulies et al., 2005,
which is incorporated herein by reference) or IonChip well (used with the Ion t
ment; rg et al., 2011, which is incorporated herein by reference) in which the
NGS chemistry can be performed. In some embodiments, solid-phase amplification may be
used to produce randomly distributed, clonally amplified clusters from nt or mate—pair
templates on a glass slide. See Metzker 2010, Fig. 1b, which is orated herein by
reference. In some embodiments, high—density forward and reverse primers are covalently
attached to the slide, and the ratio of the primers to the template on the support defines the
surface density of the amplified clusters. In some embodiments, solid-phase amplification
may be used to e 100—200 million spatially separated template clusters
(Illumina/Solexa), providing free ends to which a universal sequencing primer can be
ized to initiate the NGS reaction. See Bentley et al., 2008, which is incorporated
herein by reference.
Single-molecule templates. In some embodiments, the present invention comprises
the use of single—molecule templates or single-molecule template ation methods.
Although clonally amplified methods offer certain advantages over bacterial cloning, some of
the protocols are cumbersome to implement and require a large amount of genomic DNA
material (3—20 ug). The preparation of single-molecule tes is typically more
straightforward and requires less starting al (<1 ug). In some ments, these
methods do not require PCR, which typically create mutations in clonally amplified templates
that masquerade as sequence variants. AT—rich and GC—rich target sequences may also show
amplification bias in product yield, which results in their underrepresentation in genome
alignments and assemblies. In some embodiments, quantitative applications, such as RNA—
seq may be used. See Wang et al., 2009, which is incorporated herein by reference. Such
applications typically perform more effectively with non-amplified template sources, which
do not alter the entational abundance of mRNA les. In some embodiments, and
before the NGS reaction is carried out, single molecule templates are usually immobilized on
solid supports using one of at least three different approaches. In the first approach, which
may be used in some embodiments, lly distributed individual primer molecules are
ntly attached to the solid support (See Harris et al., 2008). The template, which may be
prepared, for example, by randomly fragmenting the starting material into small sizes (for
example, ~200—250 bp) and adding common adaptors to the nt ends, is then
ized to the immobilized primer. See Metzker 2010, Fig. 1c, which is incorporated
herein by reference. In the second approach, which may be used in in some ments,
spatially distributed single-molecule templates are covalently attached to the solid support
(See Harris et al., 2008) by priming and extending single-stranded, single-molecule templates
from immobilized primers. See Metzker 2010, Fig. 1c, which is incorporated herein by
reference. In some embodiments, a common primer is then hybridized to the template. See
Metzker 2010, Fig. 1d, which is incorporated by reference. In either approach, DNA
polymerase may be used, for example, to bind to the lized primed template
configuration to initiate the NGS reaction. In a third approach, which may be used in some
ments, lly distributed single polymerase les are ed to the solid
support (see Eid et al., 2009, which is incorporated herein by reference), to which a primed
template le is bound (see Metzker 2010, Fig. 1e, which is incorporated herein by
reference). In general, see US. Patents 7,329,492 and 6,255,083, which are incorporated
herein by reference. Larger DNA molecules (up to tens of thousands of base pairs) may be
used with this technique, for example, and, unlike the first two approaches, the third approach
can be used with real-time methods, resulting in potentially longer read lengths.
b) Sequencing and imaging
There are fundamental differences in sequencing ly amplified and single-
molecule templates. Clonal amplification may be used in some embodiments to yield
tions of identical templates, each of which has undergone the cing on.
Upon imaging, the observed signal is a consensus of the nucleotides or probes added to the
identical templates for a given cycle. Typically, this places a greater demand on the
efficiency of the addition process, as incomplete extension of the template ensemble results in
lagging-strand dephasing (also called type 2 dephasing). The addition of multiple nucleotides
or probes can also occur in a given cycle, resulting in leading—strand dephasing (also called
type 1 dephasing). The concept of dephasing was described by Cheeseman (See US. Patent
,302,509, which is incorporated herein by nce). Signal dephasing increases
fluorescence noise, causing base—calling errors and shorter reads (See Erlich et al., 2008).
Because dephasing is not an issue with single-molecule templates, the requirement for cycle
efficiency is relaxed. Single molecules, however, are susceptible to multiple nucleotide or
probe additions in any given cycle. Here, deletion errors may be observed to occur, in some
embodiments, owing to quenching s between adjacent dye molecules or no signal will
be detected because of the incorporation of dark nucleotides or probes. In the following
sections, sequencing and g strategies that use both clonally amplified and single-
le templates are sed. In some aspects of the present inventions, the reversible
ators the method of use provided herein may be applied and/or used in combination
with any one or more of the DNA polymerase-dependent gies, including, for example,
CRT, SNA, and real-time sequencing. In some embodiments, compounds of the present
invention and their method of use can be applied to and/or used in combination with the CRT
method.
There are several commercially available NGS system that imaging fluorescent
signals for single DNA molecules (See Harris et al., 2008; Bid et al., 2009, both of which are
incorporated herein by reference). Resolving single molecules on the array can be done it at
100>< magniflcation with a high sensitivity CCD camera, so long as the individual DNA
molecules are separated by a distance that approximates the diffraction limit of light (i.e., 250
nm). ions can occur that can depend on magnification and surface flatness, which
should be obvious to one of ordinary skill in the art. One technique that is widely used to
detection fluorescent signals from single molecules is total internal reflection fluorescence
(TIRF) microscopy. (See Axelrod, 1989, which is incorporated herein by reference). Other
techniques that can be used in the present invention that are known in the art include, but not
limited to, ng near—field optical microscopy (SNOM; See Moyer et al., 1993, which is
incorporated herein by reference).
F. Imaging System
illustrates an embodiment of an imaging system 100 for imaging fluorescent
signals d from clonally amplifled template. System 100 is configured to image a
microfluidic flowcell 50 that has been ed with micron beads comprising the DNA of
interest.
A range of imaging technologies, such as standard four-color imaging or color-blind
pulse-multiline excitation, may be used in various ments in combination with the 3’-
OH unblocked reversible ators. The illustrated embodiment is configured for rd
four—colored imaging.
System 100 ses an imaging device 10 (e.g., a digital camera, photocell, etc.)
configured to capture fluorescent signals derived from emPCR amplified template beads
immobilized in microfluidic flowcell 50.
Lamp 14 (e.g., a xenon lamp) creates a light path 30 between microfluidic flowcell 50
and imaging device 10. Light from lamp 14 travels to filter wheel 16. In the illustrated
embodiment, filter wheel 16 is motorized and comprises four spectral f11ters such that four-
color images may be captured. Filter wheel 16 is configured to switch between each of the
four spectral filters at each tile position so that g device 10 may capture four-color
images from the incorporated 3’—OH unblocked reversible terminators.
A portion of the light from lamp 14 travels from filter wheel 16 through objective lens
18 to microfluidic flowcell 50. Another portion of the light from lamp 14 travels from filter
wheel 16 to imaging device 10. In the illustrated embodiment, light path 30 is directed to
g device 10 using mirror 12. In other embodiments no such mirrors may be necessary,
while in still other embodiments, two or more mirrors may be required.
System 100 also comprises an ultraviolet (UV) light source 20 configured to e
UV light to flowcell 50. UV light source 20 may be a light-emitting diode (LED) in some
embodiments, or may be any other source of UV light.
G. Minimizing Ozone Contamination
In another aspect, this invention es sequences methods that minimize the s
of ozone contamination. Ozone (03) is an allotrope of oxygen and has both beneficial and
detrimental attributes to life on earth. For example, ozone is created in the stratosphere by
high energy radiation from the sun that splits molecular oxygen (02) into two atoms, which
then combine with different Oz les to form 03. This stratospheric layer ts living
organisms on the planet from harmful ultraviolet radiation produced by the sun. At the
ground level or troposphere, however, ozone is considered an air pollutant. Ozone is also
created under smog conditions where sunlight acts on the combination of oxides of nitrogen
and volatile organic compounds that are produced by industrial facilities, electric utilities,
motor vehicle t, gasoline vapors, and chemical solvents (See EPA, 2011), Ozone
levels increase during the hot summer months, and the effects of ozone damage increase with
sing relative humidity levels. Tropespherie ozone causes severe damage to crops and
forests (See Hewitt et al., 1990), numerous respiratory problems in animals and humans (See
Fairchild et al., 1959; Bhalla, 1994), as well as adverse effects with many consumer ts
including automobile tires (See Crabtree and Kemp, 1946), dyes found in e materials
(See Salvin and Walker, 1955) as well as fluorescent dyes used in molecular biology.
From a chemical perspective, ozone is an ophilic agent that is most reactive with
electron pairs commonly found in olefinic compounds (i.e., als that contain carbon-
carbon double bonds). There has been extensive research dedicated to the chemistry of
ozone, which is called ozonation. An ive two volume book series provides a
comprehensive review that describes the properties of ozone itself and the numerous
reactions it can undergo with organic ates. Volume 1 of this series is dedicated almost
exclusively to the reaction of ozone with olefinic compounds (See Bailey, 1978; Bailey
1982), for which Volume 1 and 2 are incorporated by reference.
Dyes and dye intermediates are well known in the literature and with the majority of
dye types in use today having been ered more than a century ago (Gordon and
Gregory, 1983). Dyes can be d into classes based on how they are used or based on
their chemical structures (Gordon, 2009). For the latter, dyes have been classified into the
general classes of (i) azo, (ii) anthraquinone, (iii) benzodifuranone, (iv) polycyclic aromatic
carbonyl, (v) indigoid, (vi) polymethine and related dyes (i.e., cyanine), (vii) styryl, (viii) di—
and triaryl carbonium and related dyes (i.e., fluorescein, rhodamine and their sulfonated
tives), (ix) phthalocyanine, (x) quinophthalone, (xi) sulfur, (xii) nitro and nitroso, and
(xiii) miscellaneous (i.e., coumarin and BODIPY ) (Gregory, 2009). Most, if not all, dyes
and dye intermediates have a multiplicity of carbon-carbon double bonds that make them
sensitive to ozonation. In the mid 1970s, Lofquist and colleagues taught the general
conclusion that most dyes would be susceptible to ozonolysis (See US. Patent 3,822,996;
US. Patent 3,859,045; US. Patent 3,917,499.
The effect of ozone exposure to dyed fabrics is dye fading (e.g., loss of dyefastness),
which was first ed in 1955. Salvin and Walker coined the term “O-fading”, which in
their e test of drapery fabrics revealed that ozone caused significant dye fading when
exposed to several blue anthraquinone dyes, (i.e., Eastman Blue GLF, Amacel Blue, and
Interchemical Blue B) as well as yellow and red anthraquinone dyes (See Salvin, 1955).
Other examples of anthraquinone dyes, such as C.I. Basic Blue 47 (See US. Patent
3,822,996) and Disperse Blue 3 (See US. Patent 3,859,045) have been reported to be
tible to O—fading. High humidity es O—fading (See US. Patent 3,917,449; US.
Patent 4,737,155; US. Patent 3,822,996; US. Patent 4,304,568), and for fabrics, it has been
suggested that the moisture provides the dye suff1cient mobility to diffuse to the surface of
the material where the ozonation reaction occurs (See US. Patent 155).
scent dyes, such as those belonging to the polymethine class, have also been
reported susceptibility to ozonation reactions, which reduce their fluorescent signal
intensities. For example, Cy3 and Cy5 dyes have been widely used in rray
technologies for gene expression, genotyping, and resequencing applications (See Gershon,
2004). Fare and colleagues showed results that Cy5 and its sulfonated derivative Alexa Fluor
647 were susceptible to ozone damage at exposure levels of 5 to 10 ppb within 10 to 30 sec
(See Fare et al., 2003). Fluorescent intensity levels were also reduced for Cy3 and its
sulfonated derivative Alexa Fluor 555 at higher ozone levels (>100 ppb). Kadushin and Getts
note that the ng signal can degrade to ~10% in 1-5 min (See US. Patent Appl.
Publication 2004/0110196).
There a large number of chemical reagents, which act as onants. Examples
e para-phenyldiamine, dihydroquinoline, thiourea (See US. Patent 4,737,155; U.S.
Patent 4,631,066), ted alkyl substituted thiourea, alkyl and aryl phosphites (See US.
Patent 3,822,996), ethoxylated aliphatic tertinary amines (See US. Patent 3,859,045),
substituted piperidine thiourea (See U. S. Patent 4,304,568), substituted oxadiazine thiones
and substituted thiazine thiones (See U. S. Patent 4,304,568), acrylic polymerase, copolymers
of methacrylate or ethylacrylate (See US. Patent Appl. Publication 2004/0110196), and
polythiourea (See US. Patent 3,917,449). For the present invention, thiourea may be used in
some embodiments, in one or more solutions to t the ozonolysis of fluorescent dyes in
the sequencing reaction. , which is incorporated herein by reference,
provides methods for using thiourea in combination with methods involving NGS
technologies. In some embodiments, the imaging and/or hemical cleavage steps may
be performed in the presence of thiourea, for example, at the concentrations sed in the
invention summary above.
H. Sample ation
s to prepare the DNA of interest for NGS analysis include, for example,
clonally-amplified and non—amplified (i.e., single molecule) templates. In some
embodiments, emulsion PCR (emPCR) may be used. As shown in samples may be
prepared as follows. First, genomic DNA is isolated. The DNA is then fragmented into
smaller pieces. Then, common adaptors are ligated to the ends of those fragments. The
adaptor-ligated DNA les are then separated into single strands and captured onto 1 um
size beads under conditions that favor one DNA le per bead. An oil—aqueous
on creates dual aqueous droplets that encapsulate these bead—DNA complexes.
PCR amplification is performed within these droplets to create beads containing 104—106
copies of the same template sequence. Following successful amplification and enrichment,
tens of millions to ds of millions of emPCR beads are then chemically immobilized to
microfluidic flowcell 50. In some embodiments, flowcell 50 may comprise eight ls
and may be made of glass.
1. System Operation
Once flowcell 50 is prepared with beads, flowcell 50 may be placed in the sequencing
system 100. illustrates the typical steps in one CRT cycle. A single DNA molecule
is depicted for illustrative es, but those skilled in the art would understand that this
process is performed on many DNA molecules.
First, in the incorporation step, 3’-OH reversible terminators are incorporated using
DNA polymerase (depicted as a zipper) as discussed above.
Next, fluorescently—labeled DNA molecules are imaged. Lamp 14 is activated such
that light path 30 is created from flowcell 50 to imaging device 10. Imaging device 10
captures images through the each of the four spectral filters of filter wheel 16. Using fllter-
wheel 16, each spectral channel is imaged in a tiled fashion to capture fluorescent signals
within microfluidic flowcell 50. Base calling is then performed from processed cent
intensities of dual beads (i.e., a purified blue signal may be called an “A” base as the 3’—
OH reversible terminator was labeled with a blue dye). The read length of the CRT method
is a direct function of the number cycles that are executed (see Metzker 2010; Metzker, 2005,
which are incorporated herein by reference)..
Photochemical cleavage may then be performed. Using UV light source 20, UV light
is shined upon l 50. The UV light photochemically cleaves away the ating
group and the fluorescent group. In this manner, the d nucleic acid is restored to its
native state.
A wash is then supplied, g away the terminating group and fluorescent groups.
The incorporation, imaging, cleaving and washing steps may be performed for as many CRT
cycles as desired. illustrates four—color tile images from three cycles and subsequent
base-calling from individual beads.
IV. Definitions
When used in the context of a chemical group, “hydrogen” means —H; “hydroxy”
means —OH; “oxo” means =0; “halo” means independently —F, —Cl, —Br or —I; “amino”
means —NH2; “hydroxyamino” means —NHOH; “nitro” means —NOZ; imino means =NH;
” means —CN; “isocyanate” means —N=C=O; “azido” means —N3; in a monovalent
context “phosphate” means —OP(O)(OH)2 or a deprotonated form f; in a divalent
context “phosphate” means —OP(O)(OH)O— or a deprotonated form thereof; “mercapto”
means —SH; and “thio” means =S; “sulfonyl” means —S(O)2—; and “sulflnyl” means .
In the context of chemical formulas, the symbol “—” means a single bond, “=” means
a double bond, and “E” means triple bond. The symbol “____” represents an optional bond,
which if present is either single or double. The symbol “:” represents a single bond or a
double bond. Thus, for example, the ure includes the structures 0, O,
Q, U and ©. As will be understood by a person of skill in the art, no one such
ring atom forms part of more than one double bond. The symbol “M ”, when drawn
dicularly across a bond tes a point of ment of the group. It is noted that the
point of attachment is typically only identified in this manner for larger groups in order to
assist the reader in rapidly and guously identifying a point of attachment. The symbol
“‘ ” means a single bond where the group attached to the thick end of the wedge is “out of
the page.” The symbol “ """l ” means a single bond where the group attached to the thick end
of the wedge is “into the page”. The symbol “m ” means a single bond where the
conformation (e.g., either R or 53 or the geometry is undefined (e.g., either E or Z).
Any ned valency on an atom of a structure shown in this application implicitly
represents a hydrogen atom bonded to the atom. When a group “R” is depicted as a ng
group” on a ring system, for example, in the formula:
then R may replace any hydrogen atom attached to any of the ring atoms, including a
depicted, implied, or expressly def1ned hydrogen, so long as a stable structure is formed.
When a group “R” is depicted as a “floating group” on a fused ring system, as for example in
the formula:
(FUN \”‘9’
then R may replace any hydrogen attached to any of the ring atoms of either of the fused
rings unless specified otherwise. Replaceable hydrogens include depicted hydrogens (e.g.,
the hydrogen attached to the nitrogen in the formula above), implied hydrogens (e.g., a
hydrogen of the formula above that is not shown but understood to be present), expressly
defined hydrogens, and optional hydrogens whose presence depends on the identity of a ring
atom (e.g., a hydrogen ed to group X, when X equals —CH—), so long as a stable
structure is formed. In the example depicted, R may reside on either the 5—membered or the 6—
membered ring of the fused ring system. In the formula above, the subscript letter “y”
immediately following the group “R” enclosed in parentheses, represents a numeric variable.
Unless ied otherwise, this variable can be 0, l, 2, or any integer greater than 2, only
limited by the maximum number of replaceable en atoms of the ring or ring system.
For the groups and classes below, the following parenthetical subscripts further define
the group/class as follows: “(Cn)” defines the exact number (n) of carbon atoms in the
group/class. “(CSn)” s the maximum number (n) of carbon atoms that can be in the
group/class, with the minimum number as small as possible for the group in question, e. g., it
is understood that the minimum number of carbon atoms in the group “alkenyl(cgg)” or the
class “alkene(cgg)” is two. For example, y(cg10)” designates those alkoxy groups having
from 1 to 10 carbon atoms (e.g., l, 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range derivable therein
(e.g., 3 to 10 carbon atoms). (Cn—n’) defines both the m (n) and maximum number
(n’) of carbon atoms in the group. rly, “alkyl(cz_10)” designates those alkyl groups
having from 2 to 10 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, or any range derivable
therein (e. g., 3 to 10 carbon ).
The term “saturated” as used herein means the compound or group so modified has no
carbon—carbon double and no carbon—carbon triple bonds, except as noted below. The term
does not preclude carbon—heteroatom multiple bonds, for example a carbon oxygen double
bond or a carbon nitrogen double bond. Moreover, it does not preclude a carbon—carbon
double bond that may occur as part of keto-enol tautomerism or enamine tautomerism.
The term atic” when used without the “substituted” modifier signifies that the
compound/group so modified is an acyclic or cyclic, but non—aromatic hydrocarbon
compound or group. In aliphatic compounds/groups, the carbon atoms can be joined together
in straight chains, branched chains, or omatic rings clic). Aliphatic
compounds/groups can be saturated, that is joined by single bonds (alkanes/alkyl), or
unsaturated, with one or more double bonds (alkenes/alkenyl) or with one or more triple
bonds (alkynes/alkynyl). Where the term “aliphatic” is used without the ituted”
modifier, then only carbon and hydrogen atoms are present. When the term is used with the
“substituted” modifier one or more hydrogen atom has been independently replaced by —OH,
F, Cl, Br, I, NHZ, N02, COzH, 3, —CN, —SH, —OCH3, —OCH2CH3,
—C(O)CH3, —NHCH3, —NHCH2CH3, —N(CH3)2, —C(O)NH2, —OC(O)CH3, or —S(O)2NH2.
The term “alkyl” when used t the “substituted” modifier refers to a monovalent
saturated aliphatic group with a carbon atom as the point of attachment, a linear or branched,
cyclo, cyclic or c structure, and no atoms other than carbon and hydrogen. Thus, as
used herein cycloalkyl is a subset of alkyl. The groups —CH3 (Me), —CH2CH3 (Et),
—CH2CH2CH3 (n-Pr or propyl), —CH(CH3)2 (i-Pr, Z'Pr or isopropyl), 2)2
(cyclopropyl), —CH2CH2CH2CH3 (n—Bu), —CH(CH3)CH2CH3 (sec—butyl), —CH2CH(CH3)2
(isobutyl), —C(CH3)3 (tert—butyl, t—butyl, t—Bu or tBu), —CH2C(CH3)3 (neo—pentyl), cyclobutyl,
cyclopentyl, cyclohexyl, and cyclohexylmethyl are non-limiting examples of alkyl .
The term “alkanediyl” when used without the “substituted” modifier refers to a divalent
saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of
attachment, a linear or branched, cyclo, cyclic or acyclic structure, no —carbon double
or triple bonds, and no atoms other than carbon and hydrogen. The groups, —CH2—
(methylene), —CH2CH2—, —CH2C(CH3)2CH2—, 2CH2—, and ”3 are non-
limiting examples of alkanediyl groups. The term “alkylidene” when used without the
“substituted” modifier refers to the nt group =CRR’ in which R and R’ are
independently hydrogen, alkyl, or R and R’ are taken together to represent an alkanediyl
haVing at least two carbon atoms. Non-limiting examples of alkylidene groups e:
=CH2, =CH(CH2CH3), and =C(CH3)2. When any of these terms is used with the
“substituted” modifler one or more hydrogen atom has been independently replaced by —OH,
F, Cl, Br, I, NHZ, N02, COZH, —COzCH3, —CN, —SH, —OCH3, —OCH2CH3,
—C(O)CH3, —NHCH3, CH3, —N(CH3)2, —C(O)NH2, —OC(O)CH3, or —S(O)2NH2.
The following groups are non-limiting examples of substituted alkyl groups: ,
—CH2Cl, —CF3, —CH2CN, —CH2C(O)OH, —CH2C(O)OCH3, —CH2C(O)NH2, O)CH3,
—CH20CH3, —CHZOC(O)CH3, —CH2NH2, —CH2N(CH3)2, and —CH2CH2Cl. The term
“haloalkyl” is a subset of substituted alkyl, in which one or more hydrogen atoms has been
substituted with a halo group and no other atoms aside from , hydrogen and n
are present. The group, —CH2Cl is a non-limiting example of a haloalkyl. An “alkane” refers
to the compound H—R, wherein R is alkyl. The term “fluoroalkyl” is a subset of substituted
alkyl, in which one or more hydrogen has been substituted with a fluoro group and no other
atoms aside from carbon, hydrogen and fluorine are present. The groups, —CH2F, —CF3, and
—CH2CF3 are non-limiting examples of fluoroalkyl groups. An “alkane” refers to the
compound H—R, wherein R is alkyl.
The term “alkenyl” when used without the “substituted” modifier refers to an
monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a
linear or branched, cyclo, cyclic or acyclic structure, at least one nonaromatic carbon-carbon
double bond, no carbon—carbon triple bonds, and no atoms other than carbon and hydrogen.
Non—limiting examples of alkenyl groups include: 2 (Vinyl), —CH=CHCH3,
—CH=CHCH2CH3, —CH2CH=CH2 (allyl), =CHCH3, and —CH=CH—C6H5. The term
“alkenediyl” when used without the “substituted” modif1er refers to a divalent unsaturated
aliphatic group, with two carbon atoms as points of attachment, a linear or ed, cyclo,
cyclic or acyclic structure, at least one nonaromatic carbon—carbon double bond, no carbon—
carbon triple bonds, and no atoms other than carbon and hydrogen. The groups, —CH=CH—,
s55.
—CH=C(CH3)CH2—, —CH=CHCH2—, and ‘5; are of
, non-limiting examples
diyl groups. When these terms are used with the ituted” modifier one or more
hydrogen atom has been independently replaced by OH, F, Cl, Br, I, NHz, N02,
—COzH, 3, —CN, —SH, —OCH3, —OCH2CH3, —C(O)CH3, —NHCH3, CH3,
—N(CH3)2, —C(O)NH2, —OC(O)CH3, or —S(O)2NH2. The groups, —CH=CHF, —CH=CHCl
and —CH=CHBr, are non-limiting examples of substituted alkenyl groups. An “alkene”
refers to the compound H—R, wherein R is alkenyl.
The term yl” when used without the “substituted” er refers to an
monovalent unsaturated aliphatic group with a carbon atom as the point of attachment, a
linear or branched, cyclo, cyclic or acyclic structure, at least one carbon—carbon triple bond,
and no atoms other than carbon and hydrogen. As used herein, the term l does not
de the presence of one or more non—aromatic carbon—carbon double bonds. The groups,
—CECH, —CECCH3, and —CH2CECCH3, are non-limiting examples of alkynyl groups. When
alkynyl is used with the “substituted” modifier one or more hydrogen atom has been
independently replaced by OH, F, Cl, Br, I, NHz, N02, COzH, —COzCH3, —CN,
—SH, —OCH3, —OCH2CH3, —C(O)CH3, —NHCH3, —NHCH2CH3, —N(CH3)2, —C(O)NH2,
—OC(O)CH3, or —S(O)2NH2. An “alkyne” refers to the nd H—R, wherein R is
alkynyl.
The term “aryl” when used without the “substituted” modif1er refers to a monovalent
unsaturated aromatic group with an aromatic carbon atom as the point of attachment, said
carbon atom forming part of a one or more six-membered aromatic ring structure, wherein
the ring atoms are all , and wherein the group consists of no atoms other than carbon
and hydrogen. If more than one ring is present, the rings may be fused or unfused. As used
herein, the term does not preclude the presence of one or more alkyl group (carbon number
limitation permitting) attached to the first aromatic ring or any additional aromatic ring
present. Non-limiting examples of aryl groups include phenyl (Ph), methylphenyl,
(dimethyl)phenyl, —C6H4CH2CH3 (ethylphenyl), yl, and the monovalent group derived
from biphenyl. The term “arenediyl” when used without the “substituted” modifier refers to
a divalent aromatic group with two aromatic carbon atoms as points of attachment, said
carbon atoms forming part of one or more six-membered aromatic ring structure(s) wherein
the ring atoms are all carbon, and n the monovalent group consists of no atoms other
than carbon and hydrogen. As used herein, the term does not preclude the presence of one or
more alkyl group (carbon number limitation permitting) attached to the first aromatic ring or
any additional aromatic ring present. If more than one ring is present, the rings may be fused
or unfused. Non—limiting examples of iyl groups include:
3"”,
@— —s© ad“, TOO" -%®%—.
7 7
When these terms are used with the “substituted” modifier one or more hydrogen atom has
been independently replaced by OH, F, Cl, Br, I, NHz, N02, COZH, —COzCH3,
—CN, —SH, —OCH3, —OCH2CH3, —C(O)CH3, —NHCH3, —NHCH2CH3, —N(CH3)2,
—C(O)NH2, —OC(O)CH3, or NH2. An “arene” refers to the compound H—R, wherein
R is aryl.
The term “aralkyl” when used without the ituted” modifier refers to the
monovalent group ediyl—aryl, in which the terms alkanediyl and aryl are each used in a
manner consistent with the definitions ed above. miting examples of aralkyls
are: phenylmethyl (benzyl, Bn) and 2-phenyl-ethyl. When the term is used with the
ituted” modifier one or more hydrogen atom from the alkanediyl and/or the aryl has
been independently replaced by OH, F, Cl, Br, I, NHz, N02, COZH, —COzCH3,
—CN, —SH, —OCH3, —OCH2CH3, —C(O)CH3, —NHCH3, —NHCH2CH3, —N(CH3)2,
—C(O)NH2, —OC(O)CH3, or —S(O)2NH2. Non—limiting examples of substituted aralkyls are:
(3 -chlorophenyl)-methyl, and rophenyl-eth- l -yl.
The term “heteroaryl” when used without the “substituted” modifier refers to a
monovalent aromatic group with an aromatic carbon atom or nitrogen atom as the point of
ment, said carbon atom or nitrogen atom g part of one or more aromatic ring
ures n at least one of the ring atoms is nitrogen, oxygen or sulfur, and n
the heteroaryl group consists of no atoms other than carbon, hydrogen, aromatic nitrogen,
aromatic oxygen and aromatic sulfur. As used herein, the term does not preclude the
presence of one or more alkyl, aryl, and/or aralkyl groups (carbon number limitation
permitting) attached to the aromatic ring or aromatic ring system. If more than one ring is
present, the rings may be fused or d. Non-limiting es of heteroaryl groups
e l, imidazolyl, indolyl, indazolyl (1m), isoxazolyl, methylpyridinyl, oxazolyl,
pyridinyl, pyridinyl, pyrrolyl, pyrimidinyl, pyrazinyl, quinolyl, quinazolyl,
quinoxalinyl, triazinyl, tetrazolyl, thiazolyl, thienyl, and triazolyl. The term “N-heteroaryl”
refers to a heteroaryl group with a nitrogen atom as the point of attachment. The term
“heteroarenediyl” when used without the “substituted” modifier refers to an divalent aromatic
group, with two aromatic carbon atoms, two aromatic nitrogen atoms, or one aromatic carbon
atom and one aromatic nitrogen atom as the two points of ment, said atoms forming
part of one or more aromatic ring structure(s) wherein at least one of the ring atoms is
en, oxygen or sulfur, and wherein the divalent group consists of no atoms other than
carbon, hydrogen, aromatic nitrogen, aromatic oxygen and aromatic sulfur. As used herein,
the term does not de the presence of one or more alkyl, aryl, and/or aralkyl groups
(carbon number limitation permitting) attached to the aromatic ring or aromatic ring system.
If more than one ring is present, the rings may be fused or unfused. Non-limiting examples
of heteroarenediyl groups include:
When these terms are used with the “substituted” modifier one or more hydrogen atom has
been independently replaced by OH, F, Cl, Br, I, NHz, N02, COZH, —COzCH3,
—CN, —SH, —OCH3, —OCH2CH3, —C(O)CH3, , —NHCH2CH3, —N(CH3)2,
—C(O)NH2, —OC(O)CH3, or —S(O)2NH2.
The term “heterocycloalkyl” when used without the “substituted” modifier refers to a
monovalent non-aromatic group with a carbon atom or nitrogen atom as the point of
attachment, said carbon atom or nitrogen atom forming part of one or more non-aromatic ring
structures wherein at least one of the ring atoms is en, oxygen or sulfur, and wherein
the heterocycloalkyl group consists of no atoms other than carbon, hydrogen, nitrogen,
oxygen and sulfur. As used herein, the term does not preclude the presence of one or more
alkyl groups (carbon number limitation permitting) attached to the ring or ring system. As
used herein, the term does not preclude the presence of one or more double bonds in the ring
or ring system, provided that the resulting groups remains non-aromatic. If more than one
ring is present, the rings may be fused or unfused. Non-limiting es of
heterocycloalkyl groups include aziridinyl, azetidinyl, pyrrolidinyl, dinyl, piperazinyl,
morpholinyl, thiomorpholinyl, tetrahydrofuranyl, ydrothiofuranyl, tetrahydropyranyl,
pyranyl, oxiranyl, and oxetanyl. The term “N-heterocycloalkyl” refers to a heterocycloalkyl
group with a nitrogen atom as the point of ment. When the term “heterocycloalkyl”
used with the “substituted” modif1er one or more hydrogen atom has been independently
replaced by OH, F, Cl, Br, I, NHz, N02, COZH, —COZCH3, —CN, —SH, —OCH3,
—OCH2CH3, —C(O)CH3, —NHCH3, —NHCH2CH3, —N(CH3)2, —C(O)NH2, —OC(O)CH3,
—S(O)2NH2, or —C(O)OC(CH3)3 (tert—butyloxycarbonyl, BOC).
The term “acyl” when used without the “substituted” modif1er refers to the group
—C(O)R, in which R is a hydrogen, alkyl, aryl, aralkyl or heteroaryl, as those terms are
d above. The groups, —CHO, H3 (acetyl, Ac), —C(O)CH2CH3,
—C(O)CH2CH2CH3, —C(O)CH(CH3)2, —C(O)CH(CH2)2, —C(O)C6H5, —C(O)C6H4CH3,
—C(O)CH2C6H5, —C(O)(imidazolyl) are miting examples of acyl groups. A cyl”
is defined in an ous manner, except that the oxygen atom of the group —C(O)R has
been replaced with a sulfur atom, —C(S)R. When either of these terms are used with the
“substituted” modifier one or more hydrogen atom (including a hydrogen atom directly
ed the carbonyl or thiocarbonyl group, if any) has been independently replaced by
OH, F, Cl, Br, I, NHZ, N02, COZH, —C02CH3, —CN, —SH, —OCH3, —OCH2CH3,
—C(O)CH3, —NHCH3, —NHCH2CH3, —N(CH3)2, —C(O)NH2, —OC(O)CH3, or NH2.
The groups, —C(O)CH2CF3, —COzH (carboxyl), —COzCH3 (methylcarboxyl), —COZCH2CH3,
—C(O)NH2 (carbamoyl), and —CON(CH3)2, are non-limiting examples of substituted acyl
.
The term “alkoxy” when used without the ituted” modifier refers to the group
—OR, in which R is an alkyl, as that term is defined above. Non-limiting examples of alkoxy
groups include: —OCH3 (methoxy), —OCH2CH3 (ethoxy), —OCH2CH2CH3, —OCH(CH3)2
(isopropoxy), —O(CH3)3 (tert—butoxy), H2)2, —O—cyclopentyl, and —O—cyclohexyl.
The terms “alkenyloxy”, “alkynyloxy”, “aryloxy”, “aralkoxy”, oaryloxy”,
“heterocycloalkoxy”, and “acyloxy”, when used without the “substituted” modif1er, refers to
groups, defined as —OR, in which R is alkenyl, alkynyl, aryl, aralkyl, heteroaryl,
heterocycloalkyl, and acyl, respectively. The term “alkoxydiyl” refers to the divalent group
—O—alkanediyl—, —O—alkanediyl—O—, or —alkanediyl—O—alkanediyl—. The term “alkylthio”
and hio” when used without the “substituted” modifier refers to the group —SR, in
which R is an alkyl and acyl, respectively. When any of these terms is used with the
“substituted” modifier one or more hydrogen atom has been independently replaced by —OH,
F, Cl, Br, I, NHZ, N02, COzH, —COzCH3, —CN, —SH, —OCH3, —OCH2CH3,
—C(O)CH3, —NHCH3, —NHCH2CH3, —N(CH3)2, —C(O)NH2, —OC(O)CH3, or —S(O)2NH2.
The term “alcohol” corresponds to an alkane, as defined above, wherein at least one of the
hydrogen atoms has been replaced with a hydroxy group.
The term “alkylamino” when used without the “substituted” modifier refers to the
group —NHR, in which R is an alkyl, as that term is defined above. Non-limiting examples of
alkylamino groups include: —NHCH3 and —NHCH2CH3. The term “dialkylamino” when
used without the ituted” modifier refers to the group —NRR’, in which R and R’ can be
the same or different alkyl groups, or R and R’ can be taken together to represent an
alkanediyl. Non-limiting examples of dialkylamino groups include: —N(CH3)2,
—N(CH3)(CH2CH3), and olidinyl. The terms “alkoxyamino”, “alkenylamino”,
“alkynylamino”, “arylamino”, “aralkylamino”, “heteroarylamino”, “heterocycloalkylamino”
and “alkylsulfonylamino” when used without the “substituted” r, refers to groups,
defined as —NHR, in which R is , alkenyl, alkynyl, aryl, aralkyl, heteroaryl,
cycloalkyl, and alkylsulfonyl, tively. A miting example of an arylamino
group is —NHC6H5. The term “amido” (acylamino), when used without the “substituted”
modifier, refers to the group —NHR, in which R is acyl, as that term is defined above. A non-
limiting example of an amido group is —NHC(O)CH3. The term “alkylimino” when used
without the “substituted” modifier refers to the nt group =NR, in which R is an alkyl, as
that term is defined above. The term “alkylaminodiyl” refers to the nt group
—NH—alkanediyl—, —NH—alkanediyl—NH—, or —alkanediyl—NH—alkanediyl—. When any of
these terms is used with the “substituted” modifier one or more hydrogen atom has been
independently replaced by OH, F, Cl, Br, I, NHz, N02, COZH, —COzCH3, —CN,
—SH, —OCH3, —OCH2CH3, —C(O)CH3, —NHCH3, —NHCH2CH3, )2, —C(O)NH2,
—OC(O)CH3, or —S(O)2NH2. The groups —NHC(O)OCH3 and )NHCH3 are non—
ng examples of substituted amido groups.
The term “alkylphosphate” when used without the “substituted” modifier refers to the
group —OP(O)(OH)(OR), in which R is an alkyl, as that term is defined above. Non-limiting
examples of alkylphosphate groups include: —OP(O)(OH)(OMe) and —OP(O)(OH)(OEt).
The term “dialkylphosphate” when used without the “substituted” modifier refers to the
group —OP(O)(OR)(OR’), in which R and R’ can be the same or different alkyl groups, or R
and R’ can be taken together to represent an alkanediyl. Non-limiting examples of
dialkylphosphate groups include: (OMe)2, —OP(O)(OEt)(OMe) and —OP(O)(OEt)2.
When any of these terms is used with the “substituted” r one or more en atom
has been independently replaced by OH, F, Cl, Br, I, NHz, N02, COZH, —COzCH3,
—CN, —SH, —OCH3, —OCH2CH3, —C(O)CH3, —NHCH3, —NHCH2CH3, —N(CH3)2,
—C(O)NH2, —OC(O)CH3, or —S(O)2NH2.
The terms “alkylsulfonyl” and “alkylsulfinyl” when used without the “substituted”
modifier refers to the groups R and —S(O)R, respectively, in which R is an alkyl, as
that term is defined above. The terms “alkenylsulfonyl”, “alkynylsulfonyl”, “arylsulfonyl”,
“aralkylsulfonyl”, “heteroarylsulfonyl”, and "heterocycloalkylsulfonyl” are defined in an
analogous manner. When any of these terms is used with the “substituted” modifier one or
more hydrogen atom has been independently replaced by OH, F, Cl, Br, I, NHZ,
—NOz, —COzH, —COzCH3, —CN, —SH, —OCH3, —OCH2CH3, H3, —NHCH3,
—NHCH2CH3, —N(CH3)2, —C(O)NH2, —OC(O)CH3, or —S(O)2NH2.
As used herein, a “chiral auxiliary” refers to a removable chiral group that is e
of influencing the stereoselectivity of a reaction. Persons of skill in the art are familiar with
such compounds, and many are commercially available.
The use of the word “a” or “an,” when used in conjunction with the term
“comprising” in the claims and/or the specification may mean “one,” but it is also tent
with the g of “one or more, 3’ (Cat least one,” and “one or more than one.”
Throughout this application, the term “about” is used to indicate that a value includes
the inherent variation of error for the device, the method being employed to determine the
value, or the variation that exists among the study subjects.
The terms “comprise,” “have” and “include” are open-ended linking verbs. Any
forms or tenses of one or more of these verbs, such as “comprises,3’ “comprising,” “has,”
“having,” “includes” and “including,” are also open-ended. For e, any method that
“comprises,” “has” or “includes” one or more steps is not limited to possessing only those
one or more steps and also covers other unlisted steps.
The term “effective,” as that term is used in the specification and/or claims, means
adequate to accomplish a desired, expected, or intended result. “Effective amount,”
“Therapeutically ive amount” or “pharmaceutically effective amount” when used in the
context of treating a patient or subject with a compound means that amount of the compound
which, when administered to a subject or patient for ng a disease, is sufficient to effect
such ent for the disease.
The term “hydrate” when used as a modifier to a nd means that the compound
has less than one (e.g., drate), one (e.g., monohydrate), or more than one (e.g.,
dihydrate) water molecules associated with each compound molecule, such as in solid forms
of the compound.
As used herein, the term “IC50” refers to an inhibitory dose which is 50% of the
maximum se obtained. This quantitative e indicates how much of a particular
drug or other substance (inhibitor) is needed to inhibit a given biological, biochemical or
chemical process (or component of a process, i.e. an enzyme, cell, cell receptor or
microorganism) by half.
An “isomer” of a first compound is a separate compound in which each le
contains the same constituent atoms as the first compound, but where the configuration of
those atoms in three ions differs.
As used herein, the term “patient” or “subject” refers to a living mammalian
organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or
transgenic species thereof In certain ments, the patient or t is a primate. Non-
limiting examples of human subjects are , juveniles, infants and fetuses.
As generally used herein “pharmaceutically acceptable” refers to those compounds,
materials, compositions, and/or dosage forms which are, within the scope of sound medical
judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human
beings and animals without excessive toxicity, irritation, allergic response, or other problems
or complications commensurate with a reasonable benefit/risk ratio.
“Pharmaceutically acceptable salts” means salts of compounds of the present
invention which are pharmaceutically acceptable, as d above, and which possess the
desired pharmacological activity. Such salts include acid addition salts formed with
inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid, and the like; or with organic acids such as l,2-ethanedisulfonic acid,
2-hydroxyethanesulfonic acid, 2—naphthalenesulfonic acid, ylpropionic acid,
4,4'-methylenebis(3-hydroxyene-l-carboxylic acid), 4-methylbicyclo[2.2.2]oct—2—ene—
l-carboxylic acid, acetic acid, aliphatic mono- and oxylic acids, aliphatic sulfuric acids,
aromatic sulfuric acids, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, carbonic
acid, cinnamic acid, citric acid, cyclopentanepropionic acid, ethanesulfonic acid, fumaric
acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, heptanoic acid, ic
acid, hydroxynaphthoic acid, lactic acid, laurylsulfuric acid, maleic acid, malic acid, malonic
acid, ic acid, methanesulfonic acid, muconic acid, 0-(4-hydroxybenzoyl)benzoic acid,
2012/055231
oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substituted alkanoic acids, propionic acid,
p—toluenesulfonic acid, pyruvic acid, lic acid, stearic acid, succinic acid, tartaric acid,
tertiarybutylacetic acid, trimethylacetic acid, and the like. Pharmaceutically acceptable salts
also include base addition salts which may be formed when acidic protons t are capable
of reacting with nic or c bases. Acceptable inorganic bases include sodium
hydroxide, sodium carbonate, potassium ide, aluminum hydroxide and calcium
hydroxide. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine,
tromethamine, N—methylglucamine and the like. It should be recognized that the ular
anion or cation forming a part of any salt of this invention is not critical, so long as the salt, as
a whole, is pharmacologically acceptable. Additional examples of pharmaceutically
acceptable salts and their methods of preparation and use are presented in Handbook of
Pharmaceutical Salts: Properties, and Use (P. H. Stahl & C. G. h eds., Verlag
Helvetica Chimica Acta, 2002).
The term “pharmaceutically acceptable carrier,” as used herein means a
ceutically-acceptable material, composition or vehicle, such as a liquid or solid ,
diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a
chemical agent.
“Prevention” or “preventing” includes: (1) inhibiting the onset of a e in a
subject or patient which may be at risk and/or predisposed to the disease but does not yet
experience or display any or all of the ogy or symptomatology of the disease, and/or (2)
slowing the onset of the pathology or symptomatology of a disease in a subject or patient
which may be at risk and/or predisposed to the disease but does not yet experience or display
any or all of the pathology or symptomatology of the disease.
A “stereoisomer” or “optical isomer” is an isomer of a given compound in which the
same atoms are bonded to the same other atoms, but where the configuration of those atoms
in three dimensions differs. “Enantiomers” are stereoisomers of a given compound that are
mirror images of each other, like left and right hands. “Diastereomers” are stereoisomers of a
given compound that are not enantiomers. Chiral molecules contain a chiral center, also
referred to as a center or stereogenic center, which is any point, though not necessarily
an atom, in a molecule bearing groups such that an interchanging of any two groups leads to a
stereoisomer. In organic compounds, the chiral center is typically a carbon, phosphorus or
sulfur atom, though it is also possible for other atoms to be stereocenters in organic and
inorganic compounds. A molecule can have le centers, giving it many
stereoisomers. In compounds whose stereoisomerism is due to tetrahedral stereogenic centers
(e.g., edral carbon), the total number of etically possible stereoisomers will not
exceed 2n, where n is the number of tetrahedral centers. Molecules with symmetry
frequently have fewer than the maximum possible number of stereoisomers. A 50:50 mixture
of enantiomers is referred to as a racemic mixture. Alternatively, a mixture of enantiomers
can be enantiomerically enriched so that one enantiomer is present in an amount greater than
50%. lly, enantiomers and/or diasteromers can be resolved or separated using
techniques known in the art. It is contemplated that that for any stereocenter or axis of
chirality for which stereochemistry has not been defined, that center or axis of chirality
can be present in its R form, S form, or as a mixture of the R and S forms, including racemic
and non-racemic mixtures. As used , the phrase “substantially free from other
stereoisomers” means that the composition contains 5 15%, more ably 5 10%, even
more preferably 5 5%, or most preferably 5 1% of another stereoisomer(s).
“Treatment” or “treating” includes (1) inhibiting a e in a subject or patient
experiencing or displaying the pathology or symptomatology of the disease (e.g., arresting
r development of the pathology and/or symptomatology), (2) ameliorating a disease in a
subject or patient that is experiencing or displaying the pathology or symptomatology of the
disease (e.g., reversing the pathology and/or symptomatology), and/or (3) effecting any
measurable decrease in a disease in a subject or patient that is experiencing or displaying the
pathology or symptomatology of the disease.
The terms otide base”, “nucleobase” or simply “base”, as used herein, refers to
a substituted or unsubstituted nitrogen-containing parent heteroaromatic ring of a type that is
commonly found in nucleic acids, as well as natural, substituted, modified, or engineered
variants or analogs of the same. In a l embodiment, the nucleobase is capable of
forming Watson-Crick and/or Hoogsteen hydrogen bonds with an appropriately
complementary nucleobase. Exemplary nucleobases e, but are not limited to,
purines such as 2—aminopurine, 2,6-diaminopurine, adenine (A), ethenoadenine, N6-
Az-isopentenyladenine (6iA), N6-A2-isopentenylmethylthioadenine
(2ms6iA), N6-methyladenine, guanine (G), isoguanine, NZ-dimethylguanine
(de), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG),
hypoxanthine and 06-methylguanine;
7—deaza—purines such as 7—deazaadenine (7—deaza—A), 7—deazaguanine (7—deaza—G), 7—
deazahydroxymethyladenine, 7-deazaaminomethyladenine and 7-deaza-
7—hydroxymethylguanine;
WO 40257
pyrimidines such as cytosine (C), 5—propynylcytosine, isocytosine,
oxylmethylcytosine ), 5-aminomethyl-cytosine, thymine (T),
4-thiothymine (4sT), 5,6-dihydrothymine, 04-methylthymine, uracil (U), 4-
thiouracil (4sU), 5-hydroxylmethyluracil (HOMeU), 5-aminomethyl-uracil,
and 5,6—dihydrouracil (dihydrouracil; D);
indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole;
rine; base (Y); etc.
Additional exemplary nucleobases can be found in Lehninger, 2005, which is incorporated by
reference, and the references cited therein.
The term “nucleoside” as used herein, refers to a glycosylamine consisting of a
nucleobase bound to a five—carbon sugar, typically a ribose or a deoxyribose. Examples of
these include, but are not limited to, cytidine, 2’-deoxycytidine, 5-hydroxylmethylcytidine,
2’-deoxyhydroxylmethylcytidine, 5-aminomethylcytidine, 2’-deoxy
ethylcytidine, uridine, xyuridine, 5-hydroxylmethyluridine, 2’—deoxy—5—
hydroxylmethyluridine, 5-aminomethyluridine, 2’-deoxyaminomethyluridine, adenosine,
2’—deoxyadenosine, 7—deaza—7—hydroxymethyladenosine, 2’—deoxy—7—deaza—7-
hydroxymethyladenosine, 7-deazaaminomethyladenosine, 2’-deoxydeazaamino-
methyladenosine, guanosine, 2’-deoxyguanosine, 7-deazahydroxymethyl ine, 2’-
deoxydeazahydroxymethyl, 7-deazaaminomethyl guanosine, 2’-deoxydeaza
ethyl guanosine, thymidine, and 2’-deoxythymidine.
A “nucleotide” is composed of a side with one, two, three or more phosphate
groups bound in a chain to the 5—carbon sugar of the nucleoside.
The term “dephasing” is a phenomenon that occurs with step-wise on methods,
including but not limited to CRT, SNA, and SBL methods, when growing primers move out
of synchronicity for any given cycle. Lagging strand or type 2 dephasing (for example, n — 1
from the expected cycle) result from incomplete ion, and leading strand or type 1
dephasing (for example, n + 1) result from the addition of multiple nucleotides or probes in a
population of identical templates.
The term “dark nucleotide” or “dark probe” refers to a nucleotide or probe that does
not n a fluorescent label. It can be generated from its cleavage and over from the
previous cycle or be hydrolyzed in situ from its dye—labeled counterpart in the current cycle.
Unless specified otherwise, a “linker” refers to one or more divalent groups (linking
members) that function as a covalently—bonded molecular bridge between two other groups.
A linker may contain one or more linking members and one or more types of linking
members. Exemplary linking members e: —C(O)NH C(O)O NH S
, , , , S(O)n
where n is 0, 1 or 2, —O—, —OP(O)(OH)O—, —OP(O)(O')O—, alkanediyl, alkenediyl,
alkynediyl, arenediyl, heteroarenediyl, or ations thereof. Some linkers have pendant
side chains or pendant functional groups (or both). Examples of such pendant es are
hydrophilicity modifiers, for example, solubilizing groups like, e.g., —SO3H or —S03'. In
some embodiments, a linker may connect a er to another moiety such as a chemically or
enzymatically ve group (e.g., a cleavable or eavable ating moiety). In other
embodiments, a linker connects a reporter to a biological and non-biological component, for
example, a nucleobase, a nucleoside or a nucleotide. In further embodiments, a linker
connects chemically reactive groups to a base, a nucleoside or a nucleotide. The
moiety formed by a linker bonded to a reporter may be designated —L—Reporter. Depending
on such factors as the molecules to be linked and the ions in which the method of
strand synthesis is performed, the linker may vary in length and composition for optimizing
properties such as stability, length, FRET efficiency, resistance to certain chemicals and/or
temperature parameters, and be of sufficient stereo-selectivity or size to operably link a label
to a nucleotide such that the resultant conjugate is useful in optimizing a rization
reaction. Linkers can be employed using standard chemical techniques and include but not
limited to, amine linkers for attaching labels to nucleotides (see, for example, Hobbs and
Trainor, US. Patent 5,151,507, which is incorporated herein by reference); a linker typically
contain a primary or secondary amine for operably linking a label to a nucleotide; and a rigid
hydrocarbon arm added to a nucleotide base (see, for example, Service, 1998, which is
incorporated herein by reference). Some exemplary linking methodologies for attachment of
reporters to base molecules are provided in US. Patents 4,439,356 and 5,188,934; European
Patent Appl. 87310256.0; ational Appl. PCT/US90/05565 and Barone et al., 2001, each
of which is incorporated herein by reference in its ty.
A “cleavable linker” is a linker that has one or more cleavable groups that may be
broken by the result of a reaction or condition. The term “cleavable group” refers to a moiety
that allows for e of a portion, e. g., a enic or fluorescent moiety. Such cleavage
is typically chemically, photochemically or enzymatically mediated. Exemplary
enzymatically cleavable groups e phosphates, or groups attached via a peptide bond.
As used herein, the term “IC50” refers to but not limited to the concentration of a
tide analog at which its incorporation on a primer-template complex yields equal
numbers of moles of substrate and product and/or could be defined, but not limited to,
incorporation efflciency measured by determining the tration at which the nd
incorporates on half the primer-template complexes.
As used herein, the term “oligonucleotide” refers to DNA nts of 2 to 200
covalently linked nucleotides.
As used herein, the term “reporter” refers to a chemical moiety that is able to produce
a detectable signal ly or indirectly. Examples of reporters include fluorescent dye
groups, radioactive labels or groups effecting a signal through chemiluminescent or
inescent means. Examples fluorescent dye groups include zanthene, fluorescein,
rhodamine, BODIPY, cyanine, coumarin, , phthalocyanine, phycobiliprotein, ALEXA
FLUOR® 350, ALEXA FLUOR® 405, ALEXA FLUOR® 430, ALEXA FLUOR® 488,
ALEXA FLUOR® 514, ALEXA FLUOR® 532, ALEXA FLUOR® 546, ALEXA FLUOR®
555, ALEXA FLUOR® 568, ALEXA FLUOR® 568, ALEXA FLUOR® 594, ALEXA
FLUOR® 610, ALEXA FLUOR® 633, ALEXA FLUOR® 647, ALEXA FLUOR® 660,
ALEXA FLUOR® 680, ALEXA FLUOR® 700, ALEXA FLUOR® 750, and a squaraine
dye. Additional examples, of fluorescent dye groups that may be used in some embodiments
of the present invention are disclosed throughout this Specification and in Haugland, 2005
and US. Patents 4,439,356 & 5,188,934, which are incorporated by reference herein.
Examples of radioactive labels that may be used as reporters in some embodiments of the
t invention, which are well known in the art such as 35S, 3H, 32F, or 33F. Examples of
reporters that function by chemiluminescent or bioluminescent means and that may be used
as reporters in some embodiments of the present invention are described in Nieman, 1989;
Given & Schowen, 1989; Orosz et al., 1996; and Hastings, 1983, which are incorporated by
reference herein.
As used herein, the term “template” can refer to an oligonucleotide serving as the
complimentary strand for DNA synthesis (incorporation) or a recombinant DNA le
that is made up of a known region, usually a vector or adaptor sequence to which a universal
primer can bind, and the target sequence, which is typically an unknown portion to be
sequenced.
The term “fragment templates” refers to a library of fragments that have been
ed by randomly shearing c DNA into small sizes of <1kb and ligating adaptors
to each end of the fragment. These templates lly require less DNA than would be
needed for a mate-pair library.
The term “mate-pair templates” refers to a genomic library that has ed by
circularizing sheared of fragmented DNA that has been selected for a given size, (examples
include 2 kb or 5kb or 10 kb or 20 kb or any other desired size), therefore bringing the ends
that were previously distant from one r into close proximity. Cutting these circles into
linear DNA fragments s mate-pair templates.
As used herein, the term “primer” refers to an oligonucleotide that is hybridized to a
complement sequence on the template strand (usually a known sequence) used to initiate
DNA synthesis (incorporation).
When used herein in the scientific or technical sense, the term “incorporation” refers
to a nucleotide or nucleotide analog forming a complement air with the template strand
and a covalent bond to a primer strand by a polymerase. The primer-template complex is
extended one or more bases from the initial primer strand.
As used herein, the term “cleavage” refers to the removal of the terminating group by
al cleavage, enzymatic cleavage or the like.
As used herein, the term “incorporation cycle” refers to the incorporation of a
nucleotide or nucleotide analog by a polymerase, the detection and identification of said
tide or nucleotide analog, and if a nucleotide analog, ge of the terminating group
and, if originally present on the nucleotide analog, fluorescent dye group from said .
As used herein, the term “misincorporation” refers to a nucleotide or nucleotide
analog forming a non-complement base-pair with the template strand and a covalent bond to
a primer by a polymerase. The primer—template complex is extended one or more bases from
the initial primer strand.
As used herein, the term “discrimination” refers the IC50 concentration differences for
misincorporation versus incorporation of tide or tide analogs by a polymerase.
As used herein, the term “termination” refers to the incorporation of a nucleotide or
nucleotide analog forming a complement or non-complement base-pair with the te
strand and a covalent bond to a primer by a polymerase. The primer-template complex is
extended only one base from the initial primer strand or g primer strand for any given
incorporation cycle.
The terms “terminating moiety” and “terminating group” as used herein, are
synonymous, referring to a small chemical group (e. g., <500 s, excluding any
modification, such a linker or linker/dye) that when attached to at least one part of a
nucleoside (i.e., sugar or nucleobase) or nucleotide (i.e., sugar, nucleobase, or phosphate
group) confers substantial termination properties to the nucleoside or nucleotide. In some
ments, the ating moiety is further modified with a linker and/or a linker
attached to a dye. In preferred embodiments, the ating properties of such a modified
ating group is not substantially altered.
As used herein, the term “DT50” refers to the amount of time required to cleavage
50% of the base analog incorporated in the primer-template complex.
The term “analog” as used herein, is understood as being a nce which does not
comprise the same basic carbon on and carbon functionality in its ure as a “given
compound”, but which can mimic the given compound by incorporating one or more
appropriate substitutions such as for e substituting carbon for heteroatoms.
The above definitions supersede any conflicting tion in any of the reference that
is incorporated by reference herein. The fact that certain terms are defined, however, should
not be considered as indicative that any term that is undefined is indefinite. Rather, all terms
used are believed to describe the invention in terms such that one of ordinary skill can
appreciate the scope and practice the present invention.
V. Examples
The following examples are included to demonstrate preferred embodiments of the
invention. It should be iated by those of skill in the art that the techniques disclosed in
the examples which follow represent techniques discovered by the inventor to function well
in the practice of the invention, and thus can be considered to constitute preferred modes for
its practice. However, those of skill in the art should, in light of the present disclosure,
appreciate that many changes can be made in the specific embodiments which are disclosed
and still obtain a like or similar result without departing from the spirit and scope of the
invention.
Example 1 — Methods and Materials
Reagents and als. All reagents were purchased from commercial s and
used as received, unless otherwise noted.
Spectroscopic and analytical instrumentation. 1H NMR, 13 C NMR, and 31P NMR
spectra were recorded on a Bruker DPX 400 spectrometer as previously described in Wu et
a1. (2007), which is incorporated herein by reference. Mass spectra analyses were provided
by the Mass Spectrometry Laboratory at the MD Anderson Cancer Center on, TX) and
the Core Mass Spectrometry Facility at Rice University (Houston, TX). X-ray
crystallography was performed by the X-ray Diffraction Laboratory at Texas A&M
sity (College Station, TX). UV/Vis measurements were taken using a Beckman DU-
800 spectrophotometer. Anion exchange chromatography was performed using a Q
Sepharose FF column (2.5 x 20 cm) with a linear gradient of 75% triethylammonium
bicarbonate (TEAB, 0.1 M) in 25% acetonitrile to 75% TEAB (1.5 M) in 25% acetonitrile
over 240 min at a flow rate of 4.5 mL per min. Reverse-phase high performance liquid
chromatography (RP-HPLC) was performed using a Beckman System Gold equipped with a
128 solvent module and 166 UV detector or 168 photodiode array UV/Vis detector. RP—
HPLC for nucleosides and nucleotide analogs was performed using a 4.6 mm x 250 mm
Aquapore OD—300 C18 column, with buffer A containing 100 mM triethylammonium acetate
(TEAA), pH 7.0, and buffer B containing 100 mM TEAA, pH 7.0, 70% acetonitrile (v/v).
Example 2 — Synthesis of a-substituted 2-nitr0benzyl alcohols
(R/S)-I—(2-Nitr0pheny0methyl—I-propanol.‘ Synthesis of (R/S)—1-(2-nitrophenyl)
methyl-l—propanol was previously reported. See Litosh et a]. (2011), which is incorporated
herein by nce.
(R/S)-I—(Z—NitrophenyD-Z, 2-dimethyl—I—pr0panol.‘ Synthesis of 1-(2-
nitrophenyl)-2,2—dimethyl—1—propanol was previously reported. See Litosh et a1. (2011),
which is incorporated herein by reference.
(R/S)-I—(2, trophenyD-Z—methyl—I—propanol.‘
N02 i-Pr No2
' (i)
—> HO
OZN OZN
-2,6- (R/S)- 1-(2,6-dinitropheny|)-
dinitrobenzene 2-methyIpropanol
Scheme S1. Synthesis of (R/S)(2,6-dinitrophenyl)methyl—1-pr0panol. Reagents and
conditions: (i) PhMgBr, THF, minus 50°C; O, minus 50°C to room temperature, 30%.
To a solution of 1-iodo-2,6-dinitrobenzene (Smith and Ho, 1990), which is
incorporated herein by reference) (1.55 g, 5.27 mmol) in anhydrous THF (18 mL) at minus
50 °C under a nitrogen atmosphere, phenylmagnesium bromide (2 M in THF, 3.2 mL, 6.4
mmol) was added dropwise at a rate such that the ature would not exceed minus 45°C.
Upon completion of the addition, the mixture was d at minus 50°C for five min,
followed by addition of isobutyraldehyde (0.96 mL, 11 mmol). The mixture was gradually
warmed up to room temperature, quenched with saturated NH4Cl solution (10 mL), and then
diluted with water (50 mL). The mixture was ted with CHzClz (100 mL) three times.
The combined organic phase was washed with brine (50 mL), dried over NazSO4,
concentrated in vacuo, and the residue was purified by silica gel column chromatography to
yield l-(2,6-dinitrophenyl)methyl-l-propanol (0.375 g, 30%) as a yellow oil. 1H
NMR (400 MHz, CDCZ3): 5 7.82 (d, 2 H, J= 8.0 Hz, Ph—H), 7.59 (t, l H, J: 8.0 Hz, Ph—H),
4.83 (dd, 1 H, J= 9.2 and 7.6 Hz, Ph—CH), 2.87 (d, l H, J: 7.6 Hz, OH), 2.19 (m, l H, CH),
1.12 (d, 3 H, J= 6.4 Hz, CH3), 0.76 (d, 3 H, J= 6.8 Hz, CH3).
I-(4-Meth0xynitrophenyDmethyl—I-propanol
N02 i'Pr N02
0) HO
OMe OMe
4-iodo- (R/S)- 1-(4-methoxy-
oanisole 2-nitrophenyl)methyl-
1-propanol
Scheme S2. Synthesis of (R/S)(4-methoxy-Z-nitrophenyl)methyl—1-pr0panol.
Reagents and conditions: (i) PhMgCl, THF, minus 40°C; i—PrCHO, minus 40°C to room
temperature, 67%.
To a solution of 4-iodonitroanisole (2.79 g, 10.0 mmol) in anhydrous THF (20 mL)
at minus 40°C under a nitrogen atmosphere, phenylmagnesium chloride (2 M in THF, 6.0
mL, 12 mmol) was added dropwise at a rate such that the temperature would not exceed
minus 35°C. Upon tion of the on, the mixture was stirred at minus 40°C for five
min, followed by addition of yraldehyde (1.8 mL, 20 mmol). The mixture was
gradually warmed to room temperature, quenched with saturated NH4Cl solution (5.0 mL),
diluted with CHzClz (100 mL) and washed with water (100 mL). The organic phase was
separated, and the aqueous phase was extracted with CHzClz (50 mL) three times. The
combined organic phase was washed with brine (40 mL), dried over Na2S04, concentrated in
vacuo, and the residue was purified by silica gel column chromatography to yield (R/S)-l—(4—
methoxynitrophenyl)methyl-l-propanol (1.5 g, 67%) as a light yellow oil. 1H NMR
(400 MHz, CDCZ3): 5 7.61 (d, l H, J: 8.8 Hz, Ph—H), 7.34 (d, l H, J: 2.8 Hz, Ph—H), 7.15
(dd, 1 H, J: 8.8 and 2.8 Hz, Ph—H), 4.92 (dd, 1 H, J: 5.6 and 3.2 Hz, Ph—CH), 2.46 (br s, l
H, OH), 2.00 (m, l H, CH), 0.97 (d, 3 H, J: 6.4 Hz, CH3), 0.86 (d, 3 H, J: 6.8 Hz, CH3).
(R/S)-I-(4-Meth0xynitrophenyD-Z,2-dimethyl—I—pr0panol
N02 t-Bu N02
(i) HO
OMe OMe
4-iodo- (R/S)- ethoxy-
3-nitroanisole 2-nitropheny|)-
2,2 -dimethy|-1 -propano|
Scheme S3. Synthesis of (R/S)(4-methoxynitrophenyl)-2,2-dimethylpropanol.
Reagents and conditions: (i) , THF, minus 40°C; (CH3)3CCHO, minus 40°C to room
temperature, 74%.
To a solution of 4-iodonitroanisole (2.38 g, 8.50 mmol) in anhydrous THF (10 mL)
at minus 40°C under a nitrogen atmosphere, phenylmagnesium chloride (2 M in THF, 4.7
mL, 9.4 mmol) was added se at a rate such that the temperature would not exceed
minus 35°C. Upon completion of the addition, the mixture was stirred at minus 40°C for one
hour, followed by addition of trimethylacetaldehyde (1.13 mL, 10.2 mmol). The mixture was
stirred at minus 40°C for two hours and then at room temperature for another one hour. The
reaction was quenched with brine (100 mL), and the mixture was extracted with CHzClz (40
mL) three times. The combined organic phase was dried over NaZSO4 and concentrated in
vacuo, and the residue was purified by silica gel column chromatography to yield racemic
1-(4-methoxynitrophenyl)-2,2-dimethylpropanol (1.52 g, 74%). 1H NMR (400
MHZ, .' 5 7.67 (d, 1 H, J: 9.2 Hz, Ph—H), 7.22 (d, 1 H, J: 2.4 Hz, Ph—H), 7.12 (dd, 1
H, J: 8.8 and 2.8 Hz, Ph—H), 5.27 (d, 1 H, J= 4.0 Hz, Ph—CH), 3.86 (s, 3 H, OCH3), 2.01 (d,
1 H, J: 4.0 Hz, OH), 0.86 (s, 9 H, C(CH3)3).
(R/S)-I—(5-Meth0xynitr0phenyl)-2, 2-dimethyl—I—pr0panol and (S)-I-(5-meth0xy
phenyD-2, 2-dimethyl—I-pr0panol
N02 t-Bu N02
(I). ..
HO (II)
t_Bu
OMe OMe
3-iodo- (R/S)- 1-(5-methoxy- (R/S)(5-methoxy-
4-nitroanisole 2-nitropheny|)-2,2-dimethyl- 2-nitropheny|)-2,2-dimethyl-
1-p ropanol 1-propyl (1 S)-camphanate
t-Bu N02
(III) 0 O (W)
—> —> HO
OzN t-Bu
(S)(5-meth oxy- (S)(5-methoxy-
2-nitropheny|)-2,2-dimethyl- 2-nitropheny|)-
1-propyl (1 S)-camphanate methylpropanol
Scheme S4. Synthesis of 1-(5-methoxynitrophenyl)-2,2-dimethylpropanol
and (S)(5-meth0xynitrophenyl)-2,2-dimethylpr0panol. Reagents and conditions:
(i) PhMgCl, anhydrous THF, minus 40°C; (CH3)3CCHO, minus 40°C to room temperature,
88%; (ii) (lS)—camphanic acid chloride, DMAP, CHzClz, room temperature; (iii) fractional
crystallization from ethyl e/hexane, 43%; (iv) K2CO3, MeOH, reflux, 99%.
To a solution of 3-iodonitroanisole (2.79 g, 10.0 mmol) in anhydrous THF (10 mL)
at minus 40°C under a nitrogen atmosphere, phenylmagnesium chloride (2 M in THF, 4.2
mL, 8.3 mmol) was added dropwise at a rate such that the temperature would not exceed
minus 35°C. Upon completion of the addition, the mixture was stirred at minus 40°C for two
hours, followed by addition of trimethylacetaldehyde (1.1 mL, 10 mmol). The mixture was
stirred at minus 40°C for two hours and then at room temperature for another one hour. The
reaction was then quenched with brine (100 mL), and the mixture was ted with CHzClz
(40 mL) three times. The combined organic phase was dried over NaZSO4, concentrated in
vacuo, and the e was purified by silica gel column chromatography to yield racemic
(R/S)(5-methoxynitrophenyl)-2,2-dimethyl-l-propanol (1.76 g, 88%). 1H NMR (400
MHZ, CDCZ3).' 5 7.89 (d, 1 H, J= 9.2 Hz, Ph—H), 7.27 (d, 1 H, J: 2.8 Hz, Ph—H), 6.84 (dd, 1
2012/055231
H, J: 8.8 and 2.8 Hz, Ph—H), 5.62 (d, 1 H, J= 4.0 Hz, PhCH), 3.89 (s, 3 H, OCH3), 2.08 (d,
1 H, J: 4.0 Hz, OH), 0.89 (s, 9 H, C(CH3)3).
To a solution of racemic (R/S)—1-(5-methoxynitrophenyl)-2,2-dimethyl-l-propanol
(1.75 g, 7.3 mmol) and DMAP (2.92 g, 23.9 mmol) in anhydrous CHzClz (10 mL), (1S)-
camphanic chloride (Corrie et al., 1992), which is incorporated by nce) (2.6 g, 12
mmol) was added, and the mixture was stirred overnight at room temperature under a
nitrogen atmosphere. The reaction mixture was d with CHzClz (50 mL) and washed
with saturated NaHCO3 solution (50 mL). The organic phase was dried over NaZSO4,
concentrated in vacuo, and the residue was purified by silica gel column chromatography to
yield (R/S)—1-(5-methoxynitrophenyl)-2,2-dimethyl-l-propyl (1S)-camphanate (2.5 g,
85%, 1:1 mixture of reomers). The camphanate was dissolved in ethyl acetate (30 mL)
followed by slow on of hexane (120 mL) with stirring. Needle crystals formed
gradually from the solution over a two-hour period. The crystals were collected by filtration
to yield pure single diastereomer (5-methoxynitrophenyl)-2,2-dimethyl-l-propyl
(1S)-camphanate. The filtrate was concentrated in vacuo, and the crystallization process was
repeated twice to provide additional (S)(5-methoxynitrophenyl)-2,2-dimethyl-l-propyl
amphanate (total 1.08 g, 43%). 1H NMR (400 MHZ, CDCl3): 5 8.04 (d, 1 H, J: 9.2
Hz, Ph—H), 7.27 (d, 1 H, J: 2.8 Hz, Ph—H), 6.88 (dd, 1 H, J= 2.8 and 8.8 Hz, Ph—H), 6.81 (3,
1 H, Ph—CH), 3.87 (s, 3 H, OCH3), 2.36 (m, 1 H, CH), 1.92 (m, 2 H, CH2), 1.66 (m, 1 H,
CH), 1.12 (s, 3 H, CH3), 1.06 (s, 3 H, CH3), 1.02 (s, 3 H, CH3), 0.95 (s, 9 H, C(CH3)3).
Method for obtaining X-ray crystallography data: Crystallographic measurements
were made on a l of (S)(5-methoxynitrophenyl)-2,2-dimethylpropyl (1S)-
camphanate with dimensions of 0.50 mm X 0.05 mm X 0.05 mm as described by Litosh et al
(2011), which is incorporated herein by reference. See FIG 3.
Data collection: CuKoc radiation, 1 = 1.54178 A, T = 110 :: 20K, 26max = 120.00,
32,513 reflections collected, 2,913 unique (Rim = 0.0517). Final GooF= 1.091, R] = ,
wR2 = 0.1695, R indices based on 2,913 reflections with I>2sigma(1) (refinement on F2), 290
parameters, 43 restraints. Lp and absorption corrections applied, ,a = 0.819 mm'l. Absolute
structure parameter: 0.05 :: 0.09.
X-Ray crystallography data: NO7, M = 419.46. Orthorhombic, a = 6.29, b =
.00, c = 22.27 A (a, [3, y = 90°), V= 2,099.29 A3, space group P212121, Z = 4, Dc = 1.327
g/cm'3, F(000) = 896.
WO 40257
A mixture of (S)—1-(5-methoxynitrophenyl)-2,2-dimethyl-l-propyl (1S)-
camphanate (590 mg, 1.4 mmol) and K2C03 (389 mg, 2.8 mmol) in methanol (MeOH, 25
mL) was heated to reflux for one hour, then cooled down, concentrated in vacuo, and diluted
with CHzClz (50 mL). The organic phase was washed with brine (50 mL), dried over
Na2S04, concentrated in vacuo, and the residue was purified by silica gel column
chromatography to yield enantiopure (S)(5-methoxynitrophenyl)-2,2-dimethyl
propanol (333 mg, 99%). 1H NMR was identical with that of the racemic alcohol.
(R/S)-I-(4, 5-Dimeth0xynitr0phenyl)-2, 2-dimethyl—I-pr0panol
N02 t-Bu N02
(0 ' 0M9 (ii) ' (iii) HO
3 A a
0M9 | OMe OMe OMe
OMe OMe
1,2-dimethoxy- 4,5-diiodo-1,2- 4-iodonitro- (R/S)- 1-(4,5-dimethoxy-
benzene dimethoxy- 1,2-dimethoxy- 2-nitroph eny|)-2,2-
benzene benzene dimethylpropanol
Scheme S5. Synthesis of (R/S)(4,5-dimethoxy—2-nitrophenyl)-2,2-dimethyl—1-
propanol. Reagents and conditions: (i) 1C1, CH3COOH, 100°C, 62%; (ii) HNO3,
CH3COOH, room temperature, 75%; (iii) PhMgCl, THF, minus 40°C; (CH3)3CCHO, minus
40°C to room temperature, 20%.
1,2—Dimethoxybenzene (5.0 g, 36 mmol) was ved in acetic acid (10 mL), and
the solution was cooled in an ice—water bath followed by dropwise addition of iodine de
(8.7 g, 54 mmol). After 10 min, the reaction e was heated to 100°C for two hours and
then cooled to room ature. Needle ls that precipitated from solution were
filtered and washed with acetic acid (5.0 mL) three times. The crystals were dried overnight
under high vacuum to yield 4,5—diiodo—1,2—dimethoxybenzene (8.8 g, 62%). 1H NMR (400
MHz, CDCZ3).' 5 7.28 (s, 2 H, Ph-H), 3.85 (s, 6 H, OCH3).
4,5—Diiodo—1,2—dimethoxybenzene (8.8 g, 23 mmol) was added into acetic acid (300
mL), and the mixture was heated to 100°C to dissolve the solid. The clear mixture was then
cooled to room temperature ed by dropwise addition of nitric acid (68—70%, 120 mL).
The on mixture was stirred at room temperature overnight and then poured into ice—
water (200 mL). The mixture was extracted by CHzClz (100 mL) three times. The ed
organic phase was washed with saturated NaHCO3 solution (200 mL), brine (100 mL), and
dried over Na2S04, concentrated in vacuo, and the residue was purified by silica gel column
chromatography to yield 4—iodo—5—nitro—1,2-dimethoxybenzene (5.25 g, 75%). 1H NMR (400
MHz, CDCZ3).' 5 7.60 (s, 1 H, Ph—H), 7.38 (s, 1 H, Ph—H), 3.98 (s, 3 H, OCH3), 3.92 (s, 3 H,
OCH3).
To a solution of 4-iodonitro-1,2-dimethoxybenzene (4.6 g, 15 mmol) in ous
THF (10 mL) at minus 40°C under a nitrogen atmosphere, phenylmagnesium chloride (2 M
in THF, 7.5 mL, 15 mmol) was added se at a rate such that the temperature would not
exceed minus 35°C. Upon completion of the addition, the mixture was stirred at minus 40°C
for two hours, followed by addition of trimethyl acetaldehyde (2.0 mL, 18 mmol). The
mixture was stirred at minus 40°C for two hours and then at room temperature for another
one hour. The reaction was quenched with brine (100 mL), and the mixture was extracted
with CHzClz (40 mL) three times. The combined organic phase was dried over NaZSO4 and
concentrated in vacuo, and the residue was purified by silica gel column chromatography to
yield racemic (R/S)(4,5-dimethoxynitrophenyl)-2,2-dimethylpropanol (0.8 g, 20%).
IHNMR (400 MHz, CDCZ3): 5 7.41 (1, 1 H, Ph—H), 7.21 (s, 1 H, Ph—H), 5.60 (s, 1 H, PhCH),
3.95 (s, 3 H, OCH3), 3.92 (s, 3 H, OCH3), 0.90 (s, 9 H, (CH3)3).
Example 3 — Synthesis of 7-HOMeDeaza-2'-De0xyaden0sine Triphosphate Analogs
7-(2-nitr0benzyloxy)methyl— 7-deaza-2 ’-de0xyaden0sine-5 imphosphate
| | CI
| CI
/ I N / \N
a I \N
I /
N a 2
HO N TBSO N N TBSO N N
0| (__)H
OH OTBS OTBS
1 2 3
HO 0 CI
/ \N
I / \N
A I A
TBSO N N TBSO N N
(III) 0 (iv) o (V): (VI)
OTBS OTBS
4 5
02N 02N
o NH2 0 NH2
/ \N
I / \N
A I A
HO N N N
fl»..
HO\ /0\ /o\ /O N
0 /P\\ O
_ /P\\ _ /P\\ _
O O O O O O
OH OH
6 dA.|
Scheme S6. Synthesis of 7-(2-nitr0benzyloxy)methyldeaza-2'-deoxyadenosine—5'-
triphosphate. Reagents and conditions: (i) TBSCl, imidazole, DMF, room temperature,
87%; (ii) CO, PdClz[PhCN]2, MeOH/1,4—dioxane, 50°C, 98%; (iii) LiBH4, MeOH, THF,
reflux, 45%; (iv) 2-nitrobenzyl bromide, n-Bu4NBr, CHzClz/aq. NaOH, room temperature,
50%; (v) n—Bu4NF, THF, 0°C to room temperature; (vi) NH3, oxane/MeOH, 100°C,
91% for two steps; (vii) POCl3, (MeO)3PO, minus 40 °C; (n—Bu3NH)2H2PZO7, n-Bu3N, DMF;
l M HNEt3HCO3.
Compound 1 (Seela et a]. (2005), which is incorporated herein by reference), (0.79 g,
2.0 mmol) was ated from anhydrous pyridine (2.0 mL) three times and dissolved in
anhydrous DMF (4.0 mL). tert-Butyldimethylsilyl chloride (0.90 g, 6.0 mmol) and ole
(0.82 g, 12 mmol) were added, and the mixture was stirred at room temperature for 16 hours.
The reaction was trated in vacuo and purified by silica gel chromatography to yield 9—
[B—D—3’,5’—O—bis—(tert—butyldimethylsilyl)—2’—deoxyribofuranosyl]—6—chloro—7—iodo—7—
deazapurine 2 (1.08 g, 87%) as a white foam. 1H NMR (400 MHz, CDCZ3): 5 8.61 (s, 1 H, H—
2), 7.81 (s, 1 H, H—8), 6.74 (t, 1 H, J: 6.4 Hz, H—1’), 4.56 (m, 1 H, H—4’), 4.01 (m, 1 H, H—3’),
3.87 (dd, 1 H, H-5’a), 3.79 (dd, 1 H, , 2.39 (m, 2 H, H-2’a and H—2’b), 0.96 (s, 9 H,
(CH3)3CSi), 0.91 (s, 9 H, (CH3)3CSi), 0.18 (2s, 6 H, (CH3)2Si), 0.15 (s, 6 H, (CH3)2Si).
To a solution of compound 2 (1.55 g, 2.48 mmol) in anhydrous 1,4—dioxane (42 mL)
and anhydrous MeOH (42 mL), triethylamine (0.87 mL) was added. After stirring for 10 min
under a CO atmosphere, nzonitrile)dichloropalladium(H) (0.05 g, 0.13 mmol) was
added, and the reaction was stirred at 50°C for 48 hours under a CO atmosphere. The
mixture was then concentrated in vacuo, and the e was purified by silica gel
chromatography to yield 9-[B-D-3’,5’-O-bis-(tert—butyldimethylsilyl)-2’-deoxyribofuranosyl]—
6-chloro—7-methoxycarbonyl—7—deazapurine 3 (1.36 g, 98%) as a Viscous oil. 1H NMR (400
MHz, CDC]3).' 5 8.69 (s, 1 H, H—2), 8.31 (s, 1 H, H—8), 6.77 (t, 1 H, J= 6.8 Hz, H-1’), 4.58
(m, 1 H, H—4’), 4.06 (m, 1 H, H—3’), 3.90 (s, 3 H, CH30), 3.87 (dd, 1 H, H—5’a), 3.81 (dd, 1 H,
H—5’b), 2.42 (m, 2 H, H—2’a and H—2’b), 0.93 (s, 18 H, (CH3)3CSi), 0.13 (s, 6 H, (CH3)2Si),
0.12 (s, 6 H, (CH3)2Si).
To a solution of compound 3 (0.28 g, 0.50 mmol) in anhydrous THF (4.0 mL), lithium
borohydride (44 mg, 2.0 mmol) was added, followed by MeOH (0.1 mL). The reaction
mixture was stirred at room temperature for 10 min and then heated to reflux for 45 min.
Upon cooling to room temperature, the mixture was diluted with CHzClz (20 mL) and water
(2.0 mL). The organic layer was separated, washed with brine (5.0 mL) two times, dried over
NaZSO4, and concentrated in vacuo. The e was purified by silica gel chromatography
to yield 9—[B—D—3’,5’—O—bis—(tert—butyldimethylsilyl)—2’—deoxyribofuranosyl]—6-chloro—7—
hydroxymethyldeazapurine 4 (0.12 g, 45%) as a white foam. 1HNMR (400 MHz, CDCZ3):
8.62 (s, 1 H, H-8), 7.61 (s, 1 H, H-2), 6.75 (dd, 1 H, J= 6.0 and 7.2 Hz, H—1’), 4.96 (AB d,
1 H, J: 11.6 Hz, 7—CH2a), 4.91 (AB d, 1 H, J: 11.6 Hz, 7—CH2b), 4.57 (m, 1 H, H-4’), 4.00
(m, 1 H, H—3’), 3.80 (m, 2 H, H—5’a and H—5’b), 2.44 (m, 1 H, H—2’a), 2.04 (m, 1 H, H—2’b),
0.91 (2 s, 18 H, (CH3)3CSi), 0.11 (2 s, 12 H, (CH3)2Si).
To a solution of compound 4 (30 mg, 0.057 mmol) in CHzClz (2.0 mL), n—Bu4NBr (9
mg, 0.029 mmol), 2-nitrobenzyl bromide (37 mg, 0.17 mmol) and NaOH on (1 M, 2.0
mL) were added. The reaction e was stirred Vigorously at room temperature for 48
hours in the dark. The organic layer was ted, dried over , trated in
vacuo, and the residue was purified by silica gel chromatography to yield 9—[B—D—3’,5’—0-bis—
(tert—butyldimethylsilyl)-2’-deoxyribofuranosyl]chloro(2-nitrobenzyloxy)methyl
deazapurine 5 (19 mg, 50%) as a Viscous oil. 1HNMR (400 MHz, CDCZ3).' 5 8.63 (s, 1 H, H—
2), 8.06 (dd, 1 H, J= 8.4 and 1.2 Hz, Ph—H), 7.84 (d, 1 H, J: 7.6 Hz, Ph—H), 7.64 (s, 1 H, H—
8), 7.62 (m, 1 H, Ph—H), 7.43 (t, 1 H, Ph—H), 6.75 (dd, 1 H, J= 7.2 and 6.0 Hz, H—l’), 5.03 (s,
2 H, PhCHz), 4.95 (AB d, 1 H, J: 12.0 Hz, 7—CH2a), 4.88 (AB d, 1 H, J= 12.0 Hz, 7—CH2b),
4.59 (m, 1 H, H—4’), 4.00 (m, 1 H, H—3’), 3.80 (m, 2 H, H—5’a and H—5’b), 2.48 (m, 1 H, H—2’a),
2.37 (m, 1 H, H—2’b), 0.92 (2 s, 18 H, (CH3)3CSi), 0.11 (s, 6 H, (CH3)2Si), 0.10 (s, 6 H,
(CH3)ZSi)-
A solution of n-Bu4NF (17 mg, 0.054 mmol) in THF (1.0 mL) was added to a solution
of compound 5 (18 mg, 0.028 mmol) in THF (1.0 mL) at 0°C. The reaction mixture was
lly warmed to room ature and d for two hours. The mixture was
concentrated in vacuo, dissolved in 1,4—dioxane (2.0 mL), followed by addition of NH3 in
MeOH solution (7 M, 4.0 mL). The mixture was transferred to a sealed tube and stirred at
100°C for 16 hours, then cooled to room temperature, trated in vacuo, and the residue
was purified by silica gel tography to yield 7-(2-nitrobenzyloxy)methyldeaza-2’-
denosine 6 (10 mg, 91%) as a white foam. 1HNMR (400 MHz, DMSO—dg): 5 8.08 (s,
1 H, H—2), 8.06 (m, 1 H, Ph—H), 7.75 (m, 2 H, Ph—H), 7.58 (m, 1 H, Ph—H), 7.42 (s, 1 H, H—8),
6.64 (bs, 2 H, D20 exchangeable, 6—NH2), 6.48 (dd, 1 H, J = 2.0 and 6.0 Hz, H—l’), 5.25 (d, 1
H, J = 4.0 Hz, D20 exchangeable, 3’—OH), 5.08 (t, 1 H, J = 5.6 Hz, D20 exchangeable, 5’—
OH), 4.90 (s, 2 H, PhCHz), 4.75 (AB dd, 2 H, 7—CH2), 4.33 (m, 1 H, H—3’), 3.81 (m, 1 H, H—
4’), 3.54 (m, 2 H, H—5’a and H—5’b), 2.47 (m, 1 H, H—2’a), 2.15 (m, 1 H, .
nd 6 (6 mg, 0.014mmol) was phosphorylated with POCl3 (2.6 uL, 0.028
mmol) and proton sponge (6 mg, 0.028 mmol) in trimethylphosphate (0.25 mL) at minus
40°C for four hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium
pyrophosphate (66 mg, 0.14 mmol) and tri-n-butylamine (28 uL) in anhydrous DMF (0.28
mL) was added. After 30 min of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;
1.0 mL) was added. The reaction was stirred at room temperature for one hour and then
concentrated in vacuo. The residue was dissolved in water (2.0 mL), filtered, and purified
using RP—HPLC (see above) to yield 7—(2—nitro—benzyloxy)methyl—7—deaza—2’—
deoxyadenosine—5’—triphosphate dA.I. HRMS (ESI): For the molecular ion C19H23N5015P3
[M-H]', the calculated mass was 654.0403, and the observed mass was 654.0397.
WO 40257
7-[1-(2-nitrophenyl)methyl—pr0pyloxy]methyl— 7-deaza-2 ’-deoxyaden0sine-5 ’-
triphosphate
0' ' "Pr
HO CI 0 Cl
/ |\N/ |\NN/ |\N
TBSO N N T880 T880
T:0: 1) ;0:N (ii) :0:N (iii),(iv)
OTBS OTBS OTBS
02N OZN
14°F
0 NH2 IPr
O NH2
/ \N
I 9 J
HO N N HO\ /0\ /O\ /O N
O (V) R\ IP\\ 0
_ /P\\
—> _ _
o o o o o 0
OH OH
9 dA.|||a
Scheme S7. Synthesis of 7-[1-(2-nitrophenyl)methyl-propyloxy]methyldeaza-2'-
denosine-S'-triph0sphate. Reagents and conditions: (i) TsCl, DMAP, CHzClz, room
temperature, 39%; (ii) racemic (R/S)(2-nitrophenyl)methyl-propanol, neat, 105°C,
54%; (iii) n—Bu4NF, THF, 0°C to room temperature; (iv) NH3, 1,4—dioxane/MeOH, 100°C,
76% for two steps; (v) POCl3, (MeO)3PO, minus 40°C to 0°C; (n—Bu3NH)2H2PZO7, n—Bu3N,
DMF; 1 M HNEt3HC03.
To a solution of compound 4 (0.26 g, 0.49 mmol) in anhydrous CHzClz (12 mL), 4—
ylaminopyridine (DMAP; 0.15 g, 1.2 mmol) and tosyl chloride (0.11 g, 0.58 mmol)
were added. The reaction mixture was stirred at room temperature for 18 hours and then
concentrated in vacuo. The residue was purified by silica gel chromatography to yield 9—[[3—
D-3’,5’—O—bis—(tert—butyldimethylsilyl)—2’-deoxyribofuranosyl]chlorochloromethyl
deazapurine 7 (0.103 g, 39%) as a Viscous oil. 1H NMR (400 MHz, CDCZ3).' 5 8.64 (s, 1 H,
H-2), 7.72 (s, 1 H, H-8), 6.73 (t, 1 H, J: 6.8 Hz, H—1’), 4.95 (AB d, J: 12.4 Hz, 7—CH23),
4.91 (AB d, J: 12.0 Hz, 7—CH2b), 4.58 (m, 1 H, H—3’), 4.00 (m, 1 H, H—4’), 3.82 (m, 2 H, H—
’a and , 2.41 (m, 2 H, H—2’a and H-2’b), 0.95 (s, 9 H, (CH3)3CSi), 0.93 (s, 9 H,
(CH3)3CSi), 0.12 (s, 6 H, (CH3)2Si), 0.11 (s, 6 H, (CH3)2Si).
Compound 7 (54 mg, 0.10 mmol) and racemic (R/S)(2-nitrophenyl)methyl-
propanol (191 mg, 0.98 mmol) were dissolved in anhydrous CH2Cl2 (10 mL). The solvent
was removed in vacuo, and the e was heated for one hour under a nitrogen atmosphere,
then ved in minimum amount of ethyl acetate and purified by silica gel chromatography
to yield 9—[B—D—3’,5’—O—bis—(tert—butyldimethylsilyl)-2’-deoxyribofuranosyl]chloro[1-(2-
nitro-phenyl)methyl-propyloxy]methyldeazapurine 8 (38 mg, 54%) as a 1:1 mixture of
two diastereomers. 1HNMR (400 MHz, CDCZ3) for diastereomers: 5 8.60 and 8.59 (2 s, 1 H,
H—2), 7.83 (m, 1 H, Ph—H), 7.79 (m, 1 H, Ph—H), 7.56 (m, 1 H, Ph—H), 7.48 and 7.47 (2 s, 1 H,
H—8), 7.38 (m, 1 H, Ph—H), 6.70 (m, 1 H, H—1’), 4.81 (m, 1 H, Ph—CH), 4.70 (m, 1H, ),
4.58 (m, 2 H, 7—CH2b and H—3’), 3.99 (m, 1 H, H—4’), 3.78 (m, 2 H, H—5’a and H—5’b), 2.48 (m,
1 H, H—2’a), 2.35 (m, 1 H, H—2’b), 1.96 (m, 1 H, CH), 0.98 and 0.96 (2 d, 3 H, CH3), 0.93 (2 s,
9 H, (CH3)3CSi), 0.89 (2 s, 9 H, (CH3)3CSi), 0.82 and 0.78 (2 d, 3 H, CH3), 0.12 (2 s, 6 H,
(CH3)2Si), 0.08 and 0.07 (2 s, 3 H, Si), 0.06 and 0.05 (2 s, 3 H, (CH3)2Si).
A solution of n-Bu4NF (44 mg, 0.14 mmol) in THF (2.0 mL) was added to a solution
of compound 8 (38 mg, 0.05 mmol) in THF (2.0 mL) at 0°C. The reaction was gradually
warmed to room temperature and stirred for two hours. The mixture was concentrated in
vacuo, dissolved in 1,4—dioxane (4.0 mL), followed by addition of NH3 in MeOH solution (7
M, 8.0 mL). The mixture was transferred to a sealed tube, stirred at 100°C for 24 hours,
cooled to room temperature, and then concentrated in vacuo. The residue was purified by
silica gel tography to yield 7-[1-(2-nitrophenyl)methyl-propyloxy]methyldeaza-
2’—deoxyadenosine 9 (19 mg, 76%) as a 1:1 mixture of two diastereomers. 1H NMR (400
MHZ, DMSO-dg) for diastereomers.‘ 5 8.06 and 8.04 (2 s, 1 H, H-2), 7.90 (m, 1 H, Ph-H),
7.67 (m, 2 H, Ph—H), 7.56 (m, 2 H, Ph—H), 7.19 and 7.16 (2 s, 1 H, H—8), 6.63 (bs, 2 H, D20
geable, 6—NH2), 6.39 (m, 1 H, H—1’), 5.23 (m, 1 H, D20 exchangeable, , 5.00
(m, 1 H, D20 exchangeable, 5’-OH), 4.72 (2 d, 1 H, Ph—CH), 4.45 (s, 2 H, 7—CH2): 4.30 (m, 1
H, H—3’), 3.77 (m, 1 H, H—4’), 3.49 (m, 2 H, H—5’a and H—5’b), 2.40 (m, 1 H, H—2’a), 2.12 (m, 1
H, H—2’b), 1.94 (m, 1 H, CH), 0.87 (m, 3 H, CH3), 0.74 (m, 3 H, CH3).
Compound 9 (19 mg, 0.041 mmol) was phosphorylated with POCl3 (16 uL, 0.16
mmol) and proton sponge (18 mg, 0.082 mmol) in trimethylphosphate (0.4 mL) at minus
40°C for five hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium
pyrophosphate (97 mg, 0.20 mmol) and tri-n-butylamine (40 uL) in anhydrous DMF (0.40
mL) was added. After 30 min of stirring, triethylammonium onate buffer (1 M, pH 7.5;
mL) was added. The reaction was stirred at room temperature for one hour and then
concentrated in vacuo. The residue was dissolved in water (5.0 mL), filtered, and purified by
anion exchange chromatography. The fractions containing triphosphate were combined and
lyophilized to yield 7-[l-(2-nitrophenyl)methyl-propyloxy]methyldeaza-2’-
deoxyadenosine-5’-triphosphate dA.III.a as a 1:1 mixture of two diastereomers, which were
separated using C to yield the single diastereomers dA.III.a dsl and dA.III.a ds2.
In all cases, diastereomer l (dsl) eluted faster than diastereomer 2 (ds2) by RP-HPLC.
HRMS (ESI): For the molecular ion C22H29N5015P3 [M—H]', the calculated mass was
696.0873, and the observed mass was 696.0864.
7-[1-(4-Meth0xynitrophenyl)methyl—pr0pyloxy]methyl— 7-deaza-2 ’-
deoxyadenosine-5 imphosphate
OMe OMe
OZN 02N
I i-Pr
CI 0 CI i-Pr
O NH2
/ I l / I ‘1 / I 1N
TBSO N’ RO N N/ HO N N,
| r:o: | (I) | F0: | (ii), (iii) I :o: |
OTBS OR OH
R=Hor TBS
7 1o 11
0 NH2
/ \N
. A
(IV) HO\ /o\ /o\ /O N N
—> A F\\ O
, ,Rx , ,
o o o o o o
dA.|||.b
Scheme S8. sis of 7-[1-(4-methoxynitrophenyl)methyl—propyloxy]methyl
deaza-Z'-de0xyaden0sine—5'-triph0sphate. Reagents and conditions: (i) racemic (RAB—l—
(4-methoxynitrophenyl)-2—methyl-l-propanol, 108°C; (ii) n-Bu4NF, THF, 0°C to room
temperature; (iii) NH3, 1,4—dioxane/MeOH, 100°C, 32% for three steps; (iv) POCl3,
(MCO)3PO; 00C; (n-BU3NH)2H2P207; , DMF; 1 M CO3.
Compound 7 (103 mg, 0.19 mmol) and racemic 1-(4—methoxy—2—nitrophenyl)—2—
methyl-l—propanol (428 mg, 1.9 mmol) were dissolved in anhydrous CHzClz (3.0 mL). The
solvent was removed in vacuo, and the residue was heated at 108°C for 30 min under a
nitrogen atmosphere, cooled to room temperature, dissolved in minimum amount of ethyl
e, and purified by silica gel chromatography to yield 6-chloro[1-(4-methoxy
nitrophenyl)—2—methyl-propyloxy]methyl—7—deazapurine xyribonucleosides 10. The
sample was ved in THF (8.0 mL), cooled to 0°C, and then added to a solution of n—
Bu4NF (68 mg, 0.22 mmol) in THF (2.0 mL). The reaction was gradually warmed to room
temperature and stirred for 30 min. The mixture was concentrated in vacuo, dissolved in 1,4—
dioxane (8.0 mL), followed by addition of NH3 in MeOH (7 N, 24 mL). The mixture was
transferred to a sealed tube and stirred at 100°C for 16 hours, then cooled to room
temperature, and concentrated in vacuo. The residue was purified by silica gel
chromatography to yield 7-[1-(4-methoxynitrophenyl)methyl-propyloxy]methyl
deaza—2’—deoxyadenosine 11 (30 mg, 32% for three steps) as a 1:1 mixture of two
diastereomers. 1HNMR (400 MHz, DMSO-dg) for reomers: 5 8.06 and 8.05 (2 s, 1 H,
H—2), 7.57 and 7.54 (2 d, 1 H, J: 8.8 Hz, Ph—H), 7.47 and 7.44 (2 d, 1 H, J: 2.6 Hz, Ph—H),
7.33 and 7.27 (2 dd, J: 8.8 and 2.6 Hz, 1 H, Ph—H), 7.18 and 7.15 (2 s, 1 H, H—8), 6.63 (bs, 2
H, D20 exchangeable, 6-NH2), 6.43 (m, 1 H, H-1’), 5.24 (m, 1 H, D20 exchangeable, 3’-OH),
.03 (m, 1 H, D20 exchangeable, 5’—OH), 4.55 (m, 2 H, Ph—CH, 7-CH2a), 4.30 (m, 2 H, 7—
Csz and H—3’), 3.86 and 3.84 (2 s, 3 H, MeO), 3.78 (m, 1 H, H—4’), 3.48 (m, 2 H, H—5’), 2.45
(m, 1 H, H—2’a), 2.12 (m, 1 H, , 1.93 (m, 1 H, CH(CH3)2), 0.88 (m, 3 H, CH3), 0.74
and 0.71 (2 d, J: 6.8 Hz, 3 H, CH3).
Compound 11 (28 mg, 0.06 mmol) was phosphorylated with POCl3 (11 uL, 0.12
mmol) and proton sponge (25 mg, 0.12 mmol) in trimethylphosphate (0.35 mL) at 0°C for
two hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium
pyrophosphate (237 mg, 0.50 mmol) and tri-n-butylamine (100 uL) in anhydrous DMF (1.0
mL) was added. After 10 min of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5;
mL) was added. The reaction was stirred at room temperature for one hour and then
concentrated in vacuo. The residue was dissolved in 20% s acetonitrile (10 mL),
filtered, and purified by anion exchange chromatography. The fractions containing
triphosphate were ed and lyophilized to yield 7-[1-(4-methoxynitrophenyl)
methyl-propyloxy]methyldeaza-2’-deoxyadenosine-5’-triphosphate .b as a 1:1
mixture of two diastereomers, which were separated using RP-HPLC to yield the single
diastereomers dA.III.b dsl and dA.III.b ds2. HRMS (ESI): For the molecular ion
C23H31N5016P3 [M-H]', the calculated mass was 726.0979, and the ed mass was
726.0984.
7-[1-(2, 6-Dinitr0phenyl)methyl-propyloxy]methyl— 7-deaza-2 ’-de0xyaden0sine-5 ’-
triphosphate
oZN‘ E ‘
N02 OZN No2
CI i- Pr
CI 0 CI i-Pr
o NH2
/ I] / | UN / |
TBSO N N
\JN N
I;O>I
RD N/ HO N N/
(i) |;o:| (ii), (iii) 1 :0: |
OTBS [ OR OH
R=Hor TBS
7 WE: 13
(iv) HO\ O\/O
—> /P\\
/\\ /F)\\
O O O O O O F:
dA.|||.c
Scheme S9. Synthesis of 7-[1-(2,6-dinitrophenyl)methyl-propyloxy]methyldeaza-
2'-de0xy—aden0sine—5'-triph0sphate. Reagents and conditions: (i) racemic (R/S)—1—(2,6—
ophenyl)—2—methyl—1—propanol, 108°C; (ii) n—Bu4NF, THF, 0°C to room temperature;
(iii) NH3, 1,4—dioxane/MeOH, 100°C, 38% for three steps; (iv) POCl3, (MeO)3PO, 0°C; (n—
BU3NH)2H2P207, I’Z-BU3N, DMF; l M HNEt3HCO3.
Compound 7 (109 mg, 0.20 mmol) and racemic (R/S)—1—(2,6-dinitrophenyl)—2—methyl—
l-propanol (448 mg, 1.9 mmol) were dissolved in ous CHzClz (10 mL). The solvent
was removed in vacuo, and the residue was heated at 108°C for 30 min under a nitrogen
here, then dissolved in minimum amount of ethyl acetate and purified by silica gel
chromatography to yield 6-chloro[1-(2,6-dinitrophenyl)methyl-propyloxy]methyl
deazapurine—2’—deoxyribo—nucleosides 12. The sample was dissolved in THF (5.0 mL),
cooled to 0°C, and then added a solution of n—Bu4NF (31 mg, 0.10 mmol) in THF (2.0 mL).
The on was gradually warmed to room temperature and stirred for two hours. The
mixture was concentrated in vacuo, dissolved in 1,4—dioxane (4.0 mL), followed by addition
of NH3 in MeOH (7 N, 18 mL). The mixture was transferred to a sealed tube, stirred at
100°C for 36 hours, cooled to room ature, and then concentrated in vacuo. The residue
was purified by silica gel chromatography to yield 7-[1-(2,6-dinitrophenyl)methyl-
propyloxy]methyl—7—deaza—2’-deoxyadenosine 13 (38 mg, 38% for three steps) as a 1:1
mixture of two diastereomers. 1H NMR (400 MHZ, DMSO-dg) for diastereomers: 5 8.17 (m,
1 H, Ph—H), 8.07 and 8.06 (2 s, 1 H, H—2), 7.85 (m, 1 H, Ph—H), 7.69 (m, 1 H, Ph—H), 7.20 and
7.18 (2 s, 1 H, H—8), 6.57 (bs, 2 H, D20 exchangeable, 6—NH2), 6.46 (m, 1 H, H—1’), 5.26 (d, J
= 3.6 Hz, 1 H, D20 exchangeable, 3’—OH), 5.01 (m, 1 H, D20 exchangeable, 5’—OH), 4.60 (m,
2 H, Ph—CH and 7—CH2a), 4.29 (m, 1 H, 7—CH2b), 4.13 (m, 1 H, H—3’), 3.80 (m, 1 H, H—4’),
3.51 (m, 2 H, H—5’a and H—5’b), 2.49 (m, 1 H, CH(CH3)3), 2.16 (m, 2 H, H—2’a and H—2’b),
0.91 (m, 3 H, CH3), 0.65 (m, 3 H, CH3). T0F-MS (ESI): For the molecular ion C22H27N6Og
[M+H]+, the ated mass was 503.1890, and the observed mass was 503.2029.
Compound 13 (30 mg, 0.06 mmol) was phosphorylated with POCl3 (17 uL, 0.18
mmol) and proton sponge (26 mg, 0.12 mmol) in trimethylphosphate (0.4 mL) at 0°C for four
hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium osphate
(285 mg, 0.6 mmol) and tri-n-butylamine (120 uL) in anhydrous DMF (1.2 mL) was added.
After 30 min of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5; 10 mL) was
added. The reaction was stirred for one hour at room temperature and then concentrated in
vacuo. The residue was ved in 20% aqueous acetonitrile (10 mL), filtered, and purified
by anion exchange chromatography. The fractions containing triphosphate were combined
and lyophilized to yield 7-[1-(2,6-dinitrophenyl)methyl-propyloxy]methyldeaza-2’-
deoxyadenosine-5’-triphosphate .c as a 1:1 mixture of two reomers which were
separated using RP—HPLC to yield the single diastereomers dA.III.c dsl and dA.III.c ds2.
HRMS (ESI): For the molecular ion C22H28N6017P3 [M—H]', the calculated mass was
741.0724, and the ed mass was 741.0731.
7-[(S)-I-(2-Nitr0phenyl)-2, 2-dimethyl—pr0pyloxy]methyl— 7-deaza-2 ’-deoxyaden0sine-
’-triph0sphate
OZN OZN
| t—Bu
O | t-Bu
CI O
/ I 1“ / P1
N/ N/ 715NH2
TBSO N HO N HO N N/
o (i), (ii) 0 (iii) 0
—> —P
OTBS OH OH
7 14 15
t-Bu
O NH2
/ \N
I A
(iv) HO\ ,o\ ,o\ /O N N
’ /R\ A O
, , , /F{\
O O O O O O
dA.V
Scheme S10. Synthesis of 7-[(S)(2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl
deaza-Z'-deoxyaden0sine—5'-triph0sphate. Reagents and conditions: (i) (S)—1—(2—
nitrophenyl)-2,2-dimethyl-l-propanol, 110°C; (ii) n-Bu4NF, THF, room temperature; 75% for
two steps; (iii) NH3, 1,4-dioxane/MeOH, 100°C, 93%; (iv) POC13, (MeO)3PO, 0°C; (n—
BU3NH)2H2P207, I’Z-BU3N, DMF; l M CO3.
Compound 7 (130 mg, 0.24 mmol) and (S)(2-nitrophenyl)—2,2-dimethylpropanol
(290 mg, 1.4 mmol) were heated at 110°C for 45 min under a nitrogen atmosphere. The
reaction mixture was cooled to room ature and ved in THF (10 mL) followed by
addition of n-Bu4NF (189 mg, 0.60 mmol). The mixture was stirred at room temperature for
two hours and then trated in vacuo. The residue was dissolved in CHzClz (20 mL) and
washed with brine (30 mL), and the s phase was extracted with CHzClz (20 mL) two
times. The combined organic phase was dried over NaZSO4, concentrated in vacuo, and the
residue was purified by silica gel chromatography to yield 6-chloro—9—[B-D—2’-
deoxyribofuranosyl] [(19(2-nitrophenyl)-2,2-dimethyl-propyloxy]methyldeazapurine
14 (90 mg, 75%). 1H NMR (400 MHz, CDCZ3): 5 8.51 (s, 1 H, H—2), 7.68 (m, 2 H, Ph—H),
7.45 (t, 1 H, J: 7.2 Hz, Ph—H), 7.38 (s, 1 H, H—8), 7.29 (t, 1 H, J= 7.2 Hz, Ph—H), 6.39 (dd, 1
H, J= 6.0 and 8.0 Hz, H—l’), 4.97 (s, 1 H, Ph—CH), 4.70 (m, 3 H, 7—CH2 and H—3’), 4.16 (m, 1
H, H—4’), 3.83 (m, 2 H, H—5’), 2.80 (m, 1 H, , 2.35 (m, 1 H, H—2’b), 0.82 (s, 9 H,
C(CH3)3).
Compound 14 (90 mg, 0.18 mmol) was dissolved in 1,4—dioxane (8.0 mL) followed
by addition of NH3 in MeOH (7 N, 16 mL). The mixture was transferred to a sealed tube and
stirred at 100°C for 24 hours, cooled to room temperature, and then concentrated in vacuo.
The residue was ed by silica gel chromatography to yield 7-[(S)—1-(2-nitrophenyl)-2,2-
dimethyl-propyloxy]methyl—7—deaza—2’—deoxyadenosine 15 (80 mg, 93%). 1H NMR (400
MHz, DMSO-dg): 5 8.09 (s, 1 H, H—2), 7.91 (dd, 1 H, J: 1.2 and 8.0 Hz, Ph—H), 7.71 (m, 2
H, Ph—H), 7.58 (m, 1 H, Ph—H), 7.24 (s, 1 H, H—8), 6.68 (bs, 2 H, D20 exchangeable, 6—NH2),
6.46 (dd, 1 H, J= 6.0 and 8.0 Hz, H—1’), 5.27 (d, 1 H, D20 exchangeable, 3’—OH), 5.06 (t, 1
H, D20 geable, 5’-OH), 4.87 (s, 1 H, Ph-CH), 4.65 (d, 1 H, J = 12.8 Hz, 7-CH2a), 4.49
(m, 1 H, H—3’), 4.36 (d, 1 H, 7—CH2b), 3.80 (m, 1 H, H—4’), 3.49 (m, 2 H, H—5’), 2.45 (m, 1 H,
H—2’a), 2.17 (m, 1 H, H—2’b), 0.75 (s, 9 H, C(CH3)3).
Compound 15 (25 mg, 0.053 mmol) was phosphorylated with POCl3 (22 uL, 0.24
mmol) and proton sponge (23 mg, 0.11 mmol) in trimethylphosphate (0.35 mL) at 0°C for 4.5
hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium osphate
(237 mg, 0.50 mmol) and tri-n-butylamine (100 uL) in anhydrous DMF (1.0 mL) was added.
After 10 min of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5; 10 mL) was
added. The reaction was stirred at room ature for one hour and then concentrated in
vacuo. The residue was dissolved in 20% aqueous acetonitrile (20 mL), filtered, and purified
by anion ge chromatography. The fractions containing triphosphate were combined
and lyophilized to yield 7-[(S)(2-nitrophenyl)-2,2-dimethyl-propyloxy]methyldeaza—2’-
deoxyadenosine—5’—triphosphate dA.V, which was further purified using C. HRMS
(ESI): For the molecular ion C23H31N5015P3 [M—H]', the calculated mass was 710.1029, and
the observed mass was 710.1032.
7-[(S)-I-(5-Meth0xynitrophenyD-Z,2-dimethyl—pr0pyloxy]methyl— 7-deaza-2 ’-
deoxyadenosine-5 imphosphate
/ |\NJ
TBSO N N
I T:0: I (i) (ii) 0I Oi:: II (iii) 0I II
OTBS
7 E::
t-Bu
O NH2
—" /P\\ /\\
7O O 7O O 7OP\\O
dA.V|
Scheme S11. Synthesis of 7-[(S)(5-methoxynitrophenyl)-2,2-dimethyl—
oxy]methyldeaza-2'-deoxyaden0sine—5'-triph0sphate. Reagents and conditions:
(i) (S)(5-methoxynitrophenyl)-2,2-dimethyl-l-propanol, 110°C; (ii) n-Bu4NF, THF,
room temperature; 78% for two steps; (iii) NH3, 1,4—dioxane/MeOH, 100°C, 74%; (iv)
POCl3, PO, 00C; (n-BU3NH)2H2P207, n—Bu3N, DMF; 1 M HNEt3HCO3.
Compound 7 (165 mg, 0.30 mmol) and (S)—1—(5—methoxy—2—nitrophenyl)—2,2-
yl-l-propanol (330 mg, 1.4 mmol) were heated at 110°C for 45 min under a nitrogen
atmosphere. The reaction mixture was cooled to room temperature and dissolved in THF (10
mL), followed by addition of n-Bu4NF (236 mg, 0.75 mmol). The mixture was d at room
temperature for two hours and then concentrated in vacuo. The residue was dissolved in
CHzClz (40 mL) and washed with brine (50 mL), and the s phase was extracted with
CHzClz (40 mL) two times. The combined organic phase was dried over NaZSO4,
concentrated in vacuo, and the residue was purified by silica gel chromatography to yield 6—
chloro—9—[[B—D-2’-deoxyribofuranosylH{(153- 1-(5-methoxynitrophenyl)-2,2-dimethyl-
propyloxy]methyldeazapurine 16 (122 mg, 78%). 1HNMR (400 MHZ, CDC]3).' 5 8.55 (s,
1 H, H—2), 7.79 (d, 1 H, J: 9.2 Hz, Ph—H), 7.35 (s, 1 H, H—8), 7.15 (d, 1 H, J: 3.2 Hz, Ph—H),
6.68 (dd, 1 H, J= 3.2 and 9.2 Hz, Ph—H), 6.33 (dd, 1 H, J= 5.6 and 8.8 Hz, H—l’), 5.26 (s, 1
2012/055231
H, Ph—CH), 4.85 (d, 1 H, J: 8.8 Hz, ), 4.75 (m, 1 H, H—3’), 4.70 (d, 1 H, J= 8.8 Hz,
7—CH2b), 4.13 (m, 1 H, H—4’), 3.95 (m, 1 H, H—5’a), 3.83 (s, 3 H, OCH3), 3.78 (m, 1 H, H—
’b), 2.86 (m, 1 H, H—2’a), 2.30 (m, 1 H, , 0.83 (s, 9 H, C(CH3)3).
Compound 16 (120 mg, 0.23 mmol) was dissolved in 1,4—dioxane (10 mL) followed
by addition of NH3 in MeOH (7 N, 10 mL). The mixture was transferred to a sealed tube and
stirred at 100°C for 24 hours, then cooled to room temperature, concentrated in vacuo, and
the residue was purified by silica gel chromatography to yield 7—[(S)—1—(5-methoxy—2—
nitrophenyl)—2,2—dimethyl—propyloxy]methyl—7—deaza—2’-deoxyadenosine 17 (87 mg, 74%).
IHNMR (400MHz, DMSO-dg): 5 8.06 (s, 1 H, H—2), 7.97 (d, 1 H, J: 9.2 Hz, Ph—H), 7.22 (s,
1 H, H—8), 7.08 (d, 1 H, J= 2.8 Hz, Ph—H), 7.05 (dd, 1 H, J= 2.8 and 9.2 Hz, Ph—H), 6.66 (bs,
2 H, D20 exchangeable, 6-NH2), 6.42 (dd, 1 H, J: 6.0 and 8.0 Hz, H—l’), 5.25 (d, 1 H, D20
exchangeable, 3’—OH), 5.15 (s, 1 H, Ph—CH), 5.03 (t, 1 H, D20 exchangeable, 5’-OH), 4.64
(d, 1 H, J: 12.8 Hz, 7—CH2a), 4.43 (d, 1 H, J: 12.8 Hz, 7—CH2b), 4.30 (m, 1 H, H—3’), 3.84
(s, 3 H, OCH3), 3.77 (m, 1 H, H—4’), 3.45 (m, 2 H, H—5’), 2.43 (m, 1 H, H—2’a), 2.14 (m, 1 H,
H—2’b), 0.75 (s, 9 H, C(CH3)3).
Compound 17 (21 mg, 0.042 mmol) was phosphorylated with POCl3 (40 uL, 0.43
mmol) and proton sponge (18 mg, 0.084 mmol) in trimethylphosphate (0.35 mL) at 0°C for
7.5 hours under a nitrogen atmosphere. A solution of bis—tri—n—butylammonium
pyrophosphate (237 mg, 0.50 mmol) and tri-n-butylamine (100 uL) in anhydrous DMF (1.0
mL) was added. After 10 min of stirring, triethylammonium bicarbonate buffer (0.1 M, pH
7.5; 10 mL) was added. The reaction was stirred at room temperature for one hour and then
concentrated in vacuo. The residue was dissolved in 20% aqueous acetonitrile (20 mL),
filtered, and purified by anion ge chromatography. The fractions containing
triphosphate were combined and lized to yield 7-[(S)(5-methoxynitrophenyl)-2,2-
dimethyl-propyloxy]methyldeaza-2’-deoxyadenosine-5’-triphosphate dA.VI, which was
further ed using RP-HPLC. HRMS (ESI): For the lar ion C24H33N5016P3 [M-H]'
the calculated mass was 740.1135, and the observed mass was 740.1156.
WO 40257
Example 4 — Synthesis of 7-HOMeDeaza-2'-de0xyguan0sine Triphosphate Analogs
7-(2-nitrobenzyloxy)methyl— 7-deaza-2 ’-deoxyguan0sine-5 ’-triph0sphate
I CI CI CI
I MeO
\ N \
I / | N
HO N NANHz TBSO N
. TBSO |NJ\NHTBS NANHTBS
o (I) o (H) o
—> —>
OH OTBS OTBS
1s 19 20
/ \N
I A \N
TBSO N N NHTBS TBSO *NHTBS
(III) 0 (iv)
_> _>
OTBS OTBS
21 22
OZN OZN
CI o
0 0
/ \N
/ NNH/
HO N)\NH2 HO N N)\NH2
(vi) (vu)
OH OH
23 24
/P\\ Rx R\ o
O O O O O O
dG.I
Scheme S12. Synthesis of 7-(2-nitr0benzyloxy)methyl—7-deaza-2'-deoxyguanosine-5'-
triphosphate. Reagents and ions: (i) TBSCl, imidazole, DMF, room temperature,
60%; (ii) CO, PdC12[PhCN]2, MeOH/1,4—di0xane, 50°C, 91%; (iii) LiBH4, MeOH, THF,
reflux, 54%; (iv) 2-nitr0benzy1 bromide, n-Bu4NBr, CHzClz/aq. NaOH, room temperature,
48%; (v) n—Bu4NF, THF, 0°C to room temperature, 95%; (vi) DABCO, H20, reflux, 30%;
(vii) POCl3, proton sponge, (MeO)3PO, 0°C; (n—Bu3NH)2H2PZO7, n—Bu3N, DMF; 1 M
HNEt3HCO3.
Compound 18 (Seela and Peng, 2005, which is incorporated by reference herein)
(1.35 g, 3.29 mmol) was ated from anhydrous pyridine (3.0 mL) three times and then
dissolved in anhydrous DMF (6.0 mL). tert—Butyldimethylsilyl de (5.95 g, 39.5 mmol)
and imidazole (5.37 g, 78.9 mmol) were added, and the mixture was stirred at 50°C for 48
hours with additional tert—butyldimethylsilyl chloride (2.97 g, 19.7 mmol) and imidazole
(2.69 g, 39.4 mmol) being added every six hours. The reaction mixture was concentrated in
vacuo and purified by silica gel chromatography to yield 9—[B—D—3’,5’—0—bis—(tert—
butyldimethylsilyl)-2’-deoxyribofuranosyl](tert—butyldimethylsilyl)aminochloro-7—iodo—
apurine 19 (1.48 g, 60% yield) as a white foam. 1H NMR (400 MHz, CDCZ3).' 5 7.35
(s, 1 H, H—8), 6.53 (t, 1 H, J= 6.0 Hz, H-1’), 4.70 (s, 1 H, 2-NH), 4.47 (m, 1 H, H—3’), 3.97
(m, 1 H, H-4’), 3.78 (m, 2 H, H-5’a and H-5’b), 2.23 (m, 2 H, H-2’a and H—2’b), 0.98 (s, 9 H,
(CH3)3CSi), 0.95 (s, 9 H, (CH3)3CSi), 0.90 (s, 9 H, (CH3)3CSi), 0.29 (2 s, 6 H, (CH3)2Si),
0.13 (2 s, 6 H, CH3)2Si), 0.09 (s, 6 H, CH3)2Si).
A solution of compound 19 (720 mg, 0.96 mmol) was dissolved in anhydrous 1,4—
dioxane (30 mL). Anhydrous MeOH (30 mL) and triethylamine (0.58 mL) were added, and
the mixture was stirred for 10 min under a CO atmosphere, followed by addition of
bis(benzonitrile)dichloropalladium(H) (20 mg, 0.05 mmol). The reaction was stirred at 58 0C
for 24 hours under a CO atmosphere, and then concentrated in vacuo. The residue was
purified by silica gel chromatography to yield 9-[B-D-3’,5’-O-bis-(tert—butyldimethylsilyl)-2’-
deoxyribofuranosyl]—2-(tert—butyldimethylsilyl)aminochloromethoxycarbonyl
deazapurine 20 (600 mg, 91%) as a viscous oil. 1HNMR (400 MHz, CDCZ3).' 5 7.92 (s, 1 H,
H—8), 6.57 (dd, 1 H, J= 8.0 and 6.0 Hz, H-1’), 4.78 (s, 1 H, 2—NH), 4.49 (m, 1 H, H—3’), 4.02
(m, 1 H, H—4’), 3.85 (s, 3 H, CH3), 3.81 (m, 2 H, H-5’a and H-5’b), 2.25 (m, 2 H, H-2’a and H-
2’b), 0.98 (s, 9 H, (CH3)3CSi), 0.93 (s, 9 H, (CH3)3CSi), 0.92 (s, 9 H, CSi), 0.31 (s, 6
H, (CH3)2Si), 0.13 (2 s, 6 H, Si), 0.11 (s, 6 H, Si).
To a solution of nd 20 (1.11 g, 1.63 mmol) in ous THF (56 mL),
lithium borohydride (143 mg, 6.5 mmol) was added, followed by MeOH (0.94 mL). The
reaction mixture was heated to reflux for one hour. Upon cooling to room temperature, the
reaction mixture was diluted with CHzClz (700 mL) and ed with water (70 mL). The
organic phase was separated, dried over NaZSO4, and concentrated in vacuo. The residue was
purified by silica gel chromatography to yield 9-[B-D-3’,5’-0—bis-(tert—butyldimethylsilyl)-2’-
WO 40257
deoxyribofuranosyl](tert—butyldimethylsilyl)aminochlorohydroxymethyl
deazapurine 21 (0.58 g, 54%) as a Viscous oil. 1H NMR (400 MHz, CDCZ3).' 5 7.16 (s, 1 H,
H—8), 6.56 (t, 1 H, J: 6.4 Hz, H—1’), 4.79 (AB d, J: 13.6 Hz, 7—CH2a), 4.75 (AB d, J: 13.6
Hz, 7—CH2b), 4.70 (s, 1 H, 2—NH), 4.50 (m, 1 H, H—3’), 3.96 (m, 1 H, H—4’), 3.76 (m, 2 H, H—
’a and H—5’b), 2.23 (m, 2 H, H—2’a and H—2’b), 0.98 (s, 9 H, (CH3)3CSi), 0.94 (s, 9 H,
(CH3)3CSi), 0.92 (s, 9 H, (CH3)3CSi), 0.30 (s, 3 H, (CH3)2Si), 0.29 (s, 3 H, (CH3)2Si), 0.11
(s, 6 H, Si) 0.10 (s, 6 H, (CH3)2Si).
To a solution of nd 21 (150 mg, 0.23 mmol) in CHzClz (3.0 mL), n—Bu4NBr
(37 mg, 0.12 mmol), 2-nitrobenzyl bromide (148 mg, 0.68 mmol) and NaOH solution (1 M,
3.0 mL) were added. The reaction mixture was stirred vigorously at room temperature for
two days in the dark. The organic phase was separated, dried over NaZSO4, concentrated in
vacuo, and purified by silica gel chromatography to yield 9—[B—D—3’,5’—0—bis—(tert—
butyldimethylsilyl)-2’-deoxyribofuranosyl](tert—butyldimethylsilyl)aminochloro(2-
nitrobenzyloxy)methyldeazapurine 22 (87 mg, 48%) as a Viscous oil. 1H NMR (400 MHZ,
CDCZ3).' 5 8.06 (dd, 1H, J: 8.0 and 1.2 Hz, Ph—H), 7.87 (d, 1 H, J: 7.2 Hz, Ph—H), 7.61 (dt,
1 H, J: 7.6 and 1.2 Hz, Ph—H), 7.43 (m, 1 H, Ph—H), 7.20 (s, 1 H, H—8), 6.56 (dd, 1 H, J: 7.6
and 6.0 Hz, H—1’), 4.99 (s, 2 H, PhCHz), 4.83 (AB d, 1 H, J: 11.4 Hz, 7—CH2a), 4.75 (AB d,
1 H, J: 11.4 Hz, 7—CH2b), 4.67 (s, 1 H, 2—NH), 4.50 (m, 1 H, H—3’), 3.96 (m, 1 H, H—4’), 3.77
(m, 2 H, H-5’a and H-5’b), 2.25 (m, 2 H, H-2’a and H—2’b), 0.98 (s, 9 H, (CH3)3CSi), 0.92 (s,
18 H, (CH3)3CSi), 0.30 (s, 3 H, (CH3)2Si), 0.29 (s, 3 H, (CH3)2Si), 0.09 (m, 12 H, Si).
A solution of n-Bu4NF (123 mg, 0.39 mmol) in THF (2.0 mL) was added dropwise to
a solution of nd 22 (105 mg, 0.13 mmol) in THF (3.0 mL) at 0°C. The reaction
mixture was stirred at 0°C for one hour and then at room temperature for two hours. The
mixture was trated in vacuo and purified by silica gel chromatography to yield 2—
aminochloro[B-D-2’-deoxy-ribofuranosyl](2-nitrobenzyloxy)methyldeazapurine
23 (57 mg, 95%) as a yellow foam. 1HNMR (400 MHz, DMSO-do): 5, 8.02 (m, 1 H, Ph—H),
7.74 (m, 2 H, Ph-H), 7.55 (m, 1 H, Ph-H), 7.41 (s, 1 H, H-8), 6.73 (s, 2 H, D20 exchangeable,
NHz), 6.41 (dd, 1 H, J: 8.4 and 6.0 Hz, H—1’), 5.26 (d, 1 H, D20 exchangeable, 3’-OH), 4.91
(t, 1 H, D20 exchangeable, 5’-OH), 4.88 (s, 2 H, Ph-CHz), 4.66 (dd, 2 H, J = 11.6 Hz, 7—
CH2), 4.31 (m, 1 H, H—3’), 3.78 (m, 1 H, H—4’), 3.50 (m, 2 H, H—5’), 2.38 (m, 1 H, H—2’a), 2.15
(m, 1 H, H—2’b).
A mixture of 23 (38 mg, 0.085 mmol) and 1,4-diazabicyclo[2.2.2]octane (11 mg, 0.1
mmol) in water (4.0 mL) was heated to reflux for four hours under a nitrogen atmosphere.
Water was removed in vacuo, and the residue was evaporated from MeOH (3.0 mL) three
times, and d by silica gel chromatography to yield 7-(2-nitrobenzyloxy)methyl
deaza-2’-deoxyguanosine 24 (11 mg, 30%). 1H NMR (400 MHz, DMSO-d6): 5 10.4 (s, 1 H,
D20 geable, N—H), 8.03 (dd, 1 H, J = 8.4 and 0.8 Hz, Ph—H), 7.83 (d, 1 H, J = 7.6 Hz,
Ph—H), 7.73 (m, 1 H, Ph—H), 7.55 (m, 1 H, Ph—H), 6.92 (s, 1 H, H—8), 6.28 (m, 1 H, H—l’), 6.26
(bs, 2 H, D20 exchangeable, NHz), 5.21 (d, 1 H, D20 exchangeable, 3’-OH), 4.89 (t, 1 H,
D20 exchangeable, 5’—OH), 4.88 (s, 2 H, Ph—CHz), 4.60 (dd, 2 H, 7—CH2), 4.28 (m, 1 H, H—3’),
3.74 (m, 1 H, H—4’), 3.48 (m, 2 H, H—5’), 2.32 (m, 1 H, H—2’a), 2.08 (m, 1 H, H—2’b).
Compound 24 (11 mg, 0.025 mmol) was phosphorylated with POCl3 (15 uL, 0.05
mmol) and proton sponge (11 mg, 0.05 mmol) in trimethylphosphate (0.3 mL) at 0°C for two
hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium osphate
(118 mg, 0.25 mmol) and tri—n—butylamine (50 uL) in anhydrous DMF (0.5 mL) was added.
After 30 min of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5; 5.0 mL) was
added. The reaction was d at room temperature for one hour and then concentrated in
vacuo. The residue was dissolved in water (10 mL), filtered, and purified by anion exchange
chromatography. The fractions containing triphosphate were combined and lyophilized to
give itrobenzyloxy)methyldeaza-2’-deoxyguanosine-5’-triphosphate dG.I, which
was further purified using RP-HPLC. HRMS (ESI): For the molecular ion C19H23N5016P3
[M-H]', the calculated mass was 670.0353, and the observed mass was 670.0344.
7-[1-(2-Nitr0phenyD-2, 2-dimethyl—pr0pyloxy]methyl— 7-deaza-2 ’-deoxyguan0sine-5 ’-
sphate
(jg/fir CI
\ >13\N
TBSOW NHTBS TBSO N N NHTBS
()i (“)0“)
_) O ’_>
OTBS OTBS
t—Bu
\N / NH
J\NH2 | A
Ho N N
(iv) NH2
o _,
OH OH
26
t—Bu O
HO\ VIE/15H
‘O/RO'O/O\JD /O\lP\\/O)D\\O _ DIE-IO3|
d G.V.a
Scheme 813. Synthesis of 7-[1-(2-nitr0phenyl)-2,2-dimethyl-propyloxy]methyldeaza-
2'-de0xy-guan0sine—5'-triph0sphate. Reagents and conditions: (i) MsCl, DMAP, CHzClz,
0 °C; (ii) racemic (R/S)-l-(2-nitrophenyl)—2,2-dimethyl-l-propanol, 115°C; (iii) n-Bu4NF,
THF, room temperature, 26% for three steps; (iv) ridinealdoxime, l,l,3,3—
tetramethyl ine, 1,4—dioxane/DMF, 70°C, 70%; (v) POCl3, proton sponge, (MeO)3PO,
00C; (n-BU3NH)2H2P207, n—Bu3N, DMF; 1 M HNEt3HCO3.
DMAP (148 mg, 1.2 mmol) and MsCl (71 uL, 0.9 mmol) were added to a solution of
compound 21 (200 mg, 0.30 mmol) in anhydrous CHzClz (5.0 mL) at 0°C under a nitrogen
atmosphere. The reaction was stirred at 0°C for 10 min and diluted with CHzClz (15 mL).
The solution was applied on a short silica gel plug (2 x 3 cm) and was eluted quickly with
hexane/ethyl e/triethylamine solvent system (volume ratio: 80/20/05). The eluent was
concentrated in vacuo, and the residue was mixed with racemic (R/S)—l—(2—nitrophenyl)—2,2—
dimethyl-l-propanol (500 mg, 2.4 mmol). The mixture was heated at 115°C for 45 min under
a nitrogen atmosphere, cooled to room temperature and then dissolved in THF (10 mL)
followed by addition of n—Bu4NF (283 mg, 0.90 mmol). The mixture was stirred at room
temperature for four hours and then concentrated in vacuo. The residue was dissolved in
CHzClz (25 mL) and washed with brine (25 mL), and the s phase was extracted with
CHzClz (25 mL) two times. The combined organic phase was dried over NaZSO4,
concentrated in vacuo, and the residue was purified by silica gel chromatography to yield 2—
aminochloro[B-D-2’-deoxyribofuranosyl][1-(2-nitrophenyl)-2,2-dimethyl-propyloxy]
methyl—7—deazapurine 25 (40 mg, 26% for three steps) as a 1:1 mixture of two diastereomers.
To a solution of compound 25 (40 mg, 0.08 mmol) in 1,4—dioxane (1.0 mL) and DMF
(2.0 mL), rimidinealdoxime (180 mg, 1.5 mmol) and 1,1,3,3-tetramethyl guanidine
(211 uL, 1.68 mmol) were added. The mixture was heated at 70°C overnight under a
nitrogen atmosphere. The reaction mixture was diluted with CHzClz (20 mL) and washed
sequentially with acetic acid solution (0.1 M, 30 mL), saturated NaHCO3 solution (30 mL),
and brine (30 mL). The c phase was dried over NaZSO4, concentrated in vacuo, and the
residue was purified by silica gel chromatography to yield 7-[1-(2-nitrophenyl)-2,2-dimethyl-
propyloxy]methyldeaza-2’-deoxyguanosine 26 (27 mg, 70%) as a 1:1 mixture of two
diastereomers. 1H NMR (400 MHz, MeOH—d4) for diastereomers: 5 7.79 (m, 1 H, Ph—H),
7.73 (m, 1 H, Ph—H), 7.56 (m, 1 H, Ph—H), 7.39 (m, 1 H, Ph—H), 6.87 and 6.86 (2 s, 1 H, H—8),
6.30 (m, 1 H, H—l’), 4.99 and 4.97 (2 s, 1 H, Ph—CH), 4.63—4.36 (m, 3 H, 7—CH2 and H—3’),
3.91 (m, 1 H, H—4’), 3.69 (m, 2 H, H—5’), 2.48 (m, 1 H, , 2.20 (m, 1 H, H—2’b), 0.79 and
0.77 (2 s, 9 H, (CH3)3).
Compound 26 (25 mg, 0.05 mmol) was phosphorylated with POCl3 (20 uL, 0.21
mmol) and proton sponge (21 mg, 0.1 mmol) in trimethylphosphate (0.35 mL) at 0°C for 3.5
hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate
(237 mg, 0.50 mmol) and tri-n-butylamine (100 uL) in anhydrous DMF (1.0 mL) was added.
After 10 min of stirring, triethylammonium bicarbonate buffer (0.1 M, pH 7.5; 10 mL) was
added. The reaction was stirred at room ature for one hour and then concentrated in
vacuo. The residue was dissolved in 20% aqueous acetonitrile (20 mL), filtered, and purified
by anion exchange chromatography. The fractions containing triphosphate were ed
and lyophilized to give 2-nitrophenyl)-2,2-dimethyl-propyloxy]methyldeaza-2’-
uanosine—5’—triphosphate dG.V.a as a 1:1 e of two diastereomers, which were
separated using RP—HPLC to yield the single diastereomers dG.V.a dsl and dG.V.a ds2.
HRMS (ESI): For the lar ion C23H31N5016P3 [M—H]', the calculated mass was
726.0979, and the observed mass was 726.0992.
7-[(S)-I-(2-Nitr0phenyl)-2, 2-dimethyl—pr0pyloxy]methyl— 7-deaza-2 ’-deoxyguan0sine-
’-triph0sphate
Ho—éjfcl C'
/ \N
I >IKN
TBSO N N/Jx NHTBS TBSO N N/Jx NHTBS
w (i) W (ii) (m)
_> ’_>
OTBS OTBS
02N 02N
t—Bu | t—Bu Me
O 0
/ \N
| / \N
A | A
HO N N H2 HO N N
(iv) NH2 (V) | F0: I _) o —y
OH OH
27 28
02N 02N
t—Bu O t—Bu
OEl 0
/ I r
HO N N/ NH2 HO\ /0\ /o\ ,o N N NH2
0 —>(Vl). O
_ /P\\ /P\\ /P\\
o o o o 'o 0
OH OH
29 dG.V
Scheme 814. Synthesis of 7-[(S)(2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl
deaza-Z'-de0xy-guanosine—5'-triphosphate. Reagents and conditions: (i) MsCl, DMAP,
CHzClz, 0°C; (ii) (S)—1-(2-nitrophenyl)—2,2-dimethylpropanol, 115°C; (iii) n-Bu4NF,
THF, room temperature, 35% for three steps; (iv) NaOMe, MeOH, reflux, 74%; (v) 1,4—
dioxane, 2 M NaOH, reflux, 33%; (vi) POCl3, proton sponge, PO, 0°C; (n—
BU3NH)2H2P207, 3N, DMF; 1 M HNEt3HCO3.
DMAP (224 mg, 1.8 mmol) and MsCl (107 uL, 1.4 mmol) were added to a solution
of compound 21 (300 mg, 0.46 mmol) in ous CHzClz (10 mL) at 0°C under a nitrogen
atmosphere. The reaction was stirred at 0°C for 10 min and diluted with CHzClz (20 mL).
The solution was applied on a short silica gel plug (2 x 3 cm) and was eluted quickly with
hexane/ethyl acetate/triethylamine solvent system (volume ratio 80/20/05). The eluent was
trated in vacuo, and residue was mixed with (S)(2-nitrophenyl)—2,2—dimethyl-1—
propanol (520 mg, 2.5 mmol). The mixture was heated at 115°C for 45 min under a nitrogen
atmosphere, cooled to room ature and dissolved in THF (20 mL) followed by addition
of n-Bu4NF (491 mg, 1.6 mmol). The mixture was stirred at room temperature for four hours
and then concentrated in vacuo. The residue was dissolved in CHzClz (20 mL) and washed
with brine (30 mL), and the aqueous phase was extracted with CHzClz (20 mL) two times.
The combined organic phase was dried over NaZSO4, trated in vacuo, and the residue
was purified by silica gel chromatography to yield 2-aminochloro[[3-D-2’-
deoxyribofuranosyl][(S)(2-nitrophenyl)-2,2-dimethyl-propyloxy]methyldeazapurine
27 (81 mg, 35% for three steps). 1H NMR (400 MHz, MeOH—d4): 5 7.79 (m, 2 H, Ph—H),
7.60 (dt, 1 H, J= 1.2 and 8.0 Hz, Ph—H), 7.46 (dt, 1 H, J: 1.2 and 8.0 Hz, Ph—H), 7.27 (s, 1
H, H—8), 6.47 (dd, 1 H, J= 6.4 and 8.0 Hz, H—1’), 4.98 (s, 1 H, Ph—CH), 4.71 (d, 1 H, J: 12.4
Hz, 7—CH2 a), 4.50 (m, 1 H, H—3’), 4.47 (d, 1 H, J: 12.4 Hz, 7—CH2 b), 3.96 (m, 1 H, H—4’),
3.73 (m, 2 H, H—5’), 2.59 (m, 1 H, H—2’a), 2.30 (m, 1 H, H—2’b), 0.80 (s, 9 H, (CH3)3).
Compound 27 (104 mg, 0.21 mmol) was dissolved in a solution of sodium methoxide
in MeOH (0.5 M, 10 mL), and the mixture was heated to reflux for one hour under a nitrogen
atmosphere. The reaction mixture was cooled to room ature, neutralized with acetic
acid, and then concentrated in vacuo. The residue was purified by silica gel chromatography
to yield 2-aminomethoxy[B-D-2’-deoxyribofuranosyl]—7-[(S)(2-nitrophenyl)-2,2-
dimethyl-propyloxy]methyldeazapurine 28 (75 mg, 74%). 1H NMR (400 MHZ, CDC]3).' 5
7.74 (m, 2 H, Ph—H), 7.52 (t, 1 H, J: 8.0 Hz, Ph—H), 7.36 (t, 1 H, J= 8.0 Hz, Ph—H), 6.71 (s,
1 H, H—8), 6.47 (dd, 1 H, J= 5.6 and 9.6 Hz, H—1’), 5.04 (s, 1 H, Ph—CH), 4.71 (m, 1 H, H—3’),
4.47 (dd, 2 H, J: 12 Hz, 7—CH2 ), 4.15 (m, 1 H, H—4’), 3.94 (s, 3 H, OCH3), 3.76 (m, 2 H, H—
’), 3.01 (m, 1 H, , 2.19 (m, 1 H, H—2’b), 0.82 (s, 9 H, (CH3)3).
Compound 28 (70 mg, 0.14 mmol) was dissolved in oxane (6.0 mL) followed
by addition of an aqueous solution of sodium hydroxide (2 M, 12 mL). The mixture was
heated to reflux for four days under a nitrogen atmosphere, cooled to room temperature,
neutralized with dilute hydrochloric acid (1 M), and trated in vacuo. The residue was
evaporated from MeOH (5.0 mL) three times and then purified by silica gel chromatography
to yield 7-[(S)—1-(2-nitrophenyl)-2,2-dimethyl-propyloxy]methyldeaza-2’-deoxyguanosine
29 (22 mg, 33%). Starting material 28 (42 mg, 60%) was also recovered from the on.
IHNMR (400 MHz, CDCZ3): 5 11.02 (br s, 1 H, NH), 7.69 (m, 2 H, Ph—H), 7.52 (t, 1 H, J:
7.2 Hz, Ph—H), 7.33 (t, 1 H, J: 7.2 Hz, Ph—H), 6.66 (s, 1 H, H—8), 6.13 (t, 1 H, J= 6.8 Hz, H—
1’), 6.03 (br s, 2 H, 6—NH2), 4.92 (s, 1 H, Ph—CH), 4.77 (m, 1 H, H—3’), 4.57 (d, 1 H, J: 12.8
Hz, 7—CH2 a), 4.12 (m, 1 H, H—4’), 3.05 (d, 1 H, J: 12.8 Hz, 7—CH2 b), 3.75 (m, 2 H, H—5’),
2.87 (m, 1 H, H—2’a), 2.29 (m, 1 H, H—2’b), 0.76 (s, 9 H, ).
Compound 29 (16 mg, 0.033 mmol) was phosphorylated with POCl3 (17 uL, 0.18
mmol) and proton sponge (14 mg, 0.066 mmol) in trimethylphosphate (0.35 mL) at 0°C for
four hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium
pyrophosphate (237 mg, 0.50 mmol) and butylamine (100 uL) in anhydrous DMF (1.0
mL) was added. After 10 min of stirring, triethylammonium bicarbonate buffer (0.1 M, pH
7.5; 10 mL) was added. The reaction was stirred at room temperature for one hour and then
concentrated in vacuo. The residue was dissolved in 20% aqueous acetonitrile (20 mL),
filtered, and purified by anion exchange chromatography. The fractions containing
triphosphate were ed and lyophilized to give 7-[(S)(2-nitrophenyl)-2,2-dimethyl-
propyloxy]methyldeaza-2’-deoxyguanosine-5’-triphosphate dG.V, which was further
purified using RP-HPLC ions. The ion time of dG.V was identical to that of
dG.V.a ds2 by RP—HPLC analysis using the same condition (data not shown). HRMS (ESI):
For the molecular ion C23H31N5016P3 [M—H]', the calculated mass was 726.0979, and the
observed mass was 726.0986.
7-[1-(4-Meth0xynitrophenyD-Z, 2-dimethyl—pr0pyloxy]methyl- 7-deaza-2 ’-
deoxyguanosine-5 ’-triph0sphate
HOB? C'
/ \N
| %N
TBSO N NA NHTBS TBSO N N/JxNHTBS
W (i) (ii) (m)
—> W ’—>
OTBS OTBS
OMe OMe
OZN OZN
t—Bu CI t—Bu O
0 O
/ \N
| A / | ANH
HO N NH2 HO N N
(iv) NH2
OH OH
31
/ NH
HO\ /O\ /O\ /O N N NH2
_ /F{\ R\ /P\\
O _
O O O O O
dG.V.b
Scheme SIS. sis of 7-[1-(4-methoxynitrophenyl)-2,2-dimethyl-
propyloxy]methyldeaza-2'-deoxyguan0sine—5'-triph0sphate. Reagents and conditions:
(i) MsCl, DMAP, CHzClz, 0°C; (ii) racemic (R/S)—1—(4—methoxy—2—nitrophenyl)-2,2—
dimethyl-l-propanol, 115°C; (iii) n-Bu4NF, THF, room temperature, 21% for three steps;
(iv) syn-pyridine-Z-aldoxime, l,l,3,3-tetramethyl guanidine, 1,4-dioxane/DMF, 70°C, 59%;
(v) POCl3, proton sponge, (MeO)3PO, 0°C; (n—Bu3NH)2H2PZO7, n-Bu3N, DMF; 1 M
CO3.
DMAP (346 mg, 2.8 mmol) and MsCl (165 uL, 2.1 mmol) were added to a solution
of compound 21 (470 mg, 0.72 mmol) in anhydrous CHzClz (5.0 mL) at 0°C under a nitrogen
atmosphere. The reaction was stirred at 0°C for 10 min and diluted with CHzClz (20 mL).
The on was applied on a short silica gel plug (2 x 3 cm) and was eluted quickly with
hexane/ethyl e/triethylamine solvent system e ratio 80/20/05). The eluent was
concentrated in vacuo, and the residue was mixed with racemic (R/S)—1-(4-methoxy—2—
nitrophenyl)-2,2-dimethylpropanol (1.6 g, 6.69 mmol). The mixture was heated to 115°C
for 45 min under a nitrogen atmosphere, cooled to room temperature, and dissolved in THF
(20 mL) followed by addition of n-Bu4NF (788 mg, 2.5 mmol). The mixture was stirred at
room temperature for four hours and then concentrated in vacuo. The residue was dissolved
in CHzClz (20 mL) and washed with brine (30 mL), and the aqueous phase was extracted
with CHzClz (20 mL) two times. The combined organic phase was dried over NaZSO4,
concentrated in vacuo, and the residue was purified by silica gel chromatography to yield 2—
amino—6-chloro[B-D-2’-deoxyribofuranosyl][(S)—1-(4-methoxynitrophenyl)-2,2-
dimethyl-propyloxy]-methyldeazapurine 30 (80 mg, 21% for three steps) as a 1:1 mixture
of two diastereomers.
To a solution of compound 30 (80 mg, 0.15 mmol) in 1,4—dioxane (1.0 mL) and DMF
(2.0 mL), syn-pyrimidinealdoxime (366 mg, 3.0 mmol) and 1,1,3,3-tetramethyl guanidine
(414 ”L, 3.3 mmol) were added, and the mixture was heated at 70°C overnight under a
nitrogen atmosphere. The reaction mixture was diluted with CHzClz (20 mL) and washed
sequentially with acetic acid (0.1 M, 30 mL), saturated NaHCO3 solution (30 mL), and brine
(30 mL). The c phase was dried over Na2SO4, concentrated in vacuo, and the residue
was purified by silica gel chromatography to yield 7-[(S)(4-methoxynitrophenyl)-2,2-
dimethyl-propyloxy]methyldeaza-2’-deoxy-guanosine 31 (45 mg, 59%) as a 1:1 mixture of
two diastereomers. 1H NMR (400 MHz, DMSO-dg) for diastereomers: 5 10.31 (br s, 1 H,
D20 exchangeable, NH), 7.63 and 7.62 (2 d, 1 H, J = 2,8 Hz, Ph—H), 7.41 and 7.40 (2 d, 1 H,
J: 2,8 Hz, Ph—H), 7.27 (m, 1 H, Ph—H), 6.98 and 6.96 (2 s, 1 H, H—8), 6.28 (m, 1 H, H—1’),
6.22 (br s, 2 H, D20 exchangeable, NHz), 5.22 (d, 1 H, D20 exchangeable, 3’-OH), 4.88 (t, 1
H, D20 exchangeable, 5’-OH), 4.73 and 4.71 (2 s, 1 H, Ph-CH), .24 (m, 3 H, 7-CH2
and H—3’), 3.85 and 3.83 (2 s, 3 H, OCH3), 3.74 (m, 1 H, H—4’), 3.48 (m, 2 H, H—5’), 2.28 (m,
1 H, H—2’a), 2.06 (m, 1 H, H—2’b), 0.80 and 0.78 (2 s, 9 H, (CH3)3).
nd 31 (25 mg, 0.048 mmol) was orylated with POCl3 (15 uL, 0.18
mmol) and proton sponge (21 mg, 0.10 mmol) in trimethylphosphate (0.35 mL) at 0°C for 3.5
hours under a nitrogen atmosphere. A on of bis-tri-n-butylammonium pyrophosphate
(237 mg, 0.50 mmol) and butylamine (100 uL) in anhydrous DMF (1.0 mL) was added.
After 10 min of ng, triethylammonium bicarbonate buffer (0.1 M, pH 7.5; 10 mL) was
added. The reaction was stirred at room temperature for one hour and then concentrated in
vacuo. The residue was dissolved in 20% aqueous acetonitrile (20 mL), filtered, and purified
by anion exchange chromatography. The fractions containing triphosphate were combined
and lyophilized to give 7-[l-(4-methoxynitrophenyl)-2,2-dimethyl-propyloxy]methyl
deaza—2’-deoxyguanosine—5’—triphosphate dG.V.b as a 1:1 mixture of two diastereomers,
which were separated using RP—HPLC to yield the single diastereomers dG.V.b dsl and
dG.V.b ds2. HRMS (ESI): For the molecular ion N5017P3 [M—H]', the calculated
mass was 756.1084, and the observed mass was 01.
7-[I-(5-Meth0xynitrophenyD-Z, 2-dimethyl—pr0pyloxy]methyl- 7-deaza-2 ’-
deoxyguanosine-5 ’-triph0sphate
:>1N1>1”1CICI
TBSOw NHTBS TBSO N N NHTBS
()i (“)0“)
OTBS OTBS
OMe OMe
02N 02N
t—Bu CI t—Bu O
0 O
/ |\N / I NH
A A
HO N N NH2 HO N N
ioj (iv) NH2
—> lio>l —)
OH OH
32 33
t—Bu O
HO\P/O\P/O\’P\\/O O>N1N”1HNH
_O’P\>) DIE-103'
dG.V.c
Scheme 816. Synthesis of 7-[1-(5-methoxy—2-nitrophenyl)-2,2-dimethyl—
oxy]methyldeaza-2'-deoxyguan0sine—5'-triph0sphate. ts and conditions:
(i) MsCl, DMAP, CHzClz, 0°C; (ii) racemic (R/S)—1—(5—methoxy—2—nitrophenyl)-2,2—
dimethyl-l-propanol, 115°C; (iii) n-Bu4NF, THF, room temperature, 24% for three steps;
(iv) syn-pyridinealdoxime, l,l,3,3-tetramethyl guanidine, 1,4-dioxane/DMF, 70°C, 57%;
(v) POCl3, proton sponge, (MeO)3PO, 0°C; (n—Bu3NH)2H2PZO7, n-Bu3N, DMF; 1 M
HNEt3HCO3.
DMAP (302 mg, 2.5 mmol) and MsCl (145 uL, 1.9 mmol) were added to a solution
of compound 21 (410 mg, 0.62 mmol) in anhydrous CHzClz (5.0 mL) at 0°C under a nitrogen
here. The reaction was stirred at 0°C for 10 min and d with CHzClz (20 mL).
The solution was applied on a short silica gel plug (2 x 3 cm) and was eluted quickly with a
hexane/ethyl acetate/triethylamine solvent system (volume ratio: 80/20/05). The eluent was
concentrated in vacuo, and residue was mixed with c (R/S)(5-methoxy—2—
henyl)-2,2-dimethylpropanol (800 mg, 2.2 mmol). The mixture was heated at 115°C
for 45 min under a nitrogen atmosphere, cooled to room temperature, and dissolved in THF
(10 mL) followed by addition of n—Bu4NF (683 mg, 3.3 mmol). The mixture was stirred at
room temperature for four hours and then concentrated in vacuo. The residue was ved
in CHzClz (20 mL) and washed with brine (30 mL), and the aqueous phase was ted
with CHzClz (20 mL) two times. The combined organic phase was dried over NaZSO4,
concentrated in vacuo, and the residue was purified by silica gel chromatography to yield 2—
amino—6-chloro[B-D-2 ’-deoxyribofuranosyl] [ 1 -(5 -methoxynitrophenyl)-2,2-
dimethyl-propyloxy]methyldeazapurine 32 (80 mg, 24% for three steps) as a 1:1 e
of two diastereomers. 1HNMR (400 MHz, CDCZ3) for diastereomers: 5 7.86 and 7.83 (2 d, 1
H, J: 8.8 Hz, Ph—H), 7.19 and 7.17 (2 d, 1 H, J: 2.8 Hz, Ph—H), 6.91 and 6.90 (2 s, 1 H, H—
8), 6.80 and 6.75 (2 dd, 1 H, J: .8 and 8.8 Hz, Ph—H), 6.17 (m, 1 H, H—1’), 5.23 and 5.21 (2 s,
1 H, Ph—CH), 5.01 and 5.00 (2 br s, 2 H, NHz), 4.73 (m, 1 H, H—3’), 4.65—4.49 (m, 2 H, 7—
CH2), 4.14 (m, 1 H, H—4’), 3.84 (m, 5 H, H—5’ and OCH3), 2.78 (m, 1 H, H—2’a), 2.33 (m, 1 H,
H—2’b), 0.82 and 0.81 (2 s, 9 H, (CH3)3).
To a solution of compound 32 (80 mg, 0.15 mmol) in oxane (1.0 mL) and DMF
(2.0 mL), syn-pyrimidinealdoxime (360 mg, 3.0 mmol) and 1,1,3,3-tetramethyl guanidine
(414 ”L, 3.3 mmol) were added, and the mixture was heated at 70°C ght under a
nitrogen atmosphere. The reaction mixture was diluted with CHzClz (20 mL) and washed
sequentially with acetic acid (0.1 M, 30 mL), saturated NaHCO3 solution (30 mL), and brine
(30 mL). The organic phase was dried over NaZSO4, concentrated in vacuo, and the residue
was purified by silica gel chromatography to yield 7-[1-(5-methoxynitrophenyl)—2,2-
dimethyl-propyloxy]methyldeaza-2’-deoxyguanosine 33 (43 mg, 57%) as a 1:1 mixture of
two diastereomers. 1H NMR (400 MHz, DMSO-dg) for diastereomers: 5 10.34 (br s, 1 H,
D20 exchangeable, NH), 7.92 and 7.89 (2 d, 1 H, J = 8,8 Hz, Ph—H), 7.15 (m, 1 H, Ph—H),
6.95 (m, 1 H, Ph—H), 6.82 and 6.81 (2 s, 1 H, H-8), 6.22 (m, 3 H, 2 H D20 exchangeable, H-1’
and NHz), 5.19 (d, 1 H, D20 exchangeable, , 5.12 and 5.10 (2 s, 1 H, Ph-CH), 4.84 (t,
1 H, D20 exchangeable, 5’-OH), 4.47-4.31 (m, 2 H, 7-CH2), 4.24 (m, 1 H, H-3’), 3.85 and
3.83 (2 s, 3 H, OCH3), 3.71 (m, 1 H, H—4’), 3.44 (m, 2 H, H—5’), 2.24 (m, 1 H, H—2’a), 2.01
(m, 1 H, H—2’b), 0.76 and 0.74 (2 s, 9 H, (CH3)3).
Compound 33 (20 mg, 0.04 mmol) was phosphorylated with POCl3 (25 uL, 0.27
mmol) and proton sponge (16 mg, 0.08 mmol) in trimethylphosphate (0.30 mL) at 0°C for six
hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate
(237 mg, 0.50 mmol) and tri-n-butylamine (100 uL) in ous DMF (1.0 mL) was added.
After 10 min of stirring, triethylammonium bicarbonate buffer (0.1 M, pH 7.5; 10 mL) was
added. The reaction was stirred at room temperature for one hour and then concentrated in
vacuo. The residue was dissolved in 20% aqueous acetonitrile (20 mL), filtered, and purified
by anion exchange chromatography. The fractions ning triphosphate were combined
and lyophilized to give 5-methoxynitrophenyl)-2,2-dimethyl-propyloxy]methyl
deaza—2’-deoxyguanosine—5’—triphosphate dG.V.c as a 1:1 mixture of two diastereomers,
which were separated using RP-HPLC to yield the single diastereomers dG.V.c dsl and
dG.V.c ds2. HRMS (ESI): For the molecular ion C24H33N5017P3 [M-H]', the calculated mass
was 756.1084, and the observed mass was 756.1088.
7-[I-(4, 5-Dimeth0xynitr0phenyl)-2, 2-dimethyl—pr0pyloxy]methyl— 7-deaza-2 ’-
deoxyguanosine-5 h0sphate
:5*l CI
CI5*1
TBSOw NHTBS—>TBSO N N NHTBS
(. (ii) (iii)
OTBS OTBS
OMe OMe
OMe OMe
t—Bu
\N / | ANH *NHZ HO N N
(iv) NH2
O 6
OH OH
34 35
t—Bu O
O5:rNH2
HO\P/O\JD/O\/P\\/O
_O/\\_O\B _ 0|;03|
dG.V.d
Scheme 817. Synthesis of 7-[1-(4,5-dimethoxy—2-nitrophenyl)-2,2-dimethylpropyloxy
ldeaza-2'-deoxyguan0sine—5'-triph0sphate. ts and conditions;
(i) MsCl, DMAP, CHzClz, 0°C; (ii) racemic (R/S)-l-(4,5-dimethoxynitrophenyl)-2,2-
dimethyl—l-propanol, 115°C; (iii) n—Bu4NF, THF, room ature, 23% for three steps; (iv)
syn-pyridinealdoxime, l,l,3,3-tetramethyl guanidine, dioxane/DMF, 70°C, 68%; (v)
POCl3, proton sponge, (MeO)3PO, 0°C; (n—Bu3NH)2H2P207, n-Bu3N, DMF; l M
HNEt3HCO3.
DMAP (273 mg, 2.2 mmol) and MsCl (130 uL, 1.7 mmol) were added to a solution
of compound 21 (370 mg, 0.56 mmol) in anhydrous CHzClz (5.0 mL) at 0°C under a nitrogen
atmosphere. The reaction was stirred at 0°C for 30 min and diluted with CHzClz (25 mL).
The solution was applied on a short silica gel plug (2 x 3 cm) and was eluted quickly with a
hexane/ethyl acetate/triethylamine solvent system (volume ratio: 80/20/05). The eluent was
trated in vacuo, and the residue was mixed with racemic (R/S)(4,5—dimethoxy—2—
nitrophenyl)-2,2-dimethyl-l-propanol (800 mg, 3.0 mmol). The mixture was heated at 115°C
for 45 min under a nitrogen atmosphere, cooled to room temperature and dissolved in THF
(10 mL) followed by addition of n-Bu4NF (530 mg, 1.7 mmol). The mixture was stirred at
room ature for two hours and then concentrated in vacuo. The residue was dissolved
in CHzClz (40 mL) and washed with brine (50 mL), and the aqueous phase was extracted
with CHzClz (40 mL) two times. The combined organic phase was dried over NaZSO4 and
concentrated in vacuo, and the residue was purified by silica gel chromatography to yield 2—
amino—6-chloro[B-D-2’-deoxyribofuranosyl][1-(4,5-dimethoxynitrophenyl)-2,2-
yl-propyloxy]methyldeazapurine 34 (70 mg, 23% for three steps) as a 1:1 mixture
of two diastereomers. 1H NMR (400 MHz, CDCZ3) for diastereomers: 5 7.42 and 7.39 (2 s, 1
H, Ph—H), 7.15 and 7.13 (2 s, 1 H, Ph—H), 6.89 and 6.84 (2 s, 1 H, H—8), 6.12 (m, 1 H, H—1’),
.22 and 5.16 (2 s, 1 H, Ph—CH), 5.10 and 5.08 (2 bs, 2 H, NHZ), 4.71—4.41 (m, 3 H, H—3’and
7—CH2), 4.13 (m, 1 H, H—4’), 3.94 (4 s, 7 H, OCH3 x 2 and H—5’a), 3. 78 (m, 1 H, H—5’b), 2.90
(m, 1 H, H—2’a), 2.25 (m, 1 H, H—2’b), 0.82 and 0.80 (2 s, 9 H, (CH3)3).
To a solution of compound 34 (65 mg, 0.11 mmol) in 1,4—dioxane (1.0 mL) and DMF
(2.0 mL), syn-pyrimidinealdoxime (292 mg, 2.4 mmol) and 1,1,3,3-tetramethyl guanidine
(330 uL, 2.6 mmol) were added, and the mixture was heated at 70 °C overnight under a
nitrogen atmosphere. The reaction mixture was diluted with CHzClz (40 mL) and washed
sequentially with acetic acid (0.1 M, 50 mL), saturated NaHCO3 solution (50 mL), and brine
(50 mL). The organic phase was dried over NaZSO4, concentrated in vacuo, and the residue
was purified by silica gel chromatography to yield 7-[1-(4,5-dimethoxynitrophenyl)-2,2-
dimethyl-propyloxy]methyldeaza-2’-deoxyguanosine 35 (42 mg, 68%) as a 1:1 e of
two diastereomers. 1H NMR (400 MHz, DMSO-dg) for diastereomers: 5 10.33 (br s, 1 H,
D20 exchangeable, NH), 7.47 and 7.44 (2 s, 1 H, Ph-H), 7.16 and 7.15 (2 s, 1 H, Ph-H), 6.83
and 6.82 (2 s, 1 H, H-8), 6.22 (m, 3 H, 2 H D20 exchangeable, NHz and H-l’), 5.18 (br s, 1
H, D20 exchangeable, 3’—OH), 5.06 and 5.04 (2 s, 1 H, Ph—CH), 4.83 (t, 1 H, D20
exchangeable, 5’-OH), 4.44-4.23 (m, 3 H, 7-CH2 and H—3’), 3.82 (4 s, 6 H, OCH3 x 2), 3.70
(m, 1 H, H—4’), 3.42 (m, 2 H, H—5’), 2.22 (m, 1 H, H—2’a), 2.01 (m, 1 H, , 0.77 and 0.75
(2 s, 9 H, (CH3)3).
nd 35 (40 mg, 0.073 mmol) was phosphorylated with POCl3 (14 uL, 0.15
mmol) and proton sponge (31 mg, 0.15 mmol) in trimethylphosphate (0.35 mL) at 0°C for
two hours under a nitrogen atmosphere. A on of bis-tri-n-butylammonium
pyrophosphate (237 mg, 0.50 mmol) and tri-n-butylamine (100 uL) in anhydrous DMF (1.0
mL) was added. After 10 min of stirring, triethylammonium bicarbonate buffer (0.1 M, pH
7.5; 10 mL) was added. The reaction was stirred at room temperature for one hour and then
concentrated in vacuo. The residue was dissolved in 20% aqueous acetonitrile (20 mL),
filtered, and purified by anion exchange chromatography. The fractions containing
triphosphate were combined and lyophilized to give 7-[1-(4,5-dimethoxynitrophenyl)-2,2-
dimethyl-propyloxy]methyldeaza-2’-deoxyguanosine-5’-triphosphate dG.V.d as a 1:1
e of two diastereomers, which were ted using RP-HPLC to yield the single
diastereomers dG.V.d dsl and dG.V.d ds2. HRMS (ESI): For the molecular ion
N5018P3 [M-H]', the calculated mass was 786.1190, and the observed mass was
786.1206.
7-[(S)-I-(5-Meth0xynitrophenyD-Z,2-dimethyl—pr0pyloxy]methyl— a-2 ’-
uanosine-5 ’-triph0sphate
HOMO! C'
/ IN %N
TBSO N NANHTBS TBSO N N/Jx NHTBS
k5 (i) (ii) (in)
—, k5 ’—>
OTBS OTBS
OMe OMe
OZN OZN
t—Bu | t—Bu
O O
/ \N
| A / | ANH
HO N N NH2 N
I (IV).
HO N NH2
1 (v)
o 0
—) —>
OH OH
36 37
t—Bu 0
ON13
HO\ /O\ /O\P/O N N/ NH2
/\\ O
_ /P\\ /P\\
O O O O _ O O K 9
dG.VI
Scheme 818. Synthesis of 7-[(S)(5-methoxynitrophenyl)-2,2-dimethyl-
propyloxy]methyldeaza- 2'-deoxyguan0sine—5'-triph0sphate. Reagents and conditions:
(i) MsCl, DMAP, CHzClz, 0°C; (ii) (5—methoxynitrophenyl)-2,2-dimethyl-l-
propanol, 115°C; (iii) n—Bu4NF, THF, room temperature, 27% for three steps; (iv) syn—
pyridinealdoxime, l,l,3,3-tetramethyl guanidine, 1,4-dioxane/DMF, 70°C, 76%; (v)
POCl3, proton sponge, (MeO)3PO, 0°C; (n—Bu3NH)2H2PZO7, n-Bu3N, DMF; 1 M
HNEt3HCO3.
DMAP (224 mg, 1.8 mmol) and MsCl (106 uL, 1.4 mmol) were added to a solution
of compound 21 (300 mg, 0.46 mmol) in anhydrous CHzClz (5.0 mL) at 0°C under a nitrogen
atmosphere. The reaction was stirred at 0°C for 10 min and diluted with CHzClz (20 mL).
The solution was applied on a short silica gel plug (2 x 3 cm) and was eluted quickly with a
hexane/ethyl e/triethylamine solvent system (volume ratio: 80/20/05). The eluent was
concentrated in vacuo, and residue was mixed with (S)—1—(5—methoxy—2—nitrophenyl)—2,2-
W0 2013/040257
dimethyl-l-propanol (500 mg, 2.1 mmol). The mixture was heated at 115°C for 45 min under
a nitrogen atmosphere, cooled to room temperature and dissolved in THF (10 mL) followed
by addition of n—Bu4NF (507 mg, 1.6 mmol). The mixture was stirred at room temperature
for four hours and then concentrated in vacuo. The residue was dissolved in CH2Cl2 (20 mL)
and washed with brine (30 mL), and the aqueous phase was extracted with CH2Cl2 (20 mL)
two times. The combined c phase was dried over Na2SO4, concentrated in vacuo, and
the residue was purified by silica gel chromatography to yield 2-aminochloro[[3-D-2’-
deoxyribofuranosyl][(S)(5-methoxynitrophenyl)-2,2-dimethyl-propyloxy]-methyl-7
deazapurine 36 (67 mg, 27% for three steps). 1H NMR (400 MHz, CDCZ3).' 5 7.82 (d, 1 H, J
= 8.8 Hz, Ph—H), 7.16 (d, 1 H, J= 2.8 Hz, Ph—H), 6.90 (s, 1 H, H—8), 6.72 (dd, 1 H, J= 8.8
and 2.8 Hz, Ph—H), 6.12 (dd, 1 H, J: 9.2 and 6.0 Hz, H—1’), 5.22 (s, 1 H, Ph—CH), 5.15 (br s,
2 H, NH2), 4.69—4.55 (m, 3 H, H—3’and 7—CH2), 4.11 (m, 1 H, H—4’), 3.92 (m, 1 H, H—5’a),
3.82 (s, 3 H, OCH3), 3. 73 (m, 1 H, H—5’b), 2.81 (m, 1 H, H—2’a), 2.21 (m, 1 H, H—2’b), 0.82
(s, 9 H, ).
To a on of compound 36 (65 mg, 0.12 mmol) in 1,4—dioxane (1.0 mL) and DMF
(2.0 mL), syn-pyrimidinealdoxime (292 mg, 2.4 mmol) and 1,1,3,3-tetramethyl guanidine
(331 uL, 2.6 mmol) were added, and the mixture was heated at 70°C overnight under a
nitrogen atmosphere. The reaction mixture was diluted with CH2Cl2 (20 mL) and washed
tially with acetic acid (0.1 M, 30 mL), saturated NaHCO3 solution (30 mL), and brine
(30 mL). The organic phase was dried over Na2SO4, trated in vacuo, and the residue
was purified by silica gel chromatography to yield 7-[(S)(5-methoxynitrophenyl)-2,2-
dimethyl—propyloxy]methyl—7—deaza—2’-deoxyguanosine 37 (48 mg, 76%). 1H NMR (400
MHZ, g): 5 10.37 (br s, 1 H, D2O exchangeable, NH), 7.95 (d, 1 H, J = 9.2 Hz, Ph-
H), 7.18 (d, 1 H, J= 2.8 Hz, Ph—H), 7.03 (dd, 1 H, J: 9.2 and 2.8 Hz, Ph—H), 6.84 (s, 1 H, H—
8), 6.23 (m, 3 H, 2 H D2O geable, NH2 and H-l’), 5.20 (d, 1 H, D2O exchangeable, 3’-
OH), 5.13 (s, 1 H, Ph-CH), 4.84 (t, 1 H, D2O exchangeable, 5’-OH), 4.48 (d, 1 H, J: 12.0
Hz, 7—CH2a), 4.32 (d, 1 H, J: 12.0 Hz, 7—CH2b), 4.27 (m, 1 H, H—3’), 3.88 (s, 3 H, OCH3),
3.73 (m, 1 H, H—4’), 3.46 (m, 2 H, H—5’), 2.30 (m, 1 H, H—2’a), 2.05 (m, 1 H, H—2’b), 0.77 (s, 9
H, (CH3)3).
Compound 37 (10 mg, 0.02 mmol) was phosphorylated with POCl3 (26 uL, 0.26
mmol) and proton sponge (8 mg, 0.04 mmol) in trimethylphosphate (0.3 mL) at 0°C for 6.5
hours under a en atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate
(237 mg, 0.50 mmol) and tri-n-butylamine (100 uL) in anhydrous DMF (1.0 mL) was added.
WO 40257
After 10 min of stirring, triethylammonium bicarbonate buffer (0.1 M, pH 7.5; 10 mL) was
added. The reaction was stirred at room temperature for one hour and then concentrated in
vacuo. The residue was dissolved in 20% aqueous acetonitrile (10 mL), filtered, and purified
by anion exchange chromatography. The fractions containing sphate were combined
and lyophilized to give 7-[(S)—1-(5-methoxynitrophenyl)-2,2-dimethyl-propyloxy]methyl-
7—deaza—2’—deoxyguanosine-5’-triphosphate dG.VI, which was further purified using RP-
HPLC. The retention time of dG.VI was identical to that of dG.V.c ds2 by RP—HPLC
analysis under the same condition. HRMS (ESI): For the molecular ion C24H33N5017P3 [M-
H]', the calculated mass was 756.1084, and the observed mass was 01.
Example 5 — Synthesis of 5-HOMe—2'-De0xyuridine sphate Analog
-[(S)-I-(5-Meth0xynitrophenyD-Z, 2-dimethyl—pr0pyloxy]methyl-2 ’-de0xyuridine-
’-triph0sphate
WE WE1H
A N o
TBSO N O H0
(i) (ii) (in) HO\P/O\P/o\P/O2:31:
OTBS OH OH
38 39 dU.VI
Scheme S19. Synthesis of 5-[(S)(5-methoxynitrophenyl)-2,2-dimethyl—
propyloxy]methyl-2'-deoxyuridine-5'-triph0sphate. Reagents and conditions: (i) (5—
methoxynitrophenyl)-2,2-dimethylpropanol, 110°C; (ii) NH4F, MeOH, 50°C, 56% for
two steps; (iii) POCl3, proton sponge, (MeO)3PO, 0°C; (n—Bu3NH)2H2PZO7, n—Bu3N, DMF; 1
M HNEt3HCO3.
Compound 38 (Litosh et al., 2011, which is incorporated herein by reference) (315
mg, 0.49 mmol) and (S)(5-methoxynitrophenyl)-2,2-dimethylpropanol (490 mg, 2.1
mmol) were heated at 110°C for 45 min under a nitrogen atmosphere. The mixture was
cooled down to room temperature, dissolved in MeOH (10 mL), and ed by addition of
NH4F (400 mg, 11 mmol). The e was stirred at 50°C for 12 hours, concentrated in
vacuo, ved in CHzClz (50 mL), and washed with brine (50 mL). The organic phase was
dried over NaZSO4, concentrated in vacuo, and the residue was purified by silica gel
chromatography to yield 5 -[(S)(5-methoxynitrophenyl)-2,2-dimethyl-
propyloxy]methyl-2’-deoxyuridine 39 (130 mg, 56%). 1H NMR (400 MHZ, CDC]3).' 5 9.14
(br s, 1 H, NH), 7.90 (d, 1 H, J= 9.2 Hz, Ph—H), 7.67 (s, 1 H, H—6), 7.17 (d, 1 H, J: 2.8 Hz,
Ph—H), 6.84 (dd, 1 H, J: 9.2 and 2.8 Hz, Ph—H), 6.18 (t, 1 H, J: 6.4 Hz, H—1’), 5.22 (s, 1 H,
Ph—CH), 4.56 (m, 1 H, H—3’), 4.24 (d, 1 H, J: 12.4 Hz, 5—CH2a), 4.15 (d, 1 H, J: 12.4 Hz, 5—
Csz), 4.00 (m, 1 H, H—4’), 3.90 (m, 1 H, H—5’a), 3.88 (s, 3 H, OCH3), 3.81 (m, 1 H, H—5’b),
2.35 (m, 2 H, H—2), 0.83 (s, 9 H, C(CH3)3).
Compound 39 (30 mg, 0.065 mmol) was phosphorylated with POCl3 (9 uL, 0.097
mmol) and proton sponge (28 mg, 0.13 mmol) in trimethylphosphate (0.35 mL) at 0°C for
one hour under a nitrogen atmosphere. A solution of tri-n-butylammonium pyrophosphate
(147 mg, 0.32 mmol) and tri-n-butylamine (64 uL) in anhydrous DMF (0.64 mL) was added.
After 10 min of stirring, ylammonium bicarbonate buffer (0.1 M, pH 7.5; 10 mL) was
added. The reaction was stirred at room ature for one hour and then trated in
vacuo. The e was dissolved in 20% aqueous acetonitrile (10 mL), filtered, and purified
by anion exchange chromatography. The fractions containing triphosphate were combined
and lyophilized to yield 5-[(S)—1-(5-methoxynitrophenyl)-2,2-dimethyl-propyloxy]methyl-
2’—deoxyuridine-5’-triphosphate dU.VI, which was further purified using RP-HPLC. HRMS
(ESI): For the molecular ion C22H31N3018P3 [M—H]', the calculated mass was 718.0815, and
the observed mass was 718.0824.
2012/055231
e 6 — sis of 5—HOMe-2'-De0xycytidine Triphosphate Analogs
-(2-nitr0benzyloxy)methyl-2 ’-deoxycytidine-5 ’-triph0sphate
kitNBoc
NOAELNH o I [Ii-l
TBSO RO N O HO N 0
7d "”..
—> 7d
OTBS OR OH
R = H or TBS 40
i— Pr
O\'gpI-PIM
OAELNIIOI—b 02N /\fi\I-P
TBso,
(III) OI :6“):
TBsow
OTBS OTBS
OZN NH2 OZN NH2
I\N HOD/\fiNO O | \l
N 0
TBSO HO\ /O\ /O\P/O
(vi) (W) F{\ Rx /P\\ O
—"0 0’0 0 o 0
OT BS OH OH
43 dC.|
Scheme S20. Synthesis of 5—(2-nitrobenzyloxy)methyl—2'-deoxycytidine—5'-triphosphate.
Reagents and conditions: (i) 2—nitrobenzyl alcohol, 110°C; (ii) n—Bu4NF, THF, room
temperature, 53% for two steps; (iii) TBSCl, imidazole, DMF, room temperature, 80%; (iv)
2,4,6-triisopropylbenzenesulfonyl chloride, DMAP, Et3N, CHzClz, room temperature; (v)
NH3, 1,4—dioxane, 90°C, 69% for two steps; (vi) F, THF, room temperature, 96%;
(vii) POCl3, proton sponge, (MeO)3PO, 0°C; (n—Bu3NH)2H2PZO7, n—Bu3N, DMF; 1 M
HNEt3HCO3.
Compound 38 (300 mg, 0.46 mmol) and obenzyl alcohol (500 mg, 3.3 mmol)
were heated at 110°C for 45 min under a nitrogen atmosphere. The mixture was cooled to
room temperature, dissolved in THF (20 mL) followed by addition of n-Bu4NF (362 mg, 1.2
mmol). The mixture was stirred at room temperature for four hours, concentrated in vacuo,
and the residue was purified by silica gel chromatography to yield 5-(2-
nitrobenzyloxy)methyl-2’-deoxyuridine 40 (Litosh et al., 2011, which is incorporated herein
by reference) (95 mg, 53%). IHNMR (400 MHz, CDCZ3).' 5 8.45 (br s, 1 H, NH), 8.05 (s, 1
H, H—6), 8.02 (d, J = 8.0 Hz, 1 H, Ph—H), 7.80 (d, 1 H, J = 8.0 Hz, Ph—H), 7.69 (t, 1 H, J =
8.0 Hz, Ph-H), 7.43 (t, 1 H, J = 8.0 Hz, Ph-H), 6.21 (t, 1 H, J = 6.0 Hz, H-1’), 4.94 (dd, J=
14.4 Hz, 2 H, Ph-CHZ), 4.66 (m, 1 H, H-3’), 4.35 (s, 2 H, , 3.95 (m, 3 H, H—4’ and H—
’), 2.42 (m, 1 H, H-2’a), 2.30 (m, 1 H, H-2’b).
To a solution of nd 40 (Litosh et al., 2011, which is incorporated herein by
reference) (70 mg, 0.18 mmol) in anhydrous DMF (2.0 mL), TBSCl (60 mg, 0.40 mmol) and
imidazole (54 mg, 0.80 mmol) were added. The mixture was stirred at room temperature
overnight under a nitrogen atmosphere, concentrated in vacuo, dissolved in CHzClz (20 mL),
and washed with saturated NaHCO3 on (30 mL). The organic and aqueous phases were
separated, and the aqueous phase was extracted with CHzClz (20 mL) two times. The
combined organic phase was dried with NaZSO4, concentrated in vacuo, and the e was
purified by silica gel chromatography to yield 3’,5’-0—bis-(tert—butyldimethylsilyl)(2-
nitrobenzyloxy)-methyl-2’-deoxyuridine 41 (90 mg, 80%). 1H NMR (400 MHZ, CDC]3).' 5
8.04 (d, J= 8.0 Hz, 1 H, Ph—H), 7.98 (br s, 1 H, NH), 7.80 (d, 1 H, J= 8.0 Hz, Ph—H), 7.74 (s,
1 H, H-6), 7.64 (q, 1 H, J: 8.0 Hz, Ph-H), 7.44 (t, 1 H, J: 8.0 Hz, Ph-H), 6.21 (q, 1 H, J:
6.0 Hz, H-1’), 4.95 (s, 2 H, ), 4.41 (m, 1 H, H-3’), 4.34 (dd, 2 H, J: 11.6 Hz, 5-CH2),
3.96 (m, 1 H, H-4’), 3.79 (m, 2 H, H-5’), 2.29 (m, 1 H, H-2’a), 2.05 (m, 1 H, H-2’b), 0.89 (2 s,
18 H, C(CH3)3), 0.08 (4 s, 12 H, CH3).
2,4,6—Triisopropyl benzenesulfonyl chloride (176 mg, 0.59 mmol) was added to a
solution of compound 41 (85 mg, 0.14 mmol), DMAP (19 mg, 0.16 mmol), and triethylamine
(0.18 mL, 1.3 mmol) in anhydrous CH2C12 (5.0 mL). The mixture was stirred at room
temperature overnight under a nitrogen atmosphere, concentrated in vacuo, and the residue
was dissolved in a solution of NH3 in 1,4—dioxane (0.5 M, 15 mL). The mixture was
transferred into a sealed tube and was heated at 90°C overnight. The mixture was cooled to
room temperature, concentrated in vacuo, dissolved in CHzClz (30 mL), and washed with
brine (30 mL). The c and aqueous phases were separated, and the aqueous phase was
ted with CHzClz (30 mL) two times. The combined organic phase was dried over
NaZSO4, concentrated in vacuo, and the residue was purified by silica gel column
chromatography to yield O-bis-(tert—butyldimethylsilyl)(2-nitrobenzyloxy)methyl-2’-
deoxycytidine 42 (60 mg, 69% for two steps). 1H NMR (400 MHz, CDCZ3).' 5 8.08 (d, J =
8.0 Hz, 1 H, Ph-H), 7.81 (s, 1 H, H-6), 7.65 (m, 2 H, Ph-H), 7.64 (q, 1 H, J: 8.0 Hz, Ph-H),
7.49 (m, 1 H, Ph-H), 6.29 (t, 1 H, J= 6.4 Hz, H—1’), 5.75 (br s, 1 H, NHz), 4.85 (dd, 2 H, J=
13.6 Hz, Ph—CHz), 4.41 (s, 2 H, 5—CH2), 4.34 (m, 1 H, H—3’), 3.95 (m, 1 H, H—4’), 3.89 (dd, 1
H, J= 2.8 Hz, H—5’a), 3.76 (dd, 1 H, J: 2.8 Hz, , 2.46 (m, 1 H, H—2’a), 1.98 (m, 1 H,
, 0.92 and 0.89 (2 s, 18 H, C(CH3)3), 0.11—0.08 (4 s, 12 H, CH3).
To a solution of compound 42 (55 mg, 0.09 mmol) in THF (10 mL), F (63 mg,
0.20 mmol) was added. The mixture was stirred at room temperature for four hours and
concentrated in vacuo, and the residue was purified by silica gel column chromatography to
yield 5-(2-nitrobenzyloxy)methyl-2’-deoxycytidine 43 (34 mg, 96%). 1H NMR (400 MHZ,
DMSO-dg): 5 8.05 (d, J = 8.0 Hz, 1 H, Ph—H), 7.89 (s, 1 H, H—6), 7.74 (m, 2 H, Ph—H), 7.55
(m, 1 H, Ph—H), 7.39 (br s, 1 H, D20 exchangeable, NHz), 6.74 (br s, 1 H, D20 exchangeable,
NHz), 6.12 (t, 1 H, J = 6.4 Hz, H—1’), 5.21 (br s, 1 H, D20 exchangeable, 3’—OH), 4.99 (br s, 1
H, D20 exchangeable, 5’-OH), 4.81 (s, 2 H, Ph-CHz), 4.30 (dd, 2 H, J = 11.6 Hz, 5—CH2),
4.20 (m, 1 H, H—3’), 3.76 (m, 1 H, H—4’), 3.55 (m, 2 H, H—5’), 2.11 (m, 1 H, H—2’a), 1.95 (m, 1
H, H—2’b).
nd 43 (32 mg, 0.081 mmol) was phosphorylated with POCl3 (30 uL, 0.32
mmol) and proton sponge (35 mg, 0.16 mmol) in trimethylphosphate (0.35 mL) at 0°C for
three hours under a nitrogen atmosphere. A solution of tri-n-butylammonium pyrophosphate
(237 mg, 0.50 mmol) and tri-n-butylamine (100 uL) in anhydrous DMF (1.0 mL) was added.
After 10 min of stirring, triethylammonium bicarbonate buffer (0.1 M, pH 7.5; 10 mL) was
added. The on was stirred at room temperature for one hour and then concentrated in
vacuo. The residue was dissolved in 20% aqueous acetonitrile (20 mL), filtered, and d
by anion exchange chromatography. The fractions containing triphosphate were combined
and lyophilized to give 5-(2-nitrobenzyloxy)methyl-2’-deoxycytidine-5’-triphosphate dC.I,
which was further purified using RP-HPLC. HRMS (ESI): For the lar ion
C17H22N4016P3 [M-H]', the calculated mass was 631.0244, and the observed mass was
63 1.025 8.
-[(S)-I-(2-nitr0phenyZ)-2, 2-dimethyl—pr0pyloxy]methyl-2 ’-deoxycytidine-5 ’-
triphosphate
OTBS OTBS OTBS
38 44 45
OZN NH2 OZN NH2
tBu o Iii tBu o It];
(m) TBSO N 0 (iv) HO N 0 (v)
—y 0 —> O —)-
OTBS OH
46 47
02N NH2
tBu o \N
I N’go
HO\ /O\ /O\P/o
/\\ O
, /P\\ _ /P\\
o o 7
o o o 0
dC.V
Scheme 821. Synthesis of 5-[(S)(2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-2'-
deoxycytidine-S'-triph0sphate. Reagents and conditions: (i) (S)-1—(2—nitrophenyl)-2,2—
dimethyl-l-propanol, 110°C, 21%; (ii) 2,4,6-triisopropylbenzenesulfonyl chloride, DMAP,
Et3N, CHzClz, room ature, 31%; (iii) NH3, 1,4-dioxane, 90°C, 91%; (iv) n—Bu4NF,
THF, room temperature, 82%; (V) POCl3, proton sponge, (MeO)3PO, 0°C; (n—
BU3NH)2H2P207, I’Z-BU3N, DMF, l M HNEt3HCO3.
Compound 38 (Litosh et al., 2011, which is incorporated herein by reference) (520
mg, 0.80 mmol) and (S)(2-nitrophenyl)—2,2-dimethyl-l-propanol (580 mg, 2.8 mmol) were
heated at 110°C for one hour under a nitrogen atmosphere. The mixture was cooled down to
room temperature, ved in a minimum amount of ethyl acetate, and purified by silica gel
tography to yield 3’,5’—0-bis-(tert—butylsimethylsilyl)—5-[(5)—1—(2—nitrophenyl)—2,2-
dimethyl-propyloxy]methyl-2’-deoxyuridine 44 (115 mg, 21%). (3’ or 5’)—0—(tert—
imethylsilyl)-5 -[(S)(2-nitrophenyl)—2,2-dimethyl-propyloxy]methyl-2’-deoxyuridine
(78 mg, 17%) and 5-[(S)—1-(2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-2’-deoxyuridine
(16 mg, 4%) was also obtained from the on. 1H NMR (400 MHZ, .' 5 8.97 (s, 1
H, NH), 7.76 (d, 2 H, J= 8.0 Hz, Ph—H), 7.60 (m, 2 H, Ph—H and H—6), 7.41 (s, 1 H, Ph—H),
6.29 (dd, 1 H, J= 6.0 and 7.6 Hz, H—l’), 4.97 (s 1 H, Ph-CH), 4.42 (m, 1 H, H-3’), 4.28 (AB
d, 1 H, J: 12.0 Hz, 5—CH2a), 4.06 (AB d, 1 H, J: 12.0 Hz, 5—CH2b), 3.92 (m, 1 H, H-4’),
3.76 (m, 2 H, H-5’), 2.30 (m, 1 H, H—2’a), 2.05 (m, 1 H, H—2’b), 0.95 (s, 9 H, (CH3)3CSi), 0.90
(s, 9 H, (CH3)3CSi), 0.83 (s, 9 H, (CH3)3C), 0.12 (s, 3 H, , 0.09 (s, 3 H, CH3Si), 0.07
(s, 3 H, CH3Si), 0.06 (s, 3 H, CH3Si).
2,4,6—Triisopropyl esulfonyl chloride (61 mg, 0.20 mmol) was added to a
solution of compound 44 (110 mg, 0.16 mmol), DMAP (20 mg, 0.17 mmol), and
triethylamine (63 uL, 0.45 mmol) in anhydrous CHzClz (3.0 mL). The mixture was stirred at
room temperature for 36 hours under a en atmosphere, concentrated in vacuo, and the
residue was purified by silica gel column chromatography to give 3’,5’—0—bis—(tert—
butylsimethylsilyl)[(S)(2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-O4-(2,4,6-
triisopropylbenzenesulfonyl)—2’-deoxyuridine 45 (47 mg, 31%). 1HNMR (500 MHZ, CDC]3).'
8.08 (s, 1 H, H—6), 7.80 (dd, 1 H, J= 1.2 and 8.0 Hz, Ph—H), 7.78 (dd, 1 H, J= 1.6 and 8.0
Hz, Ph—H), 7.67 (m, 1 H, Ph—H), 7.46 (m, 1 H, Ph—H), 7.20 (s, 2 H, Ph—H), 6.09 (t, 1 H, J:
6.4 Hz, H-l’), 4.98 (s, 1 H, Ph—CH), 4.35 (m, 1 H, H-3’), 4.25 (AB d, 1 H, J: 11.6 Hz, 5—
CHza), 4.11 (AB d, 1 H, J: 11.6 Hz, 5—CH2b), 3.97 (m, 1 H, H—4’), 3.79 (dd, 1 H, J= 3.6 and
11.6 Hz, H—5’a), 3.74 (dd, 1 H, J=11.6 and 3.6 Hz, H—5’b), 2.90 (m, 1 H, CH), 2.50 (m, 2 H,
H-2’), 1.98 (m, 2 H, CH), 1.31 - 1.22 (m, 18 H, (CH3)2CH x 3), 0.88 (2 s, 18 H, (CH3)3CSi x
2), 0.87 (s, 9 H, (CH3)3C), 0.07 (s, 6 H, (CH3)2Si), 0.06 (s, 6 H, (CH3)2Si).
A solution of NH3 in 1,4—dioxane (0.5 M, 2.0 mL) was added to a solution of
compound 45 (47 mg, 0.05 mmol) in anhydrous 1,4—dioxane (2.0 mL). The mixture was
transferred into a sealed tube and was heated at 90°C for ten hours. The mixture was cooled
to room temperature, concentrated in vacuo and the e was purified by silica gel column
chromatography to yield 3’,5’-O-bis-(tert—butyldimethylsilyl)[(S)(2-nitrophenyl)-2,2-
dimethyl-propyloxy]-methyl-2’-deoxycytidine 46 (31 mg, 91%). 1H NMR (400 MHZ,
CDCZ3).' 5 7.67 (m, 3 H, Ph—H), 7.53 (s, 1 H, H—6), 7.45 (m, 1 H, Ph—H), 6.30 (t, 1 H, J= 6.6
Hz, H-l’), 5.72 (br s, 2 H, NHz), 4.88 (s, 1 H, Ph—CH), 4.32 (m, 1 H, H-3’), 4.28 (AB d, 1 H, J
= 12.8 Hz, 5—CH2a), 4.08 (AB d, 1 H, J: 12.8 Hz, 5—CH2b), 3.87 (m, 1 H, H—4’), 3.74 (dd, 1
H, J= 3.6 and 14.8 Hz, H—5’a), 3.66 (dd, 1 H, J: 3.6 and 11.3 Hz, H—5’b), 2.41 (m, 1 H, H—
2’a), 2.03 (m, 1 H, H—2’b), 0.90 (s, 9 H, (CH3)3CSi), 0.87 (s, 9 H, (CH3)3CSi), 0.83 (s, 9 H,
C(CH3)3), 0.09 (2 s, 6 H, (CH3)2Si), 0.06 (2 s, 6 H, (CH3)2Si).
13 1
A solution of n-Bu4NF (28 mg, 0.09 mmol) in THF (1.0 mL) was added to a solution
of nd 46 (20 mg, 0.03 mmol) in THF (2.0 mL). The mixture was stirred at room
temperature for 30 min and concentrated in vacuo, and the residue was purified by silica gel
column tography to yield 5-[(S)(2-nitrophenyl)—2,2-dimethyl-propyloxy]methyl-2’-
deoxycytidine 47 (11 mg, 82%). 1H NMR (400 MHz, CD30D): 5 7.87 (s, 1 H, H—6), 7.82
(dd, 1 H, J: 1.2 and 8.4 Hz, Ph—H), 7.76 (dd, 1 H, J: 1.6 and 8.0 Hz, Ph—H), 7.68 (m, 1 H,
Ph—H), 7.51 (m, 1 H, Ph—H), 6.23 (t, 1 H, J: 6.6 Hz, H—1’), 4.94 (s, 1 H, Ph—CH), 4.44 (AB d,
1 H, J: 13.2 Hz, 5—CH2a), 4.34 (m, 1 H, H—3’), 4.11 (AB d, 1 H, J= 13.2 Hz, 5—CH2b), 3.88
(m, 1 H, H—4’), 3.71 (dd, 1 H, J: 3.2 and 12.0 Hz, H—5’a), 3.63 (dd, 1 H, J= 4.0 and 12.0 Hz,
H—5’b), 2.35 (m, 1 H, H—2’a), 2.14 (m, 1 H, H—2’b), 0.80 (s, 9 H, C(CH3) 3).
Compound 47 (11 mg, 0.025 mmol) was phosphorylated with POC13 (7 uL, 0.075
mmol) and proton sponge (11 mg, 0.05 mmol) in trimethylphosphate (0.3 mL) at 0°C for
three hours under a nitrogen atmosphere. A solution of tri-n-butylammonium pyrophosphate
(59 mg, 0.125 mmol) and tri-n-butylamine (30 uL) in anhydrous DMF (0.25 mL) was added.
After 5 min of stirring, triethylammonium bicarbonate buffer (1 M, pH 7.5; 5.0 mL) was
added. The reaction was stirred at room temperature for one hour and then lized to
dryness. The residue was dissolved in water (5.0 mL), filtered, and purified by anion
exchange chromatography. The fractions containing triphosphate were combined and
lyophilized to give 5- [(19(2-nitrophenyl)-2,2-dimethyl-propyloxy]methyl-2 ’-
ytidine—5’—triphosphate dC.V, which was further purified using by RP—HPLC. HRMS
(ESI): For the molecular ion C21H30N4016P3 [M—H]', the calculated mass was 70, and
the observed mass was 687.0873.
-[(S)-I-(5-Meth0xynitrophenyD-Z, 2-dimethyl—pr0pyloxy]methyl-2 ’-deoxycytidine-
’-triph0sphate
OMe OMe OMe o
O\\/I
/S I-Pr.
OZN O OZN O OZN O
t—Bu o/kaH
HO N O
TBSO N 0 TBSO N 0
(i) ..
O (II)
—> 0 —y o
OH OTBS OTBS
39 48
OZN E /OMe NH2 02N NH2
_ \N t—Bu o \N
OTBS OH
49 50
OZN NH2
tBu o I :5:
, /\ /P\\ /\
o o o o o p
dC.V|
Scheme 822. Synthesis of 5-[(S)(5-methoxynitrophenyl)-2,2-dimethyl-
propyloxy]methyl-2'-deoxycytidine-5'-triph0sphate. Reagents and conditions: (i) TBSCl,
imidazole, DMF, room temperature, 70%; (ii) 2,4,6-triisopropylbenzenesulfonyl chloride,
DMAP, Et3N, CHzClz, room temperature; (iii) NH3, 1,4—dioxane, 90°C, 65% for two steps;
(iv) n—Bu4NF, THF, room temperature, 82%; (v) POCl3, proton sponge, (MeO)3PO, 0°C; (n—
BU3NH)2H2P207, n—Bu3N, DMF; 1 M HNEt3HCO3.
To a solution of nd 39 (235 mg, 0.49 mmol) in anhydrous DMF (3.0 mL),
TBSCl (320 mg, 0.8 mmol) and imidazole (109 mg, 1.6 mmol) were added. The mixture was
stirred at room temperature for six hours, concentrated in vacuo, dissolved in CHzClz (20
mL), and washed with ted NaHCO3 solution (50 mL). The organic and aqueous phases
were separated, and the s phase was extracted with CHzClz (30 mL) three times. The
combined organic phase was dried with NaZSO4, trated in vacuo, and the residue was
W0 2013/040257
d by silica gel chromatography to yield 3’,5’-O-bis-(tert—butyldimethylsilyl)-5{(5)
(5-methoxynitrophenyl)-2,2-dimethyl-propyloxy]methyl-2’-deoxyuridine 48 (245 mg,
70%). IHNMR (400 MHz, CDCZ3).' 5 8.00 (br s, 1 H, NH), 7.88 (d, J: 9.2 Hz, 1 H, Ph—H),
7.60 (s, 1 H, H—6), 7.22 (d, 1 H, J= 2.8 Hz, Ph—H), 6.84 (dd, 1 H, J: 2.8 and 8.0 Hz, Ph—H),
6.25 (dd, 1 H, J= 5.6 and 8.0 Hz, H—l’), 5.23 (s, 1 H, Ph-CH), 4.40 (m, 1 H, H—3’), 4.26(d, 1
H, J: 12 Hz, ), 4.11 (d, 1 H, J: 12 H2b), 3.89 (m, 4 H, OCH3 and H—4’), 3.78
(m, 2 H, H-5’), 2.27 (m, 1 H, H-2’a), 2.04 (m, 1 H, H—2’b), 0.90 and 88 (2 s, 18 H,
SiC(CH3)3), 0.84 (s, 9 H, C(CH3)3), 0.08 (3 s, 12 H, CH3).
2,4,6—Triisopropyl benzenesulfonyl chloride (363 mg, 1.2 mmol) was added to a
solution of compound 48 (170 mg, 0.24 mmol), DMAP (32 mg, 0.26 mmol), and
triethylamine (0.34 mL, 2.4 mmol) in anhydrous CHzClz (8.0 mL). The mixture was stirred
at room temperature overnight under a nitrogen atmosphere, concentrated in vacuo, and the
residue was dissolved in a solution of NH3 in 1,4—dioxane (0.5 M, 20 mL). The mixture was
erred into a sealed tube and was heated at 90°C overnight. The mixture was cooled to
room ature, concentrated in vacuo, dissolved in CHzClz (20 mL), and washed with
brine (50 mL). The organic and aqueous phases were ted, and the aqueous phase was
extracted with CHzClz (30 mL) three times. The combined organic phase was dried over
NaZSO4, concentrated in vacuo, and the residue was purified by silica gel column
chromatography to yield 3’,5’-O-bis-(tert—butyldimethylsilyl)[(S)(5-methoxy
nitrophenyl)-2,2-dimethyl-propyloxy]methyl-2’-deoxycytidine 49 (110 mg, 65% for two
steps). 1H NMR (400 MHz, DMSO-dg): 5 7.96 (d, J= 8.8 Hz, 1 H, Ph—H), 7.50 (br s, 1 H,
NHz), 7.38 (s, 1 H, H—6), 7.08 (dd, 1 H, J= 2.8 and 8.8 Hz, Ph—H), 7.04 (d, 1 H, J: 2.8 Hz,
Ph—H), 6.80 (br s, 1 H, NHz), 6.13 (t, 1 H, J: 6.4 Hz, H—1’), 5.09 (s, 1 H, Ph-CH), 4.31 (m, 1
H, H—3’), 4.25 (d, 1 H, J: 12.8 Hz, 5—CH2a), 4.08 (d, 1 H, J= 12.8 Hz, 5—CH2b), 3.87(s, 3 H,
OCH3), 3.76 (m, 1 H, H—4’), 3.64 (m, 2 H, H—5’), 3.76 (dd, 1 H, J: 2.8 Hz, , 2.10 (m, 1
H, H-2’a), 2.00 (m, 1 H, , 0.87 (s, 9 H, C(CH3)3), 0.78 and 0.76 (2 s, 18 H, SiC(CH3)3),
0.07, 0.06, —0.01, and —0.04 (4 s, 12 H, SiCH3).
To a solution of compound 49 (130 mg, 0.18 mmol) in THF (10 mL), n—Bu4NF (141
mg, 0.44 mmol) was added. The mixture was stirred at room temperature for four hours,
concentrated in vacuo, and the residue was purified by silica gel column chromatography to
yield 5 -[(S)(5 -methoxynitrophenyl)-2,2-dimethyl-propyloxy]methyl-2’-deoxycytidine
50 (72 mg, 82%). IHNMR (400 MHZ, DMSO-dg): 5 7.99 (d, J= 8.0 Hz, 1 H, Ph—H), 7.65 (s,
1 H, H-6), 7.42 (br s, 1 H, D20 exchangeable, NHza), 7.06 (m, 2 H, Ph-H), 6.72 (br s, 1 H,
D20 exchangeable, Nsz), 6.11 (t, 1 H, J = 6.4 Hz, H-l’), 5.17 (d, 1 H, D20 exchangeable,
3’-OH), 5.12 (s, 1 H, Ph-CH), 4.78 (t, 1 H, D20 exchangeable, 5’-OH), 4.25 (d, 1 H, J: 12.4
Hz, ), 4.15 (m, 1 H, H—3’), 4.05 (d, 1 H, J: 12.4 Hz, 5—CH2b), 3.87(s, 3 H, OCH3),
3.72 (m, 1 H, H—4’), 3.44 (m, 2 H, H—5’), 3.76 (dd, 1 H, J: 2.8 Hz, H—5’b), 2.08 (m, 1 H, H—
2’a), 1.95 (m, 1 H, H—2’b), 0.77 (s, 9 H, C(CH3)3).
Compound 50 (20 mg, 0.043 mmol) was phosphorylated with POCl3 (24 uL, 0.26
mmol) and proton sponge (19 mg, 0.086 mmol) in hylphosphate (0.3 mL) at 0°C for six
hours under a nitrogen here. A solution of tri-n-butylammonium osphate (237
mg, 0.50 mmol) and tri-n-butylamine (100 uL) in anhydrous DMF (1.0 mL) was added.
After 10 min of stirring, triethylammonium bicarbonate buffer (0.1 M, pH 7.5; 10 mL) was
added. The reaction was stirred at room temperature for one hour and then concentrated in
vacuo. The residue was dissolved in 20% aqueous acetonitrile (20 mL), filtered, and purified
by anion exchange chromatography. The fractions containing triphosphate were combined
and lyophilized to give 5-[(S)—1-(5-methoxynitrophenyl)-2,2-dimethyl-propyloxy]methyl-
2’—deoxycytidine-5’-triphosphate dC.VI, which was r purified using RP-HPLC. HRMS
(ESI): For the molecular ion C22H32N4017P3 [M—H]', the calculated mass was 719.0975, and
the observed mass was 719.0983.
Example 7 —Synthesis of (R/S)(4-iodo-S-methoxy-Z-nitr0phenyl)-2,2-dimethyl—1-
propane] and (S)(4-i0d0-5—methoxy-Z-nitrophenyl)-2,2-dimethyl—1-propanol
N02 t-BU N02
|\Q |
(i) (ii) HO (III)
t-Bu
| I
OMe OMe OMe MeO N02
3-iodoaniso le 3,_6-dii0_d 0 (R/S)- 1-(4-iodomethoxy- (R/S)(4-iodomethoxy-
nltroanlsole ophenyl)-2,2-dimethyl- 2-nitrophenyl)-2,2-dimethyl-
1-propan ol 1-propyl (1 S)-camphanate
t-Bu N02
(W) (V) HQ
—> t-Bu —>
MeO N02
(S)(4-iodomethoxy- (S)(4-iodomethoxy-
2-nitrophenyl)-2,2-d l- 2-nitrop henyl)-2,2-dimethyl-
1-propyl (1 S)-camphanate 1-propanol
Scheme S23. Synthesis of (R/S)—1—(4—iodo—5—methoxy—2—nitrophenyl)—2,2—dimethyl—1—
propanol and (S)-l-(4-iodomethoxynitrophenyl)-2,2-dimethyl-l-propanol. (i) NaNOz,
CH3COOH, HNO3, room temperature; 12, 60 0C, 25% for two steps (80% pure); (ii) PhMgCl,
(CH3)3CCHO, THF, minus 40°C to room temperature, 72%; (iii) amphanic acid
chloride, DMAP, CHzClz room temperature, 80%; (iii) fractional
, crystallization from
l, 63%; (iv) K2CO3, MeOH, reflux.
Nitric acid (68—70%, 125 mL) was slowly mixed with glacial acetic acid (125 ml) at
room temperature, followed by addition of NaNOz (400 mg, 5.8 mmol) and 3—iodoanisole (10
g, 42.7 mmol). After the on was stirred at room temperature for 24 hours, 12 (10.8 g,
42.7 mmol) was added and the mixture was stirred at 60°C overnight. The reaction mixture
was poured into ice—water (500 ml) and extracted by CHzClz (100 ml) three times. The
combined organic phase was neutralized with saturated NaHCO3 solution (500 ml), washed
with aqueous solution of Na2S203 (20%, 100 ml), dried over NaZSO4, and concentrated in
vacuo. The residue was purified by silica gel column chromatography to yield crude 3,6—
—4-nitroanisole (5.4 g), which is mixed with one unknown by—product (20%) and used
in the next step without further purification. 1HNMR (400 MHz, CDCZ3): 5 8.42 (s, l H, Ph-
H), 7.33 (s, 1 H, Ph-H), 3.97 (s, 3 H, OCH3).
To a solution of crude 3,6—diiodo—4—nitroanisole (770 mg, 80% purity, 1.52 mmol) in
anhydrous THF (10 mL) at minus 40°C under a nitrogen atmosphere, phenylmagnesium
chloride (2 M in THF, 0.46 mL, 0.92 mmol) was added dropwise at a rate such that the
temperature would not exceed minus 35°C. Upon completion of the on, the mixture
was stirred at minus 40°C for two hours, followed by addition of trimethylacetaldehyde (0.22
mL, 1.97 mmol). The mixture was stirred at minus 30°C for two hours and then at room
temperature for another one hour. The reaction was then quenched with brine (1.0 mL),
diluted with CHzClz (100 mL), and the solution was washed with CH3COOH (0.1 N, 50 ml)
and brine (50 ml) tially. The organic phase was dried over NaZSO4, concentrated in
vacuo, and the residue was purified by silica gel column chromatography to yield c
(R/S)(4-iodomethoxynitrophenyl)-2,2-dimethylpropanol (399 mg, 72%). 1H NMR
(400 MHz, CDC]3).' 5 8.32 (s, 1 H, Ph—H), 7.17 (s, 1 H, Ph—H), 5.60 (d, 1 H, J: 4.0 Hz,
PhCH), 3.98 (s, 3 H, OCH3), 2.12 (d, 1 H, J: 4.0 Hz, OH), 0.89 (s, 9H,C(CH3)3).
To a solution of racemic (R/S)—1-(4-iodomethoxynitrophenyl)-2,2-dimethyl
propanol (395 mg, 1.1 mmol) and DMAP (263 mg, 2.16 mmol) in anhydrous CHzClz (5.0
mL), (lS)—camphanic chloride (Corrie et al., 1992, which is orated by nce) (350
mg, 1.62 mmol) was added, and the e was stirred overnight at room temperature under
a nitrogen atmosphere. The reaction mixture was diluted with CHzClz (50 mL) and washed
with saturated NaHCO3 solution (50 mL). The organic phase was dried over NaZSO4,
concentrated in vacuo, and the residue was purified by silica gel column chromatography to
yield (R/S)(4-iodomethoxynitrophenyl)-2,2-dimethylpropyl (1S)-camphanate (490
mg, 80%, 1:1 e of diastereomers).
1-(4-Iodo-5 -methoxynitrophenyl)-2,2-dimethylpropyl (1S)-camphanate
(3.4 g) was dissolved in boiling ethanol (150 ml), the solution was kept in a warm oil bath
and slowly cooled to room temperature and stood overnight. Needle ls were formed
gradually and collected by filtration to yield pure single diastereomer (R)—1—(4—iodo—5—
methoxynitrophenyl)-2,2-dimethylpropyl (1S)—camphanate (870 mg, 51%). The
remaining mother liquor was concentrated in vacuo, and the residue was dissolved again in
boiling ethanol (150 ml), and the solution was quickly cooled to room temperature and needle
crystals were formed within two hours. The ls were collected by ion to yield pure
single diastereomer (S)—1-(4-iodomethoxynitrophenyl)-2,2-dimethylpropyl (1S)-
camphanate. The crystallization process was repeated twice to yield additional pure (S)—
diastereomer (total 1.07 g, 63 %). 1HNMR (400 MHZ, CDCZ3) for (R)-I—(4-i0d0meth0xy-
2-nitr0phenyD-2,2-dimethyl—I—propyl (IS)-camphanate: 5 8.48 (s, 1 H, Ph—H), 6.94 (s, 1 H,
Ph—H), 6.84 (s, 1 H, Ph—CH), 3.93 (s, 3 H, OCH3), 2.42 (m, 1 H, CH), 2.11 (m, 1 H, CH), 1.92
(m, 1 H, CH2), 1.75 (m, 1 H, CH2), 1.11 (s, 3 H, CH3), 1.05 (s, 3 H, CH3), 0.97 (s, 9 H,
3), 0.86 (s, 3 H, CH3). 1H NMR (400 MHZ, CDCZ3) for (4-i0d0meth0xy
nitrophenyD-Z,2-dimethyl—I—pr0pyl (IS)-camphanate: 5 8.48 (s, 1 H, Ph—H), 6.95 (s, 1 H, Ph-
H), 6.80 (s, 1 H, Ph—CH), 3.96 (s, 3 H, OCH3), 2.37 (m, 1 H, CH), 1.92 (m, 2 H, CH2), 1.66
(m, 1 H, CH), 1.14 (s, 3 H, CH3), 1.07 (s, 3 H, CH3), 1.06 (s, 3 H, CH3), 0.98 (s, 9 H,
C(CH3)3).
A mixture of (S)—1-(4-iodomethoxynitrophenyl)—2,2-dimethyl-l-propyl (15)-
camphanate (1.1 g, 2.0 mmol) and K2C03 (552 mg, 4.0 mmol) in methanol (50 mL) was
heated to reflux for one hour, then cooled down, concentrated in vacuo, and diluted with
CHzClz (50 mL). The organic phase was washed with brine (50 mL), dried over NaZSO4,
concentrated in vacuo, and the residue was purified by silica gel column chromatography to
yield enantiopure (S)—1-(4-iodomethoxynitrophenyl)—2,2-dimethyl-l-propanol (720 mg,
98%). IHNMR (400 MHz, CDCZ3): 5 8.32 (s, 1 H, Ph—H), 7.17 (s, 1 H, Ph—H), 5.60 (d, 1 H,
J: 4.0 Hz, PhCH), 3.98 (s, 3 H, OCH3), 2.12 (d, 1 H, J= 4.0 Hz, OH), 0.89 (s, 9H,C(CH3)3).
Example 8 — Dye-labeled OMe-Z-nitrobenzyl Alkylated Hydroxymethyl
Nucleotides sis
Synthesis of (R/S)-I—(4-i0d0meth0xynitrophenyD-Z, 2-dimethyl—I—pr0panol and
(S)-I—(4-i0d0meth0xynitrophenyD-Z, 2-dimethyl—I—pr0panol
N02 t-BU N02
|g |
(i) (ii) HO (m)
t-Bu
| I
OMe OMe OMe MeO N02
3-iodoanisole 3,_6-diio_d 0 (R/S)(4-iodomethoxy- (R/S)(4-iodomethoxy-
nItroanIsole ophenyl)-2,2-dimethyl- 2-nitrophenyl)-2,2-dimethyl-
1-propanol 1-propyl (1S)-camphanate
t-Bu N02
(W) (V) HO
—> t-Bu —>
MeO N02
(S)(4-iod om ethoxy- (S)(4-iodomethoxy-
2-nitrophenyl)-2,2-d imethyl- 2-nitrop henyl)-2,2-dimeth yl-
1-propyl (1 S)-camphanate 1-propanol
Scheme S24. Synthesis of (R/S)—1—(4—iodo—5—methoxy—2—nitrophenyl)—2,2—dimethyl—1—
ol and (S)(4-iodomethoxynitrophenyl)—2,2-dimethylpropanol. (i) NaNOz,
H, HNO3, room temperature; 12, 60 0C, 25% for two steps (80% pure); (ii) PhMgCl,
(CH3)3CCHO, THF, minus 40°C to room temperature, 72%; (iii) amphanic acid
chloride, DMAP, CHzClz room temperature,
, 80%; (iii) fractional crystallization from
ethanol, 63%; (iv) K2C03, MeOH, reflux, 98%.
Nitric acid (68—70%, 125 mL) was slowly mixed with glacial acetic acid (125 ml) at
room ature, followed by addition of NaNOz (400 mg, 5.8 mmol) and 3—iodoanisole (10
g, 42.7 mmol). After the reaction was stirred at room temperature for 24 hours, 12 (10.8 g,
42.7 mmol) was added and the mixture was stirred at 60°C overnight. The reaction mixture
was poured into ice—water (500 ml) and ted by CHzClz (100 ml) three times. The
combined organic phase was neutralized with saturated NaHCO3 solution (500 ml), washed
with aqueous solution of Na2S203 (20%, 100 ml), dried over NaZSO4, and concentrated in
vacuo. The residue was purified by silica gel column chromatography to yield crude 3,6—
diiodo—4-nitroanisole (5.4 g), which is mixed with one unknown by—product (20%) and used
in the next step without further purification. 1HNMR (400 MHz, CDCZ3): 5 8.42 (s, 1 H, Ph-
H), 7.33 (s, 1 H, Ph—H), 3.97 (s, 3 H, OCH3).
To a solution of crude 3,6—diiodo—4—nitroanisole (770 mg, 80% purity, 1.52 mmol) in
anhydrous THF (10 mL) at minus 40°C under a nitrogen atmosphere, magnesium
chloride (2 M in THF, 0.46 mL, 0.92 mmol) was added dropwise at a rate such that the
temperature would not exceed minus 35°C. Upon completion of the addition, the mixture
was stirred at minus 40°C for two hours, followed by addition of trimethylacetaldehyde (0.22
mL, 1.97 mmol). The mixture was stirred at minus 30°C for two hours and then at room
temperature for another one hour. The reaction was then quenched with brine (1.0 mL),
diluted with CHzClz (100 mL), and the solution was washed with CH3COOH (0.1 N, 50 ml)
and brine (50 ml) sequentially. The organic phase was dried over NaZSO4, concentrated in
vacuo, and the residue was purified by silica gel column chromatography to yield racemic
(R/S)(4-iodomethoxynitrophenyl)-2,2-dimethylpropanol (399 mg, 72%). 1H NMR
(400 MHz, CDCZ3).' 5 8.32 (s, 1 H, Ph—H), 7.17 (s, 1 H, Ph—H), 5.60 (d, 1 H, J: 4.0 Hz,
PhCH), 3.98 (s, 3 H, OCH3), 2.12 (d, 1 H, J= 4.0 Hz, OH), 0.89 (s, H3)3).
To a solution of racemic (R/S)—1-(4-iodomethoxynitrophenyl)-2,2-dimethyl
propanol (395 mg, 1.1 mmol) and DMAP (263 mg, 2.16 mmol) in anhydrous CHzClz (5.0
mL), (lS)—camphanic chloride (Corrie et al., 1992, which is orated by reference) (350
mg, 1.62 mmol) was added, and the mixture was stirred ght at room temperature under
a nitrogen atmosphere. The reaction mixture was diluted with CHzClz (50 mL) and washed
with saturated NaHCO3 on (50 mL). The organic phase was dried over Na2SO4,
concentrated in vacuo, and the residue was purified by silica gel column chromatography to
yield (R/S)(4-iodomethoxynitrophenyl)-2,2-dimethylpropyl amphanate (490
mg, 80%, 1:1 mixture of reomers).
(R/S)(4-Iodo-5 -methoxynitrophenyl)-2,2-dimethylpropyl (1S)-camphanate
(3.4 g) was dissolved in boiling ethanol (150 ml), the solution was kept in a warm oil bath
and slowly cooled to room temperature and stood overnight. Needle ls were formed
gradually and collected by filtration to yield pure single diastereomer (4—iodo—5—
methoxynitrophenyl)-2,2-dimethylpropyl (1S)—camphanate (870 mg, 51%). The
remaining mother liquor was concentrated in vacuo, and the residue was ved again in
boiling ethanol (150 ml), and the solution was quickly cooled to room temperature and needle
crystals were formed within two hours. The crystals were ted by filtration to yield pure
single diastereomer (S)—1-(4-iodomethoxynitrophenyl)-2,2-dimethylpropyl (1S)-
camphanate. The crystallization process was repeated twice to yield additional pure (S)—
diastereomer (total 1.07 g, 63 %). 1HNMR (400 MHZ, CDCZ3) for (R)-I—(4-i0d0meth0xy-
2-nitr0phenyD-2,2-dimethyl—I—propyl (IS)-camphanate: 5 8.48 (s, 1 H, Ph—H), 6.94 (s, 1 H,
Ph—H), 6.84 (s, 1 H, Ph—CH), 3.93 (s, 3 H, OCH3), 2.42 (m, 1 H, CH), 2.11 (m, 1 H, CH), 1.92
(m, 1 H, CH2), 1.75 (m, 1 H, CH2), 1.11 (s, 3 H, CH3), 1.05 (s, 3 H, CH3), 0.97 (s, 9 H,
C(CH3)3), 0.86 (s, 3 H, CH3). 1H NMR (400 MHZ, CDCZ3) for (S)-I—(4-i0d0meth0xy
henyD-Z,2-dimethyl—I—pr0pyl (IS)-camphanate: 5 8.48 (s, 1 H, Ph—H), 6.95 (s, 1 H, Ph-
H), 6.80 (s, 1 H, Ph—CH), 3.96 (s, 3 H, OCH3), 2.37 (m, 1 H, CH), 1.92 (m, 2 H, CH2), 1.66
(m, 1 H, CH), 1.14 (s, 3 H, CH3), 1.07 (s, 3 H, CH3), 1.06 (s, 3 H, CH3), 0.98 (s, 9 H,
C(CH3)3).
A mixture of (S)—1-(4-iodomethoxynitrophenyl)—2,2-dimethyl-l-propyl (15)-
camphanate (1.1 g, 2.0 mmol) and K2C03 (552 mg, 4.0 mmol) in methanol (50 mL) was
heated to reflux for one hour, then cooled down, concentrated in vacuo, and d with
CHzClz (50 mL). The organic phase was washed with brine (50 mL), dried over NaZSO4,
concentrated in vacuo, and the residue was purified by silica gel column chromatography to
yield enantiopure (S)—1-(4-iodomethoxynitrophenyl)—2,2-dimethyl-l-propanol (720 mg,
98%). IHNMR (400 MHz, CDCZ3): 5 8.32 (s, 1 H, Ph—H), 7.17 (s, 1 H, Ph—H), 5.60 (d, 1 H,
J: 4.0 Hz, PhCH), 3.98 (s, 3 H, OCH3), 2.12 (d, 1 H, J: 4.0 Hz, OH), 0.89 (s, 9H,C(CH3)3).
Synthesis of dye labeled 7-{(S)-I—[4-(3-amin0-I-pr0pynyl)meth0xynitr0phenyl]-
2,2-dimethyl—pr0pyloxy}methyl— a-2 ’-de0xyaden0sine-5 ’-triph0sphate and 5 ’-a-
thiotriphosphate
02N OZN
CI t'BU
HO O NH2
TBSO 0| O:HO:N HO N N/)
:O:N (ii) (iii) (iv) 0
OTBS OH
>—NH H2N
OMe OMe
OZN 02N
t-Bu
O NH2 t—Bu
O NH2
/ \ N
l \)N / I
(V) HO N N/ (Vi) HO\ /O\ /O\P/O N N/J
—> O —> /F{\ R\ ,\\ O
_ O O _O O _O R
0“ OH
53 54a R = 0
54b R = S
Scheme 825a. Synthesis of dye labeled 7-{(S)[4-(3-aminopropynyl)methoxy—2-
nitrophenyl]-2,2-dimethyl-propyloxy}methyldeaza-2'-de0xyadenosine-5'-
triphosphate and hiotriph0sphate. Reagents and conditions: (i) MsCl, DMAP,
CHzClz, (ii) (S)(4-iodomethoxynitropheny1)-2,2—dimethy1—1-propanol, 110°C; (iii) n-
Bu4NF, THF, room temperature; 29% for three steps; (iv) NH3, 1,4—dioxane/MeOH, 100°C,
80%; (v) N—propargyltrifluoroacetamide, Pd(PPh3)4(0), CuI, Et3N, DMF; 98%; (vi) For 5421:
POC13, proton sponge, (MeO)3PO, 0°C; (n—Bu3NH)2H2PZO7, n-Bu3N, DMF; 1 M
CO3; NH4OH. For 54b: PSC13, 2,4,6—Collidine, (EtO)3PO, 0°C to room temperature;
(n-BU3NH)2H2P207; I’Z-BU3N; DMF; 1 M HNEt3HCO3; NH4OH.
(El—N
(vi)
t-Bu
O NH2
N |\)N
Ho\P/o\ /O\P/O N/
x\\ /F{\ /\\ O
_ o o _o o _ o R
55aR=O
55bR=S
Scheme S25b. (vi) Alexa Fluor 488 NHS, 0.1 M NazCO3/NaHCO3 buffer (pH 9.2).
DMAP (463 mg, 3.80 mmol) and MsCl (177 uL, 2.28 mmol) were added to a solution
of compound 4 (400 mg, 0.76 mmol) in anhydrous CHzClz (5.0 mL) at 0°C under a nitrogen
atmosphere. The reaction was stirred at 0°C for 10 mins and at room temperature for another
3 hours. The reaction was then diluted with CHzClz (20 mL). The solution was applied on a
short silica gel plug (2 x 3 cm) and was eluted quickly with a hexane/ethyl
acetate/triethylamine solvent system (80 mL, volume ratio: 05). The eluent was
concentrated in vacuo, and the residue was mixed with (S)—1—(4—iodo—5—methoxy
henyl)-2,2-dimethylpropanol (500 mg, 1.37 mmol). The mixture was heated at
115°C for 45 min under a nitrogen atmosphere, cooled to room temperature and dissolved in
THF (10 mL). n-Bu4NF (526 mg, 1.67 mmol) was added and the e was stirred at room
temperature for 12 hours and then concentrated in vacuo. The residue was dissolved in
CHzClz (50 mL) and washed with brine (50 mL), and the aqueous phase was extracted with
CHzClz (20 mL) two times. The combined organic phase was dried over NaZSO4,
concentrated in vacuo, and the residue was purified by silica gel chromatography to yield 6—
chloro—9—(B—D—2 ’ ribofuranosyl)[(S)—1-(4-iodo-5 xynitrophenyl)-2,2-
dimethyl—propyloxy]—methyl—7—deazapurine 51 (135 mg, 29% for three steps). 1H NMR (400
MHZ, CDCZ3).' 5 8.56 (s, 1 H, H—2), 8.21 (s, 1 H, Ph—H), 7.34 (s, 1 H, H—8), 7.02 (s, 1 H, Ph—
H), 6.35 (dd, 1 H, J= 6.0 and 8.8 Hz, H—1’), 5.20 (s, 1 H, Ph—CH), 4.76 (dd, 2 H, J: 12.4 and
36.4 Hz, 7—CH2), 4.74 (m, 1 H, H—3’), 4.13 (m, 1 H, H—4’), 3.96 (m, 1 H, H—5’a), 3.92 (s, 3 H,
OCH3), 3.80 (m, 1 H, , 2.85 (m, 1 H, H—2’a), 2.30 (m, 1 H, H—2’b), 0.83 (s, 9 H,
Compound 51 (135 mg, 0.22 mmol) was dissolved in 1,4—dioxane (10 mL) followed
by addition of NH3 in MeOH (7 N, 20 mL). The e was transferred to a sealed tube and
stirred at 100°C for 24 hours, then cooled to room temperature, concentrated in vacuo, and
the residue was purified by silica gel chromatography to yield 7—[(S)—1—(4—iodo—5—methoxy—2—
nitrophenyl)—2,2-dimethyl-propyloxy]methyl—7—deaza—2’—deoxyadenosine 52 (110 mg, 80%).
1H NMR (400 MHz, CDCZ3): 5 8.24 (s, 1 H, H—2), 8.22 (s, 1H, Ph—H), 6.96 (s, 1 H, Ph—H),
6.79 (s, 1 H, H—8), 6.14 (dd, 1 H, J= 6.0 and 7.6 Hz, H—1’), 5.21 (s, 1 H, Ph—CH), 4.74 (m, 1
H, H—3’), 4.56 (dd, 2H, J: 13.2 and 24.8 Hz, 7—CH2), 4.17 (m, 1 H, H—4’), 3.93 (m, 1H, H—
’a), 3.83 (s, 3H, OCH3), 3.79 (m, 1H, H—5’b), 2.96 (m, 1 H, H—2’a), 2.21 (m, 1 H, H—2’b),
0.83 (s, 9 H, C(CH3)3).
A solution of compound 52 (183 mg, 0.29 mmol), N—propargyltrifluoroacetylamide
(435 mg, 2.9 mmol), tetrakis(triphenylphosphine)-palladium(0) (66 mg, 0.057 mmol), CuI
(21 mg, 0.11 mmol), and Et3N (170 uL, 1.22 mmol) in anhydrous DMF (4.0 mL) was stirred
at 50 0C for 24 hours. The mixture was concentrated in vacuo and purified by silica gel
column chromatography to yield [5-methoxy(3-trifluoroacetamidopropynyl)-
2—nitrophenyl]—2,2-dimethyl-propyloxy}methyl—7—deaza—2’—deoxyadenosine 53 (185 mg,
98%). IHNMR (400 MHz, MeOH—d4): 5 8.06 (s, 1 H, H—2), 7.89 (s, 1 H, Ph—H), 7.17 (s, 1 H,
H—8), 7.15 (s, 1 H, Ph—H), 6.37 (dd, 1 H, J= 6.0 and 7.6 Hz, H—1’), 5.23 (s, 1 H, , 4.68
(d, 1 H, J: 12.8 Hz, 7—CH2a), 4.49 (m, 1 H, H—3’), 4.34 (s, 2H, CH2), 3.97 (m, 1 H, H—4’),
3.86 (s, 3 H, OCH3), 3.70 (m, 2 H, H—5’), 2.59 (m, 1 H, H—2’a), 2.2.8 (m, 1 H, H—2’b), 0.85 (s,
9 H, C(CH3)3).
Compound 53 (52 mg, 0.08 mmol) was phosphorylated with POCl3 (14 uL, 0.15
mmol) and proton sponge (34 mg, 0.16 mmol) in trimethylphosphate (0.5 mL) at 0°C for 3
hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate
(237 mg, 0.5 mmol) and tri-n-butylamine (100 uL) in ous DMF (1.0 mL) was added.
After 10 min of stirring, triethylammonium bicarbonate solution (TEAB, 0.1 M, pH 7.5; 10
mL) was added. The reaction was stirred at room temperature for one hour and then
concentrated in vacuo. The residue was dissolved in 75% 0.1 M TEAB/25% acetonitrile (20
mL), filtered, and purified by anion exchange tography using a Q Sepharose FF
column (2.5 X 20 cm). Mobile phase: A, 75% 0.1 M TEAB/25% acetonitrile; B, 75% 1.5 M
TEAB/25% acetonitrile. The fractions containing triphosphate were combined and
lyophilized to dryness. The residue was dissolved in water (10 mL) and treated with
concentrated ammonium hydroxide (10 mL, 27%) at room temperature for one hour to yield
7- {(19- 1-[4-(3 -aminopropynyl)methoxynitrophenyl]-2,2-dimethyl-
propyloxy}methyldeaza-2’-deoxyadenosine-5’-triphosphate 5421, which was further
purified by reverse phase HPLC on a PerkinElmer re OD-300 column (7 pm, 250 x
4.6 mm). Mobile phase: A, 0.1 M TEAB; B, acetonitrile. HRMS (ESI): For the molecular
ion C27H36N6016P3 [M—H]', the ated mass was 793.1401, and the observed mass was
793.1426.
Compound 53 (91 mg, 0.14 mmol) was thiophosphorylated with PSCl3 (14 uL, 0.14
mmol) and 2,4,6-collidine (34 mg, 0.28 mmol) in triethylphosphate (1.0 mL) at 0°C for 1
hour under a nitrogen here. A solution of i-n-butylammonium pyrophosphate
(332 mg, 0.7 mmol) and tri-n-butylamine (140 uL) in anhydrous DMF (1.4 mL) was added.
After 2 min of stirring, triethylammonium bicarbonate solution (TEAB, 1 M, pH 7.5; 20 mL)
was added. The reaction was stirred at room ature for one hour and then concentrated
in vacuo. The residue was dissolved in 75% 0.1 M TEAB/25% acetonitrile (20 mL), filtered,
and purified by anion exchange chromatography using a Q ose FF column (2.5 X 20
cm). Mobile phase: A, 75% 0.1 M TEAB/25% acetonitrile; B, 75% 1.5 M TEAB/25%
acetonitrile. The fractions containing thiotriphosphate were ed and lyophilized to
dryness. The e was dissolved in water (10 mL) and treated with concentrated
ammonium hydroxide (10 mL, 27%) at room temperature for one hour to yield 7-{(S)—1—[4—
(3 -aminopropynyl)-5 -methoxynitrophenyl] -2,2-dimethyl-propyloxy} methyldeaza-
xyadenosine—5let—thiotriphosphate 54b, which was further purified by reverse phase
HPLC on a PerkinElmer Aquapore OD—300 column (7 pm, 250 x 4.6 mm). Mobile phase: A,
0.1 M TEAB; B, acetonitrile. HRMS (ESI): For the molecular ion C27H36N6015P3S [M-H]',
the calculated mass was 809.1172, and the observed mass was 809.1155.
A solution of Alexa Fluor 488 NHS (5 mg, 7.8 umol) in anhydrous DMSO (200 uL)
was added to a on of triphosphate 5421 (1.6 umol) in NaHCO3/Na2CO3 buffer (0.1 M,
pH 9.2, 0.4 mL). The mixture was left at room temperature in dark for one hour. The mixture
was first purified by anion ge HPLC on a Dionex DNApac PA200 column (250 x 4
mm). Mobile phase: A, 75% 0.1 M TEAB/25% acetonitrile; B, 75% 1.5 M TEAB/25%
acetonitrile. The fractions containing dye labeled triphosphate 5521 were ed and
concentrated to small volume, and the product was further purified by reverse phase HPLC
on a PerkinElmer Aquapore OD—300 column (7 pm, 250 x 4.6 mm). Mobile phase: A, 0.1 M
TEAB; B, acetonitrile.
A solution of Alexa Fluor 488 NHS (5 mg, 7.8 umol) in anhydrous DMSO (200 11L)
was added to a on of iphosphate 54b (4.1 umol) in NaHCO3/Na2CO3 buffer (0.1
M, pH 9.2, 1.0 mL). The mixture was left at room temperature in dark for one hour. The
mixture was first purified by anion exchange chromatography using a Q Sepharose FF
column (2.5 X 10 cm). Mobile phase: A, 75% 0.1 M TEAB/25% acetonitrile; B, 75% 1.5 M
% acetonitrile. The fractions containing dye labeled thiotriphosphate 55b were
combined and lyophilized to dryness, and the product was further purified by reverse phase
HPLC on a PerkinElmer Aquapore OD—300 column (7 pm, 250 x 4.6 mm). Mobile phase: A,
0.1 M TEAB; B, acetonitrile.
Synthesis of dye labeled 7-{(S)-I—[4-(3-amin0-I-pr0pynyl)meth0xynitr0phenyl]-
2,2-dimethyl—pr0pyloxy}methyl— a-2 ’-de0xyguan0sine-5 ’-triph0sphateand 5 ’-a-
thiotriphosphate
0' C'
HO Cl
/ I ‘1 / I ‘1
TBSO N N/ NHTBS TBSO N N/ NHTBS
I (i) (m)
(H) ’
, :0: I ,
OTBS OTBS
| |
OMe OMe
02N OZN
t—Bu CI t—Bu O
0 O
/ \N
HO N N/J\NH2 NNH
HO N N/)\NH2
I (M
I (V)
:0: O
’ >
OH OH
56 57
>—NH H2N
OMe OMe
02N OZN
t—Bu O t—Bu O
O O
NH NH
HO N N NH2 N N NH2
_ HO\P/O\P/O\P,O 0 (VI) /\\ /\\ O
_ /\\
> O O _ O O 'O R
0” OH
58 59a R=O
59bR=S
Scheme 826a. Synthesis of dye labeled 7-{(S)[4-(3-aminopropynyl)methoxy—2-
nitrophenyl]-2,2-dimethyl—propyloxy}methyldeaza-2 '-de0xyguan0sine-5 '-
triphosphate and 5’-a-thi0triph0sphate. Reagents and conditions: (i) MsCl, DMAP,
CHzClz, 0°C; (ii) (S)—1—(4-i0d0meth0xynitr0pheny1)-2,2-dimethy1—1-pr0pan01, 115°C;
(iii) n—Bu4NF, THF, room ature; 18% for three steps; (iv) syn—pyridine—Z-aldoxime,
1,1,3 ,3 -tetramethy1 guanidine, 1,4-di0xane/DMF, 70°C, 72%; (v) N—
gyltrifluoroacetamide, Pd(PPh3)4(0), CuI, Et3N, DMF, 96%; (vi) For 5921: POC13,
proton sponge, PO, 0°C; (n—Bu3NH)2H2PZO7, n-Bu3N, DMF; 1 M HNEt3HCO3;
NH4OH. For 59b: PSCl3, 2,4,6—collidine, PO, 0°C to room temperature; (n—
BU3NH)2H2P207, I’Z-BU3N, DMF; l M HNEt3HCO3; NH4OH.
Scheme SZ6b. (vii) Alexa Fluor 594 NHS, 0.1 M NazCO3/NaHCO3 buffer (pH 9.2).
DMAP (502 mg, 4.1 mmol) and MsCl (238 uL, 3.1 mmol) were added to a solution
of compound 18 (680 mg, 1.0 mmol) in anhydrous CHzClz (6.0 mL) at 0°C under a nitrogen
atmosphere. The reaction was stirred at 0°C for 10 min and then diluted with CHzClz (20
mL). The solution was applied on a short silica gel plug (2 x 3 cm) and was eluted quickly
with a hexane/ethyl acetate/triethylamine t system (80 mL, volume ratio: 80/20/05).
The eluent was concentrated in vacuo, and the residue was mixed with (S)—l—(4—iodo—5—
methoxynitrophenyl)—2,2-dimethyl-l-propanol (500 mg, 2.1 mmol). The mixture was
heated at 115°C for 45 min under a nitrogen atmosphere, cooled to room temperature and
dissolved in THF (10 mL). n—Bu4NF (1.07 g, 3.40 mmol) was added and the mixture was
stirred at room temperature for 12 hours and then concentrated in vacuo. The residue was
ved in CHzClz (50 mL) and washed with brine (50 mL), and the aqueous phase was
extracted with CHzClz (20 mL) two times. The combined organic phase was dried over
NaZSO4, trated in vacuo, and the residue was purified by silica gel chromatography to
yield 2-aminochloro(B-D-2 ’ -deoxyribofuranosyl)-7{(5)(4-iodomethoxy
nitrophenyl)-2,2-dimethyl-propyloxy]-methyldeazapurine 56 (125 mg, 18% for three
steps). IHNMR (400 MHz, CDC]3).' 5 8.24 (s, 1 H, Ph—H), 7.04 (s, 1 H, Ph—H), 6.91 (s, 1 H,
H—8), 6.17 (dd, 1 H, J: 6.0 and 8.4 Hz, H—l’), 5.18 (s, 1 H, Ph—CH), 5.11 (br s, 2 H, NHz),
4.71 (m, 1 H, H—3’), 4.59 (dd, 2 H, J: 12.4 and 24.4 Hz, 7—CH2), 4.13 (m, 1 H, H—4’), 3.96
(s, 3 H, OCH3), 3.88 (m, 1 H, H—5’a), 3. 79 (m, 1 H, H—5’b), 2.76 (m, 1 H, H—2’a), 2.32 (m, 1
H, H—2’b), 0.81 (s, 9 H, (CH3)3).
To a solution of compound 56 (100 mg, 0.16 mmol) in 1,4—dioxane (1.5 mL) and
DMF (3.0 mL), syn-pyrimidinealdoxime (389 mg, 3.2 mmol) and 1,1,3,3-tetramethyl
guanidine (439 ”L, 3.5 mmol) were added, and the mixture was heated at 70°C overnight
under a nitrogen atmosphere. The reaction mixture was diluted with CHzClz (20 mL) and
washed tially with acetic acid solution (0.1 M, 50 mL), saturated NaHCO3 solution (50
mL), and brine (50 mL). The organic phase was dried over NaZSO4, concentrated in vacuo,
and the residue was purified by silica gel chromatography to yield 7-[(S)—1-(4-iodo
y—2—nitrophenyl)—2,2-dimethyl—propyloxy]methyl—7—deaza—2’—deoxyguanosine 57 (70
mg, 72%). 1H NMR (400 MHz, MeOH—d4): 5 8.20 (s, 1 H, Ph—H), 7.17 (s, 1 H, Ph—H), 6.82
(s, 1 H, H—8), 6.18 (m, 1 H, H—1’), 5.23 (s, 1 H, Ph—CH), 4.71 (d, 1 H, J: 12.0 Hz, 7—CH2a),
4.52 (d, 1 H, J: 12.0 Hz, 7—CH2b), 4.43 (m, 1 H, H—3’), 3.97 (s, 3 H, OCH3), 3.91 (m, 1 H,
H—4’), 3.71 (m, 2 H, H—5’), 2.49 (m, 1 H, H—2’a), 2.19 (m, 1 H, H—2’b), 0.85 (s, 9 H, (CH3)3).
A solution of compound 57 (50 mg, 0.08 mmol), N—propargyltrifluoroacetylamide
(117 mg, 0.8 mmol), tetrakis(triphenylphosphine)-palladium(0) (18 mg, 0.02 mmol), CuI (5.9
mg, 0.03 mmol), and Et3N (48 uL, 0.34 mmol) in anhydrous DMF (3.0 mL) was stirred at 50
0C for 12 hours. The mixture was concentrated in vacuo and the e was purified by
silica gel column chromatography to yield 7-{(S)[5-methoxy(3-trifluoroacetamido
propynyl)nitrophenyl]—2,2—dimethyl—propyloxy}methyl—7—deaza—2’—deoxyguanosine 58 (50
mg, 96%). 1H NMR (400 MHz, MeOH—d4): 5 7.87 (s, 1 H, Ph—H), 7.26 (s, 1 H, Ph—H), 6.84
(s, 1 H, H—8), 6.20 (m, 1 H, H—1’), 5.25 (s, 1 H, Ph—CH), 4.67 (d, 1 H, J: 12.0 Hz, 7—CH2a),
4.54 (d, 1 H, J: 12.0 Hz, 7—CH2b), 4.43 (m, 1 H, H—3’), 4.33 (s, 2 H, CH2), 3.95 (s, 3 H,
OCH3), 3.89 (m, 1 H, H—4’), 3.70 (m, 2 H, H—5’), 2.46 (m, 1 H, H—2’a), 2.18 (m, 1 H, H—2’b),
0.86 (s, 9 H, ).
Compound 58 (52 mg, 0.08 mmol) was phosphorylated with POCl3 (27 uL, 0.3
mmol) and proton sponge (33 mg, 0.16 mmol) in trimethylphosphate (0.35 mL) at 0°C for 4
hours under a nitrogen atmosphere. A on of i-n-butylammonium pyrophosphate
(237 mg, 0.5 mmol) and tri-n-butylamine (100 uL) in anhydrous DMF (1.0 mL) was added.
After 10 min of stirring, triethylammonium bicarbonate solution (TEAB, 0.1 M, pH 7.5; 10
mL) was added. The reaction was d at room temperature for one hour and then
concentrated in vacuo. The residue was dissolved in 75% 0.1 M TEAB/25% acetonitrile (20
mL), filtered, and purified by anion ge chromatography using a Q Sepharose FF
column (2.5 X 20 cm). Mobile phase: A, 75% 0.1 M TEAB/25% acetonitrile; B, 75% 1.5 M
TEAB/25% acetonitrile. The fractions containing triphosphate were combined and
lyophilized to dryness. The residue was dissolved in water (10 mL) and treated with
concentrated um ide (10 mL, 27%) at room temperature for one hour to yield
7- {(191-propynyl)methoxynitrophenyl]-2,2-dimethyl-
propyloxy}methyldeaza-2’-deoxyguanosine-5’-triphosphate 5921, which was further
purified by e phase HPLC on a PerkinElmer Aquapore OD-300 column (7 pm, 250 X
4.6 mm). Mobile phase: A, 0.1 M TEAB; B, acetonitrile. HRMS (ESI): For the molecular
ion C27H36N6017P3 [M—H]', the calculated mass was 809.1350, and the observed mass was
809.1360.
Compound 59 (50 mg, 0.075 mmol) was thiophosphorylated with PSCl3 (9 uL, 0.09
mmol) and 2,4,6-collidine (18 mg, 0.15 mmol) in triethylphosphate (0.5 mL) at room
temperature for 2.5 hours under a nitrogen atmosphere. A solution of bis-tri-n-
butylammonium pyrophosphate (237 mg, 0.5 mmol) and tri-n-butylamine (100 uL) in
anhydrous DMF (1.0 mL) was added. After 2 min of stirring, triethylammonium bicarbonate
solution (TEAB, 1 M, pH 7.5; 20 mL) was added. The reaction was stirred at room
temperature for one hour and then concentrated in vacuo. The residue was ved in 75%
0.1 M TEAB/25% acetonitrile (20 mL), filtered, and purified by anion exchange
chromatography using a Q Sepharose FF column (2.5 X 20 cm). Mobile phase: A, 75% 0.1
M TEAB/25% acetonitrile; B, 75% 1.5 M TEAB/25% acetonitrile. The fractions containing
thiotriphosphate were combined and lyophilized to s. The residue was ved in
water (10 mL) and treated with concentrated ammonium hydroxide (10 mL, 27%) at room
temperature for one hour to yield 7-{(5')[4-(3-aminopropynyl)methoxy
nitrophenyl]—2,2—dimethyl-propyloxy}methyl—7—deaza—2’—deoxyguanosine—5’—0t—
thiotriphosphate 59b, which was further purified by reverse phase HPLC on a PerkinElmer
Aquapore OD—300 column (7 pm, 250 X 4.6 mm). Mobile phase: A, 0.1 M TEAB; B,
acetonitrile. HRMS (ESI): For the molecular ion C27H36N6016P3S [M-H]', the calculated
mass was 21, and the observed mass was 825.1103.
A solution of Alexa Fluor 594 NHS (4.2 mg, 5.2 umol) in anhydrous DMSO (170 uL)
was added to a on of triphosphate 5921 (2.2 umol) in NaHCO3/Na2CO3 buffer (0.1 M,
pH 9.2, 0.5 mL). The mixture was left at room temperature in dark for one hour. The mixture
was first purified by anion exchange HPLC on a Dionex DNApac PA200 column (250 x 4
mm). Mobile phase: A, 75% 0.1 M TEAB/25% acetonitrile; B, 75% 1.5 M TEAB/25%
acetonitrile. The fractions containing dye d triphosphate 6021 were combined and
concentrated to small volume, and the product was r purified by reverse phase HPLC
on a PerkinElmer re OD—300 column (7 pm, 250 X 4.6 mm). Mobile phase: A, 0.1 M
TEAB; B, acetonitrile.
A solution of Alexa Fluor 594 NHS (5 mg, 6.2 umol) in anhydrous DMSO (200 uL)
was added to a solution of thiotriphosphate 59b (4.45 umol) in NaHCO3/Na2CO3 buffer (0.1
M, pH 9.2, 0.78 mL). The mixture was left at room temperature in dark for one hour. The
mixture was first purified by anion exchange chromatography using a Q Sepharose FF
column (2.5 X 10 cm). Mobile phase: A, 75% 0.1 M TEAB/25% acetonitrile; B, 75% 1.5 M
TEAB/25% acetonitrile. The fractions containing dye labeled iphosphate 60b were
combined and concentrated to small volume, and the product was further purified by reverse
phase HPLC on a PerkinElmer Aquapore OD-300 column (7 pm, 250 x 4.6 mm). Mobile
phase: A, 0.1 M TEAB; B, acetonitrile.
Synthesis of dye labeled -I—[4-(3-amin0-I-pr0pynyl)meth0xynitr0phenyl]-
2,2-dimethyl—pr0pyloxy}methyl-2’-de0xyuridine-5 ’-triph0sphate and 5 ’-0t-thi0triph0sphate
>\—H
OMe OMe
o OZN o OZN 0
Br NBOC t-Bu OA6NH O NH
I * t—Bu A6
TBSO N O HO N O HO N O
(I) (N)
O 0 (III)
—> —>
38 61 62
OZN o
t—Bu OAELNH
N O
63aR=0
63bR=S
Scheme 8273. Synthesis of dye d 5-{(S)[4-(3-aminopropynyl)-5—methoxy—2-
nitrophenyl]-2,2-dimethyl—propyloxy}methyl-2'-deoxyuridine-5'-triph0sphate. Reagents
and conditions: (i) (S)-1—(4—i0d0—5—meth0xy—2-nitr0pheny1)-2,2-dimethy1—1-pr0pan01, 110°C;
(ii) NH4F, MeOH, 50°C, 28% for two steps; (iii) N—propargyltrifluoroacetamide,
Pd(PPh3)4(0), CuI, Et3N, DMF; 90%; (iv) For 6321: POC13, proton sponge, (MeO)3PO, 0°C;
(n-BU3NH)2H2P207; I’Z-BU3N; DMF; 1 M HNEt3HCO3; NH4OH. For 63]). PSC13, 2,6—lutidine,
(EtO)3PO, 0°C to room temperature; (n-Bu3NH)2H2PZO7, n-Bu3N, DMF; 1 M HNEt3HCO3;
NH4OH.
WO 40257
OZN O
t—Bu o NH
I NAG
(V) HO\ /O\ /O\ /O
’ /F{\ /F<\ /F<\ O
O O _ _ O O _O R
64a R=O
64bR=S
Scheme S27b. (v) Alexa Fluor 532 NHS, 0.1 M NaHCO3/Na2CO3 buffer (pH 9.2).
Compound 38 (350 mg, 0.54 mmol) and (S)-1—(4—iodo—5—methoxynitrophenyl)—2,2-
dimethyl-l-propanol (720 mg, 1.97 mmol) were heated at 110°C for 45 min under a nitrogen
atmosphere. The mixture was cooled down to room temperature, dissolved in MeOH (10
mL), and followed by addition of NH4F (400 mg, 11.1 mmol). The mixture was stirred at 50
°C for 12 hours, concentrated in vacuo, dissolved in CH2C12 (50 mL), and washed with brine
(50 mL). The c phase was dried over NaZSO4, concentrated in vacuo, and the residue
was purified by silica gel chromatography to yield 5-[(S)(4-iodomethoxy
nitrophenyl)-2,2-dimethyl-propyloxy]methyl-2'-deoxyuridine 61 (90 mg, 28%). 1HNMR (400
MHz, CDCZ3).' 5 8.34 (s, 1 H, Ph—H), 7.65 (s, 1 H, H—6), 7.12 (s, 1 H, Ph—H), 6.17 (t, 1 H, J:
6.8 Hz, H—1’), 5.18 (s, 1 H, Ph—CH), 4.59 (m, 1 H, H—3’), 4.27 (d, 1 H, J: 12.0 Hz, 5—CH2a),
4.15 (d, 1 H, J: 12.0 Hz, 5-CH2b), 4.00 (m, 1 H, H—4’), 3.97 (s, 3 H, OCH3), 3.95 (m, 1 H,
H—5’a), 3.82 (m, 1 H, H—5’b), 2.34 (m, 2 H, H—2), 0.84 (s, 9 H, C(CH3)3).
A solution of compound 61 (80 mg, 0.13 mmol), N—propargyltrifluoroacetylamide
(196 mg, 1.30 mmol), tetrakis(triphenylphosphine)-palladium(0) (30 mg, 0.026 mmol), CuI
(9.9 mg, 0.052 mmol), and Et3N (80 uL) in anhydrous DMF (3.0 mL) was stirred at 50°C for
12 hours. The mixture was concentrated in vacuo and the residue was d by silica gel
column chromatography to yield 5 - {(19[5 -methoxy(3 -trifluoroacetamidopropynyl)-
2-nitrophenyl]-2,2-dimethyl-propyloxy}methyl-2’-deoxyuridine 62 (75 mg, 90%). 1H NMR
(400 MHZ, MeOD-d4): 5 8.11 (s, 1 H, H—6), 8.08 (s, 1 H, Ph—H), 7.36 (s, 1 H, Ph—H), 6.27 (t,
1 H, J: 6.4 Hz, H—1’), 5.33 (s, 1 H, Ph—CH), 4.47 (m, 1 H, H—3’), 4.44 (s, 2 H, 5—CH2), 4.32
(d, 2 H, J: 2.0 Hz, CH2), 4.08 (s, 3 H, OCH3), 3.99 (m, 1 H, H—4’), 3.87 (m, 1 H, H—5’a),
3.79 (m, 1 H, H—5’b), 2.30 (m, 2 H, H—2), 0.93 (s, 9 H, C(CH3)3).
Compound 62 (40 mg, 0.064 mmol) was phosphorylated with POCl3 (21 uL, 0.22
mmol) and proton sponge (27 mg, 0.13 mmol) in trimethylphosphate (0.35 mL) at 0°C for 4
hours under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate
(237 mg, 0.5 mmol) and tri-n-butylamine (100 uL) in anhydrous DMF (1.0 mL) was added.
After 10 min of stirring, triethylammonium bicarbonate solution (TEAB, 0.1 M, pH 7.5; 10
mL) was added. The reaction was stirred at room temperature for one hour and then
concentrated in vacuo. The residue was dissolved in 75% 0.1 M TEAB/25% acetonitrile (20
mL), filtered, and purified by anion exchange chromatography using a Q Sepharose FF
column (2.5 X 20 cm). Mobile phase: A, 75% 0.1 M TEAB/25% acetonitrile; B, 75% 1.5 M
TEAB/25% itrile. The fractions containing triphosphate were combined and
lyophilized to dryness. The residue was ved in water (10 mL) and treated with
concentrated ammonium hydroxide (10 mL, 27%) at room temperature for one hour to yield
- -[4-(3 -aminopropynyl)methoxynitrophenyl]-2,2-dimethyl-
propyloxy}methyl-2’-deoxyuridine-5’-triphosphate 6321, which was further purified by
e phase HPLC on a Elmer Aquapore OD-300 column (7 pm, 250 x 4.6 mm).
Mobile phase: A, 0.1 M TEAB; B, acetonitrile.
Compound 62 (130 mg, 0.21 mmol) was thiophosphorylated with PSCl3 (26 ”L, 0.25
mmol) and 2,6-lutidine (89 mg, 0.84 mmol) in triethylphosphate (0.6 mL) at room
temperature for 1 hour under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium
pyrophosphate (474 mg, 1.0 mmol) and tri-n-butylamine (200 uL) in ous DMF (2.0
mL) was added. After 2 min of stirring, triethylammonium bicarbonate solution (TEAB, 1
M, pH 7.5; 20 mL) was added. The reaction was stirred at room temperature for one hour
and then concentrated in vacuo. The residue was dissolved in 75% 0.1 M TEAB/25%
acetonitrile (20 mL), ed, and purified by anion exchange chromatography using a Q
Sepharose FF column (2.5 X 20 cm). Mobile phase: A, 75% 0.1 M TEAB/25% acetonitrile;
B, 75% 1.5 M TEAB/25% acetonitrile. The ons containing thiotriphosphate were
combined and lyophilized to s. The residue was dissolved in water (10 mL) and
treated with concentrated ammonium hydroxide (10 mL, 27%) at room ature for one
hour to yield 5-{(5')[4-(3-aminopropynyl)methoxynitrophenyl]-2,2-dimethyl-
propyloxy}methyl-2’-deoxyuridine-5’-0t-thiotriphosphate 63b, which was further purified by
reverse phase HPLC on a PerkinElmer re OD-300 column (7 pm, 250 x 4.6 mm).
Mobile phase: A, 0.1 M TEAB; B, acetonitrile. HRMS (ESI): For the molecular ion
C25H34N4017P3S , the calculated mass was 787.0853, and the ed mass was
787.0884.
A solution of Alexa Fluor 532 NHS (2 mg, 2.76 umol) in anhydrous DMSO (80 uL)
was added to a solution of triphosphate 6221 (1.07 umol) in NaHCO3/Na2CO3 buffer (0.1 M,
pH 9.2, 0.3 mL). The mixture was left at room temperature in dark for one hour. The mixture
was first purified by anion exchange HPLC on a Dionex DNApac PA200 column (250 x 4
mm). Mobile phase: A, 75% 0.1 M TEAB/25% acetonitrile; B, 75% 1.5 M TEAB/25%
acetonitrile. The fractions containing dye labeled triphosphate 6321 were combined and
concentrated to small volume, and the product was further purified by reverse phase HPLC
on a Elmer Aquapore OD—300 column (7 pm, 250 x 4.6 mm). Mobile phase: A, 0.1 M
TEAB; B, acetonitrile.
A solution of Alexa Fluor 532 NHS (2.5 mg, 3.45 umol) in anhydrous DMSO (100
uL) was added to a solution of thiotriphosphate 62b (1.03 umol) in NaHCO3/Na2C03 buffer
(0.1 M, pH 9.2, 0.15 mL). The mixture was left at room ature in dark for one hour.
The mixture was first purified by anion exchange chromatography using a Q Sepharose FF
column (2.5 X 10 cm). Mobile phase: A, 75% 0.1 M TEAB/25% acetonitrile; B, 75% 1.5 M
TEAB/25% acetonitrile. The fractions containing dye labeled thiotriphosphate 63b were
combined and lized to dryness, and the product was further purified by reverse phase
HPLC on a PerkinElmer Aquapore OD—300 column (7 pm, 250 x 4.6 mm). Mobile phase: A,
0.1 M TEAB; B, acetonitrile.
2012/055231
Synthesis of dye labeled 5-{(S)-I—[4-(3-amin0-I-pr0pynyl)meth0xynitr0phenyl]-
2,2-dimethyl—pr0pyloxy}methyl-2’-de0xycytidine-5 ’-triph0sphate and 5 ’-0t-thi0triph0sphate
' ' ' i—Pr
OMe OMe OMe O
O\\ II
/S i-Pr
02N O OZN O OZN O
HO N 0
TBSO N 0 TBSO N 0
Koj (I) (H)
—> Koj —> Koj
OH OTBS OTBS
61 65
| |
OMe OMe
OZN NHZ 02N NH2
t-Bu o \N t-Bu
| CAKE;
N 0
(m) TBSO NAG
(iv) HO (v)
OTBS OH
>_NH HZN
OMe OMe
OZN NH2 OZN NH2
t-Bu o \N t-Bu
| AG
HO NAG
(vi)
0 —> HO\P/O\P/O\P\/O'o’\0‘o’\o-0’R\ \ \ R o J
OH OH
67 68a R=O
68bR=S
Scheme 8283. Synthesis of dye labeled 5-{(S)[4-(3-aminopropynyl)methoxy—2-
nitrophenyl]-2,2-dimethyl-propyloxy}methyl-2 '-de0xycytidine—5 '-triphosphate.
Reagents and conditions: (i) TBSCl, imidazole, DMF, room temperature, 96%; (ii) 2,4,6—
triisopropylbenzenesulfonyl de, DMAP, Et3N, CHzClz, room temperature; (iii) NH3,
1,4—dioxane, 90°C; (iv) n—Bu4NF, THF, room temperature, 83% for three steps; (v)
N—propargyltrifluoroacetamide, Pd(PPh3)4(0), Cul, Et3N, DMF, 91%; (vi) For 6821: POC13,
proton sponge, (MeO)3PO, 0°C; (n—Bu3NH)2H2PZO7, n-Bu3N, DMF; 1 M HNEt3HCO3;
NH4OH. For 68]). PSC13, 2,6—lutidine, (EtO)3PO, 00C; (n-BU3NH)2H2P207, I’Z-BU3N, DMF, 1
M HNEt3HCO3; NH4OH.
Scheme S28b. (vii) Cy5 NHS, 0.1 M NazCO3/NaHCO3 buffer (pH 9.2).
To a solution of compound 61 (295 mg, 0.49 mmol) in anhydrous DMF (5.0 mL),
TBSCl (185 mg, 1.23 mmol) and imidazole (160 mg, 2.35 mmol) were added. The mixture
was stirred at room temperature for 12 hours, trated in vacuo, and dissolved in CHzClz
(50 mL). The solution was washed with saturated NaHCO3 solution (50 mL) and the aqueous
phase was extracted with CHzClz (30 mL) three times. The combined organic phase was
dried with NaZSO4, concentrated in vacuo, and the residue was purified by silica gel
chromatography to yield O-bis-(tert—butyldimethylsilyl)[(S)—l-(4-iodomethoxy
nitrophenyl)-2,2-dimethyl-propyloxy]methyl-2'-deoxyuridine 65 (400 mg, 96%). 1H NMR
(400 MHZ, CDCZ3).' 5 8.32 (s, 1 H, Ph—H), 7.64 (s, 1 H, H—6), 7.12 (s, 1 H, Ph—H), 6.12 (t, 1
H, J: 6.8 Hz, H—l’), 5.20 (s, 1 H, Ph—CH), 4.60 (m, 1 H, H—3’), 4.25 (d, 1 H, J: 12.0 Hz, 5—
CHza), 4.14 (d, 1 H, J: 12.0 Hz, 5—CH2b), 4.02 (m, 1 H, H—4’), 3.97 (s, 3 H, OCH3), 3.94 (m,
1 H, H—5’a), 3.83 (m, 1 H, H—5’b), 2.34 (m, 2 H, H—2), 0.90 and 88 (2 s, 18 H, SiC(CH3)3),
0.84 (s, 9 H, C(CH3)3), 0.08 (3 s, 12 H, CH3).
2,4,6-Triisopropyl benzenesulfonyl chloride (581 mg, 1.92 mmol) was added to a
solution of compound 65 (400 mg, 0.48 mmol), DMAP (64 mg, 0.53 mmol), and
ylamine (0.60 mL, 4.32 mmol) in anhydrous CH2C12 (15 mL). The mixture was stirred
at room temperature overnight under a nitrogen atmosphere, concentrated in vacuo, and the
residue was dissolved in a solution of NH3 in 1,4—dioxane (0.5 M, 20 mL). The mixture was
transferred into a sealed tube and was heated at 90°C ght. The reaction was then
cooled to room temperature, concentrated in vacuo, and the residue was dissolved in THF
(8.0 mL) ed by addition of n—Bu4NF (333 mg, 1.06 mmol). The mixture was stirred at
room temperature for four hours, concentrated in vacuo, and the e was purified by silica
gel column chromatography to yield —1-(4-iodomethoxynitrophenyl)-2,2-
dimethyl-propyloxy]methyl-2'-deoxycytidine 66 (240 mg, 83% for three steps in total). 1H
NMR (400 MHz, MeOD-d4): 5 8.33 (s, 1 H, Ph—H), 7.91 (s, 1 H, H—6), 7.10 (s, 1 H, Ph—H),
6.16 (t, 1 H, J: 6.4 Hz, H—1’), 5.19 (s, 1 H, Ph—CH), 4.41 (d, 1 H, J: 12.4 Hz, 5—CH2a), 4.33
(m, 1 H, H—3’), 4.26 (d, 1 H, J: 12.4 Hz, 5—CH2b), 3.97(s, 3 H, OCH3), 3.88 (m, 1 H, H—4’),
3.70 (m, 2 H, H—5’), 2.34 (m, 1 H, H—2’a), 2.17 (m, 1 H, H—2’b), 0.84 (s, 9 H, C(CH3)3).
A solution of compound 66 (245 mg, 0.4 mmol), N—propargyltrifluoroacetylamide
(603 mg, 4.0 mmol), tetrakis(triphenylphosphine)-palladium(0) (92 mg, 0.8 mmol), CuI (30
mg, 0.16 mmol), and Et3N (240 uL, 1.7 mmol) in anhydrous DMF (5.0 mL) was stirred at
50°C for 12 hours. The mixture was concentrated in vacuo and purified by silica gel column
chromatography to yield 5-{(S)[5-methoxy(3-trifluoroacetamidopropynyl)
nitrophenyl]-2,2-dimethyl-propyloxy}methyl-2’-deoxycytidine 67 (230 mg, 91%). 1H NMR
(400 MHz, 4): 5 7.98 (s, 1 H, Ph—H), 7.92 (s, 1 H, H—6), 7.19 (s, 1 H, Ph—H), 6.15 (t,
1 H, J: 6.4 Hz, H—1’), 5.21 (s, 1 H, , 4.41 (d, 1 H, J: 13.2 Hz, 5—CH2a), 4.33 (m, 3
H, H—3’ and CH2), 4.27 (d, 1 H, J: 13.2 Hz, 5—CH2b), 3.96 (s, 3 H, OCH3): 3.88 (m, 1 H, H—
4’), 3.72 (m, 2 H, H—5’), 2.30 (m, 1 H, H—2’a), 2.12 (m, 1 H, H—2’b), 0.84 (s, 9 H, C(CH3)3).
Compound 67 (45 mg, 0.072 mmol) was phosphorylated with POCl3 (15 uL, 0.16
mmol) and proton sponge (31 mg, 0.14 mmol) in trimethylphosphate (0.35 mL) at 0°C for 4
hours under a nitrogen atmosphere. A solution of i-n-butylammonium pyrophosphate
(237 mg, 0.5 mmol) and tri-n-butylamine (100 uL) in anhydrous DMF (1.0 mL) was added.
After 10 min of stirring, triethylammonium onate on (TEAB, 0.1 M, pH 7.5; 10
mL) was added. The reaction was stirred at room temperature for one hour and then
concentrated in vacuo. The residue was dissolved in 75% 0.1 M TEAB/25% acetonitrile (20
mL), filtered, and purified by anion exchange chromatography using a Q Sepharose FF
column (2.5 X 20 cm). Mobile phase: A, 75% 0.1 M TEAB/25% acetonitrile; B, 75% 1.5 M
TEAB/25% acetonitrile. The fractions ning triphosphate were combined and
lyophilized to dryness. The residue was dissolved in water (10 mL) and treated with
concentrated ammonium hydroxide (10 mL, 27%) at room temperature for one hour to yield
- {(19- 1-[4-(3 -aminopropynyl)methoxynitrophenyl]-2,2-dimethyl-
propyloxy}methyl-2’-deoxycytidine-5’-triphosphate 6821, which was further d by
reverse phase HPLC on a PerkinElmer Aquapore OD-300 column (7 pm, 250 X 4.6 mm).
Mobile phase: A, 0.1 M TEAB; B, acetonitrile. HRMS (ESI): For the molecular ion
C25H35N5017P3 [M-H]', the calculated mass was 770.1241, and the observed mass was
770.1234.
Compound 67 (118 mg, 0.19 mmol) was thiophosphorylated with PSCl3 (24 uL, 0.23
mmol) and 2,6-lutidine (80 mg, 0.75 mmol) in ylphosphate (0.5 mL) at 0°C for 1 hour
under a nitrogen atmosphere. A solution of bis-tri-n-butylammonium pyrophosphate (474
mg, 1.0 mmol) and tri-n-butylamine (200 uL) in anhydrous DMF (2.0 mL) was added. After
2 min of stirring, triethylammonium bicarbonate solution (TEAB, 1 M, pH 7.5; 20 mL) was
added. The reaction was stirred at room temperature for one hour and then concentrated in
vacuo. The residue was dissolved in 75% 0.1 M TEAB/25% acetonitrile (20 mL), filtered,
and purified by anion exchange tography using a Q Sepharose FF column (2.5 X 20
cm). Mobile phase: A, 75% 0.1 M 5% acetonitrile; B, 75% 1.5 M TEAB/25%
acetonitrile. The fractions containing thiotriphosphate were combined and lyophilized to
dryness. The residue was dissolved in water (10 mL) and treated with concentrated
ammonium hydroxide (10 mL, 27%) at room temperature for one hour to yield 5-{(S)—1—[4—
(3 -aminopropynyl)-5 -methoxynitrophenyl] imethyl-propyloxy} methyl-2 ’ -
deoxycytidine—5let—thiotriphosphate 68b, which was further purified by reverse phase HPLC
on a PerkinElmer Aquapore OD—300 column (7 pm, 250 X 4.6 mm). Mobile phase: A, 0.1 M
TEAB; B, acetonitrile. HRMS (ESI): For the molecular ion C25H35N5016P3S [M-H]', the
calculated mass was 786.1012, and the observed mass was 83.
A solution of Cy5 NHS (5 mg, 6.3 umol) in anhydrous DMSO (200 uL) was added to
a solution of triphosphate 6821 (1.59 umol) in NaHCO3/Na2CO3 buffer (0.1 M, pH 9.2, 0.35
mL). The mixture was left at room temperature in dark for one hour. The dye labeled
triphosphate was first purified by anion exchange HPLC on a Dionex DNApac PA200
column (250 X 4 mm). Mobile phase: A, 75% 0.1 M TEAB/25% acetonitrile; B, 75% 1.5 M
% acetonitrile. The ons containing dye d triphosphate 6921 were
combined and concentrated to small volume, and the product was further purified by reverse
phase HPLC on a Elmer Aquapore OD-300 column (7 pm, 250 x 4.6 mm). Mobile
phase: A, 0.1 M TEAB; B, acetonitrile.
A solution of Cy5 NHS (5 mg, 6.3 umol) in anhydrous DMSO (200 uL) was added to
a solution of thiotriphosphate 68b (2.96 umol) in NaHCO3/Na2C03 buffer (0.1 M, pH 9.2,
0.53 mL). The mixture was left at room temperature in dark for one hour. The dye labeled
thiotriphosphate was first purified by anion exchange chromatography using a Q Sepharose
FF column (2.5 X 10 cm). Mobile phase: A, 75% 0.1 M TEAB/25% acetonitrile; B, 75% 1.5
M TEAB/25% acetonitrile. The ons containing dye labeled thiotriphosphate 69b were
combined and lized to dryness, and the product was further purified by reverse phase
HPLC on a PerkinElmer Aquapore OD—300 column (7 pm, 250 x 4.6 mm). Mobile phase: A,
0.1 M TEAB; B, acetonitrile.
****************
All of the methods disclosed and claimed herein can be made and executed without
undue experimentation in light of the present disclosure. While the compositions and
methods of this invention have been described in terms of certain embodiments, it will be
nt to those of skill in the art that variations may be d to the methods and in the
steps or in the ce of steps of the method described herein without departing from the
concept, spirit and scope of the invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be substituted for the
agents described herein while the same or similar results would be achieved. All such similar
substitutes and modifications apparent to those skilled in the art are deemed to be within the
, scope and concept of the invention as defined by the appended claims.
Example 9 — UV Photocleavage Studies
The rate of UV photocleavage was found to be dependent on a number of
experimental factors including light intensity. See McCray et a]. (1980) and McGall et a].
(1997), which are both incorporated herein by reference. To compare the rates of
photochemical cleavage between the nucleotide analogs described here, a protocol was
ped to deliver a daily light intensity output of 0.70 :: 0.01 W/cm2 to samples, see
below. A custom—designed UV deprotector used in these studies has been previously
described in Wu et a]. (2007), which is orated herein by nce, and the protocol
implemented is described below.
UV ector set-up: The power supply was turned on for about 30 min prior to
that of the lamp and recirculation bath as described by the manufacturer. The IR liquid filter
was cooled to 9°C. Light intensity was ined using a model PM100 power meter
(Thorlabs), a 1000 um pinhole d Optics), a modified 0.5 mL Eppendorf tube cut in
half, and a 3-axis manual translation stage (Newport), see The half cut Eppendorf
tube was positioned in front of the pinhole and power meter detector head to account for the
geometric shape distortion of the light as it passes through a reaction solution. The
ation stage was then used to align the tube/pinhole/detector device with the highest
intensity from the arc beam.
ity ment to 0. 7W/cmZ: To stabilize its output, the lamp was left on for one
hour prior to ity measurements. Thereafter, the measured power was adjusted by
increasing the current from the power supply. In order to achieve intensity (I) of 0.70 W/cmz,
the measured power (P) was adjusted to ~55 mW, according to the equation:
where r is the radius of pinhole. Power gs are recorded over a five minute period (in
one second intervals) and were converted into intensity readings, which ranged over a six
week period between 0.68 :: 0.01 and 0.72 :: 0.02 W/cmz.
Beam alignment with the 0.5 mL tube holder: The ed Eppendorf tube, pin hole
and power meter were then removed from the UV deprotector, and the rotating sample holder
was installed with the height being 67.18 :: 0.25 mm. The beam was then focused by placing
an 0.5 mL Eppendorf tube into the sample holder, the tube of which was modified with an
internal alignment card to provide reference lines for volume heights of 10 uL and 20 uL, see
The reference lines enabled the beam to be centered for a given reaction volume.
Beam alignment was further verified by observing the mercury arc image of the lamp
produced by the rear reflector. A second alignment card was placed into the ng sample
holder to view the image, which when properly d using the reflector would produce an
inverted arc image on the arc gap itself This step ensured that are hotspots were not
superimposed, which could cause overheating while maintaining a power output of ~5.5 mW.
The speed of the rotating sample holder was ed within a range of 1,200 — 1,350 rpm
using a Nova—Strobe DA Plus stroboscope (Monarch Instrument) by adjusting the motor’s
torque with an adjustment screw.
Photochemical cleavage : Nucleotide analogs were incorporated using 10 uL
reactions, as described for the PEP assays, at a final tration of 100 nM. See Litosh et
al. (2011), which is incorporated herein by reference. OligoTemplate-2, oligoTemplate-5,
and oligoTemplate-4 each hybridized with BODIPY-FL labeled primer—1 were used for C7-
HOMedA, C7—HOMedG, and HOMedU analogs, respectively. OligoTemplate-8 hybridized
with BODIPY—FL labeled primer-3 was used to assay HOMedC analogs. Incorporated
reactions were quenched with either 1 mM sodium azide solution; 1 mM sodium azide, 50
mMDTT solution; or ts C—G (see key in , exposed to 365 nm ultraviolet (UV)
light for various time points using our UV ector, and then placed on ice. Ten uL of
stop solution (98% deionized formamide; 10 mM NazEDTA, pH 8.0; 25 mg/mL Blue
Dextran, MW 2,000,000) was added, and samples were analyzed using an AB model 377
DNA sequencer. Cleavage assays were performed in triplicate to calculate the average DT50
value :: lSD, as described in Litosh et a]. (2011), which is incorporated herein by reference.
WO 40257
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Claims (115)
1. A compound of the formula: R5 R5 R6 OMe R6 OMe R6 OMe O2N R4 O2N R4 O2N R4 H NH2 H O R3 O NH H R3 NH2 R3 O N N O R1 N O O R1 R1 N N O O OH R2 OH R2 OH R2 (I), (II), (III), R5 R5 R6 OMe R6 OMe O2N R4 O2N R4 H H R3 O O R3 NH NH N N R1 N N NH2 R1 N N O O OH R2 (IV), OH R2 (V), R5 R6 OMe R6 OMe O2N R4 O2N R4 H R3 NH R3 O N N N O R1 N N NH2 R1 O O OH R2 (VI), or OH R2 (VII), R1 is hydroxy, monophosphate, diphosphate, triphosphate, α-thiotriphosphate or polyphosphate; R2 is hydrogen or hydroxy; R3 is alkyl(C8) or substituted C8); R4 is hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto; alkyl(C6), acyl(C6), alkoxy(C6), acyloxy(C6), alkylamino(C6), lamino (C6), amido(C6), or a substituted version of any of these groups; R5 and R6 are each independently: hydrogen, hydroxy, halo, amino, nitro, cyano, azido or mercapto; alkyl(C6), alkenyl(C6), alkynyl(C6), aryl(C6), aralkyl(C8), heteroaryl(C6), acyl(C6), alkoxy(C6), acyloxy(C6), alkylamino(C6), dialkylamino (C6), amido(C6), or a substituted version of any of these groups; a group of formula: H2N X n , H2N N O , or H2N Y N X Y N O m n , wherein X is −O−, −S−, or −NH−; or alkanediyl(C12), diyl(C12), alkynediyl(C12), or a substituted version of any of these ; Y is −O−, −NH−, diyl(C12) or substituted alkanediyl (C12); n is an integer from 0–6; and m is an integer from 0–6; or a −linker−reporter, wherein the reporter is a fluorophore; and wherein the linker is a group of either of the following two formulas: N X H n , or H H N Y N X Y N O m n , wherein X is −O−, −S−, or −NH−; or alkanediyl(C12), alkenediyl(C12), alkynediyl(C12), arenediyl(C12), heteroarenediyl(C12), or a substituted version of any of these ; Y is −O−, −NH−, alkanediyl(C12) or substituted alkanediyl(C12); n is an integer from 0–6; and m is an integer from 0–6. or a salt, tautomer, or optical isomer thereof.
2. The nd of claim 1, further defined as a compound of formula I.
3. The compound of claim 1, further defined as a compound of formula II.
4. The compound of claim 1, r defined as a nd of formula III.
5. The compound of claim 1, r defined as a compound of a IV.
6. The compound of claim 1, further defined as a compound of formula V.
7. The compound of claim 1, further defined as a compound of formula VI.
8. The compound of claim 1, further defined as a compound of formula VII.
9. The compound according to any one of claims 1-8, wherein R1 is hydroxy.
10. The compound ing to any one of claims 1-8, wherein R1 is a monophosphate.
11. The compound according to any one of claims 1-8, wherein R1 is a diphosphate.
12. The compound according to any one of claims 1-8, wherein R1 is a triphosphate.
13. The compound according to any one of claims 1-8, wherein R1 is an α-thiotriphosphate.
14. The compound ing to any one of claims 1-8, n R1 is a polyphosphate.
15. The compound according to any one of claims 1-14, wherein R2 is hydrogen.
16. The compound according to any one of claims 1-14, wherein R2 is hydroxy.
17. The compound according to any one of claims 1-16, wherein R3 is alkyl(C8).
18. The compound of claim 17, wherein R3 is alkyl(C3-4).
19. The compound of claim 18, wherein R3 is isopropyl.
20. The compound of claim 18, wherein R3 is tert-butyl.
21. The compound according to any one of claims 1-20, wherein R4 is hydrogen.
22. The nd according to any one of claims 1-20, wherein R4 is nitro.
23. The compound according to any one of claims 1-22, wherein R5 is hydrogen.
24. The compound according to any one of claims 1-22, wherein R5 is iodo.
25. The compound according to any one of claims 1-22, wherein R5 is alkoxy(C6).
26. The compound of claim 25, wherein R5 is y.
27. The compound according to any one of claims 1-22, wherein R5 is a group of formula: H2N X X is −O−, −S−, or −NH−; or alkanediyl(C12), alkenediyl(C12), alkynediyl(C12), arenediyl(C12), heteroarenediyl(C12), or a tuted version of any of these groups; n is an integer from 0–6.
28. The compound of claim 27, wherein X is alkynediyl(C2-8).
29. The compound of claim 28, wherein X is −C≡C−.
30. The compound according to any of claims 27-29, wherein n is zero.
31. The compound ing to any of claims 1-22, wherein R5 is a group of formula: H2N Y N X Y N O m n , wherein X is −O−, −S−, or −NH−; or alkanediyl(C12), alkenediyl(C12), alkynediyl(C12), arenediyl(C12), heteroarenediyl(C12), or a substituted version of any of these groups; Y is −O−, −NH−, alkanediyl(C12) or substituted alkanediyl(C12); n is an integer from 0–6; and m is an integer from 0–6.
32. The compound of claim 31, wherein X is alkynediyl(C2-8).
33. The compound of claim 32, wherein X is −C≡C−.
34. The compound according to any one of claims 31-33, wherein Y is −CH2−.
35. The compound according to any one of claims 31-34, n n is zero.
36. The nd according to any one of claims 31-35, wherein m is zero.
37. The compound according to any one of claims 1-22, wherein R5 is a −linker−reporter, wherein the reporter is a fluorophore.
38. The compound of claim 37, wherein the linker is: N X H n wherein X is −O−, −S−, or −NH−; or alkanediyl(C12), alkenediyl(C12), alkynediyl(C12), iyl(C12), heteroarenediyl(C12), or a substituted version of any of these groups; n is an integer from 0–6.
39. The compound of claim 38, wherein X is alkynediyl(C2-8).
40. The compound of claim 39, wherein X is −C≡C−.
41. The compound according to any one of claims 38-40, wherein n is zero.
42. The compound of claim 37, n the linker is: H H N Y N X Y N O m n , wherein X is −O−, −S−, or −NH−; or alkanediyl(C12), alkenediyl(C12), alkynediyl(C12), arenediyl(C12), arenediyl(C12), or a substituted version of any of these groups; Y is −O−, −NH−, alkanediyl(C12) or substituted alkanediyl(C12); n is an integer from 0–6; and m is an integer from 0–6.
43. The compound of claim 42, wherein X is alkynediyl(C2-8).
44. The compound of claim 43, wherein X is −C≡C−.
45. The compound ing to any one of claims 42-44, n Y is −CH2−.
46. The compound according to any one of claims 42-45, wherein n is zero.
47. The compound according to any one of claims 42-46, wherein m is zero.
48. The compound according to any one of claims 37-47, wherein the fluorophore is zanthene, fluorescein, rhodamine, BODIPY, cyanine, coumarin, pyrene, phthalocyanine, phycobiliprotein, or a squaraine dye.
49. The compound according to any of claims 37-47 wherein the phore is: Cl Cl O O OH O O OH N O N H3CO OCH3 HOOC HOOC OOC C C C O , O , O , CH3 CH3 N O N H3C CH3 OOC H2N O O C HO3S C C O , CH3 , O O HO3S OCH2 C N C (CH2)5 O O N CH3 HO3S SO3H , CF3 CH2SO3H , SO3 SO3H SO3 SO3H H2N H H O N CH3 N O N CH3 H3C CH3 H3C CH3 HOOC CH3 CH3 H3C O , O , SO3 SO3H H H H H H3C H3C N N CH3 O N CH3 O N H3C CH3 H3C CH3 CH2SO3H H3C HOC Cl CH3 O3SH2C O 6 Cl SCH2 C NH(CH2)5 C 5 C Cl O O , O , CH3 CH3 CH3 CH3 H3C N O N CH3 H3C N O N CH3 H3C CH3 H3C CH3 O3SH2C CH2SO3H HOC O3SH2C HOC Cl CH2SO3H O O 5 C Cl SCH2 C NH(CH2)5 C O , Cl O O , or O3S SO3 N N
50. The compound according to any one of claims 1-49, wherein R6 is hydrogen.
51. The compound according to any one of claims 1-50, wherein the starred carbon atom is in the S configuration.
52. The compound ing to any one of claims 1-50, wherein the starred carbon atom is in the R configuration.
53. The nd of claim 1, further defined as: OMe OMe O2N O2N t-Bu O NH2 t-Bu O NH2 N N HO N N HO O O O N N O P P P O O O O O O O OH , OH , OMe OMe O2N O2N t-Bu O O t-Bu O O NH NH HO N N NH2 HO O O O N N NH2 O P P P O O O O O O O OH , OH , OMe OMe O2N O2N t-Bu O t-Bu O O O NH NH HO N N NH2 HO O O O N N NH2 O P P P O O O O O O O OH , OH , OMe OMe OMe OMe O2N O2N t-Bu O t-Bu O O O NH NH HO N N NH2 HO O O O N N NH2 O P P P O O O O O O O OH , OH , OMe OMe O2N O O2N O t-Bu O NH t-Bu O NH N O N O HO HO O O O O P P P O O O O O O O OH , OH , OMe OMe O2N NH2 O2N NH2 t-Bu O N t -Bu O N HO N O HO N O O O O O P P P O O O O O O O OH , or OH , or a salt and/or protonated form of any of these formulas.
54. The nd of claim 1, further defined as: , , , or , wherein R is =O or =S, or a salt and/or ated form of any of these formulas.
55. The compound of claim 1, further defined as: , , , or wherein R is =O or =S, or a salt and/or ated form of any of these formulas.
56. The nd of claim 1, further defined as: SO3 SO3 H2N O NH2 O O OH HOOC HOOC H H C N N OMe OMe O2N O2N t-Bu O NH2 t-Bu O NH2 N N HO O O O N N HO O O O N N P P P O P P P O O O O O O O O O O O O O OH , OH , NH(CH2CH2O)8CH2CH2 C SO3H O S O O H2N O NH COOH O2N t-Bu O NH2 HO O O O N N P P P O O O O O O O OH , SO3 SO3H H H Cl Cl N O N H3C CH3 O O OH H3C CH3 CH3 H3C H3CO OCH3 HOOC H H N N OMe OMe O2N O O2N O t-Bu O NH t-Bu O NH HO O O N O HO O N O O O O P P P O P P P O O O O O O O O O O O O O OH , OH , O3S SO3 N N O2N O t-Bu O NH HO O O O N O P P P O O O O O O O OH , CH3 CH3 H3C N O N CH3 H3C CH3 O3SH2C HOC CH2SO3H O 6 5 C N t-Bu O O HO O O O N N NH2 P P P O O O O O O O OH , O3S SO3 N O N N N O O O2N OMe t-Bu O O O2N NH2 NH t-Bu O N HO O O O N N NH2 O P P P HO O O O N O P P P O O O O O O O O O O O O O OH , or OH , or a salt and/or protonated form of any of these formulas.
57. A method of cing a target nucleic acid comprising the following steps: (i) attaching the 5′-end of a primer to a solid surface; (ii) hybridizing a target c acid to the primer attached to the solid surface to form a hybridized primer/target nucleic acid complex; (iii) obtaining a polymerase and one or more compounds selected from the group consisting of the following formulas and salts thereof: R5 R5 R6 OMe R6 OMe R6 OMe O2N R4 O2N R4 O H O2N R4 NH2 H R3 O NH H R3 O NH2 R3 O N N O R1 N O O R1 R1 N N O O OH R2 OH R2 OH R2 (I), (II), (III), R5 R5 R6 OMe R6 OMe O2N R4 O2N R4 H H R3 O O R3 NH NH N N R1 N N NH2 R1 N N O O OH R2 (IV), OH R2 (V), R5 R6 OMe R6 OMe O2N R4 O2N R4 H R3 NH R3 O N N N O R1 N N NH2 R1 O O OH R2 (VI), or OH R2 (VII), wherein: R1 is triphosphate, α-thiotriphosphate or polyphosphate; R2 is en or hydroxy; R3 is alkyl(C8) or substituted alkyl(C8); R4 is hydrogen; R5 is a −linker−reporter, n the reporter is based on a fluorophore; and the linker is a group of either of the ing two formulas: N X H n , or H H N Y N X Y N O m n , wherein X is −O−, −S−, or −NH−; or alkanediyl(C12), alkenediyl(C12), alkynediyl(C12), arenediyl(C12), heteroarenediyl(C12), or a substituted version of any of these groups; Y is −O−, −NH−, alkanediyl(C12) or substituted alkanediyl(C12); n is an integer from 0–6; and m is an integer from 0–6; and R6 is hydrogen; with the proviso that compounds of different formulas I-VII have different fluorophores; (iv) reacting the hybridized primer/target nucleic acid complex with a polymerase and one or more of the compounds of step (iii) to form a growing primer strand via a polymerase on; (v) imaging the growing primer strand to identify the incorporated compound of step (iv) via its fluorophore; (vi) exposing the solid surface with the growing primer strand to a light source to remove a leavable terminating moiety of the formula: R6 OMe O2N R4 R3 , with the variables as defined in step (iii), resulting in an extended primer; and (vii) repeating steps (iv) through (vi) one or more times to identify a plurality of bases in the target nucleic acid, where the extended primer of step (vi) of the previous cycle reacts in place of the hybridized primer/target nucleic acid complex in step (iv) of the subsequent cycle.
58. The method of claim 57, wherein step (vi) is conducted in the ce of sodium azide.
59. The method of claim 58, where the sodium azide concentration is from 0.1 mM to 10 mM.
60. The method of claim 59, where the sodium azide concentration is about 1 mM.
61. The method of claim 57, n step (vi) is conducted in the presence of sodium acetate.
62. The method of claim 61, where the sodium e concentration is from 0.1 mM to 10
63. The method of claim 61, where the sodium acetate concentration is about 1 mM.
64. The method according to any of claims 57-63, wherein steps (v) or (vi) is conducted in the presence of thiourea.
65. The method of claim 64, where the thiourea concentration is from 10 mM to 500 mM.
66. The method of claim 64, where the thiourea concentration is about 100 mM.
67. The method of claim 58, wherein step (vi) is conducted in the presence of dithiothreitol (DTT).
68. A method of sequencing a target nucleic acid comprising the following steps: (i) attaching the 5′-end of a nucleic acid to a solid surface; (ii) hybridizing a primer to the nucleic acid attached to the solid surface to form a hybridized primer/target nucleic acid complex; (iii) obtaining a polymerase and one or more compounds ed from the group consisting of the following as and salts thereof: R5 R5 R6 OMe R6 OMe R6 OMe O2N R4 O2N R4 O H O2N R4 NH2 H R3 O NH H R3 O NH2 R3 O N N O R1 N O R1 N O R1 N O O OH R2 OH R2 OH R2 (I), (II), (III), R5 R5 R6 OMe R6 OMe O2N R4 O2N R4 H H R3 O O R3 NH NH N N R1 N N NH2 R1 N N O O OH R2 OH R2 (IV), (V), R5 R6 OMe R6 OMe O2N R4 O2N R4 H R3 NH R3 O N N N O R1 N N NH2 R1 O O OH R2 OH R2 (VI), or (VII), wherein: R1 is triphosphate, α-thiotriphosphate or polyphosphate; R2 is hydrogen or y; R3 is alkyl(C8) or substituted alkyl(C8); R4 is en; R5 is a −linker−reporter, wherein the reporter is based on a fluorophore; and the linker is a group of either of the following two formulas: N X H n , or H H N Y N X Y N O m n , X is −O−, −S−, or −NH−; or alkanediyl(C12), alkenediyl(C12), diyl(C12), arenediyl(C12), heteroarenediyl(C12), or a substituted n of any of these groups; Y is −O−, −NH−, alkanediyl(C12) or substituted alkanediyl(C12); n is an integer from 0–6; and m is an integer from 0–6; and R6 is hydrogen; with the proviso that compounds of different formulas I-VII have different fluorophores; (iv) reacting the hybridized primer/target nucleic acid complex with a polymerase and one or more of the compounds of step (iii) to form a growing primer strand via a polymerase reaction; (v) imaging the growing primer strand to identify the incorporated compound of step (iv) via its fluorophore; (vi) exposing the solid surface with the growing primer strand to a light source to remove a photocleavable terminating moiety of the formula: R6 OMe O2N R4 R3 , with the variables as defined in step (iii), resulting in an ed primer; and (vii) ing steps (iv) through (vi) one or more times to identify a plurality of bases in the target c acid, where the extended primer of step (vi) of the previous cycle reacts in place of the hybridized primer/target nucleic acid complex in step (iv) of the subsequent cycle.
69. The method of claim 68, wherein step (vi) is conducted in the presence of sodium azide.
70. The method of claim 69, wherein step (vi) is conducted in the presence of dithiothreitol (DTT).
71. The method according to either one of claims 57 and 68, wherein the incorporation of at least one compound according to step (iv) occurs at about 70% to about 100% of the efficiency of incorporation of its natural nucleotide counterpart.
72. The method of claim 71, wherein the incorporation efficiency occurs at about 85% to about 100%.
73. The method according to either one of claims 57 and 68, n the polymerase is selected from the group consisting of reverse transcriptase, terminal transferase, and DNA polymerase.
74. The method according to either one of claims 57 and 68, wherein about 85% to about 100% of the photocleavable terminating moieties are removed by exposure to a light source in step (vi).
75. The method according to either one of claims 57 and 68, wherein incorporation of at least one compound ing to step (iv) is followed by termination of strand growth at an efficiency of from about 90% to about 100%.
76. The method according to either one of claims 57 and 68, wherein a pulsed multiline excitation detector is used for g in step (v).
77. The method according to either one of claims 57 and 68, further comprising washing the growing primer strand prior after step (iv).
78. The method according to either one of claims 57 and 68, further comprising washing the extended primer after step (vi).
79. The method according to either one of claims 57 and 68, r comprising prior to step (iv) capping any primers or growing primer strands that did not react in step (iv).
80. The method according to either one of claims 57 and 68, further comprising sequencing multiple target c acids synchronistically.
81. A method of converting a non-naturally occurring component in a nucleic acid molecule into a naturally-occurring component comprising: (i) orating a compound selected from the group consisting of the following formulas and salts thereof: R5 R5 R6 OMe R6 OMe R6 OMe O2N R4 O2N R4 O H O2N R4 NH2 H R3 O NH H R3 O NH2 R3 O N N O R1 N O O R1 R1 N N O O OH R2 OH R2 OH R2 (I), (II), (III), R5 R5 R6 OMe R6 OMe O2N R4 O2N R4 H H R3 O O R3 NH NH N N R1 N N NH2 R1 N N O O OH R2 (IV), OH R2 (V), R5 R6 OMe R6 OMe O2N R4 O2N R4 H R3 NH R3 O N N N O R1 N N NH2 R1 O O OH R2 (VI), or OH R2 (VII), R1 is triphosphate, α-thiotriphosphate or polyphosphate; R2 is en or hydroxy; R3 is alkyl(C8) or substituted alkyl(C8); R4 is hydrogen; R5 is a −linker−reporter, wherein the reporter is based on a fluorophore; and the linker is a group of either of the following two formulas: N X H n , or H H N Y N X Y N O m n , wherein X is −O−, −S−, or −NH−; or alkanediyl(C12), alkenediyl(C12), alkynediyl(C12), arenediyl(C12), heteroarenediyl(C12), or a substituted version of any of these groups; Y is −O−, −NH−, alkanediyl(C12) or substituted alkanediyl(C12); n is an integer from 0–6; and m is an integer from 0–6; and R6 is hydrogen; (ii) exposing the resulting c acid to a light source to remove a photocleavable terminating moiety of the formula: R6 OMe O2N R4 R3 , with the variables as defined in step (i), from the nucleic acid.
82. The method of claim 81, further comprising converting non-naturally occurring components in multiple nucleic acid molecules into naturally-occurring components synchronistically.
83. The method of claim 82, further comprising terminating multiple nucleic acid syntheses synchronistically.
84. A method of terminating a nucleic acid synthesis sing the step of placing a 3′-OH ked nucleotide or nucleoside according to claim 1 in the environment of a polymerase and allowing incorporation of the 3′-OH unblocked nucleotide or nucleoside into a nucleic acid molecule.
85. The method of claim 84, wherein the efficiency of termination of DNA synthesis upon incorporation of the 3′-OH unblocked nucleotide or nucleoside ranges from about 90% to about 100%.
86. The method of claim 84, n the efficiency of incorporation of the 3′-OH unblocked nucleotide or nucleoside ranges from about 70% to about 100% ed to the ency of incorporation of a naturally-occurring nucleotide or nucleoside with the same base as the 3′-OH unblocked nucleotide or nucleoside.
87. A method of performing Sanger or Sanger-type sequencing comprising using a compound ing to any one of claims 1-56 as a terminating tide .
88. The method according to any one of claims 57, 68, 81, 84 and 87, where the compound is further defined as a compound of a (I).
89. The method according to any one of claims 57, 68, 81, 84 and 87, where the compound is r defined as a compound of formula (II).
90. The method according to any one of claims 57, 68, 81, 84 and 87, where the compound is further defined as a compound of formula (III).
91. The method according to any one of claims 57, 68, 81, 84 and 87, where the compound is further defined as a compound of formula (IV).
92. The method according to any one of claims 57, 68, 81, 84 and 87, where the compound is further defined as a compound of formula (V).
93. The method according to any one of claims 57, 68, 81, 84 and 87, where the compound is further defined as a compound of formula (VI).
94. The method ing to any one of claims 57, 68, 81, 84 and 87, where the compound is further defined as a compound of formula (VII).
95. A method of determining the sequence of a target nucleic acid comprising (i) adding a target nucleic acid to a Sanger or Sanger-type sequencing apparatus, (ii) adding one or more compounds selected from the group consisting of the following formulas and salts thereof: R5 R5 R6 OMe R6 OMe R6 OMe O2N R4 O2N R4 O H O2N R4 NH2 H R3 O NH H R3 O NH2 R3 O N N O R1 N O O R1 R1 N N O O OH R2 OH R2 OH R2 (I), (II), (III), R5 R5 R6 OMe R6 OMe O2N R4 O2N R4 H H R3 O O R3 NH NH N N R1 N N NH2 R1 N N O O OH R2 OH R2 (IV), (V), R5 R6 OMe R6 OMe O2N R4 O2N R4 H R3 NH R3 O N N N O R1 N N NH2 R1 O O OH R2 OH R2 (VI), or (VII), wherein: R1 is triphosphate, α-thiotriphosphate or polyphosphate; R2 is hydrogen or y; R3 is alkyl(C8) or substituted alkyl(C8); R4 is hydrogen; R5 is a −linker−reporter, wherein the reporter is based on a phore; and the linker is a group of either of the following two formulas: N X H n , or H H N Y N X Y N O m n , X is −O−, −S−, or −NH−; or alkanediyl(C12), alkenediyl(C12), alkynediyl(C12), arenediyl(C12), heteroarenediyl(C12), or a substituted version of any of these groups; Y is −O−, −NH−, diyl(C12) or tuted alkanediyl(C12); n is an integer from 0–6; and m is an integer from 0–6; and R6 is hydrogen; to the sequencing apparatus, with the proviso that compounds of different formulas I-VII have ent fluorophores; (iii) adding a complementary primer and a polymerase enzyme, (iv) performing a polymerase reaction to incorporate at least one of the compounds of step (ii) into a growing nucleic acid strand, and (v) analyzing the result of the Sanger sequencing reaction with fluorescence sequencing instrumentation or by pulsed multiline excitation fluorescence, wherein steps (i)-(iii) can be performed in any order.
96. The method according to claim 95, wherein incorporation of at least one compound ing to step (iv) is followed by termination of strand growth at an efficiency of from about 90% to about 100%.
97. The method according to claim 95, wherein the incorporation of at least one compound according to step (iv) occurs at about 70% to about 100% of the efficiency of incorporation of a native substrate with the same base in the polymerase reaction.
98. The method according to claim 97, n the incorporation efficiency occurs at about 85% to about 100%.
99. The method according to claim 95, wherein the polymerase is ed from the group ting of reverse transcriptase, terminal transferase, and DNA polymerase.
100. A method of incorporating a non-naturally occurring component into a nucleic acid comprising: (i) adding a target nucleic acid to a sequencing apparatus; (ii) adding one or more compounds selected from the group consisting of the following formulas and salts thereof: R5 R5 R6 OMe R6 OMe R6 OMe O2N R4 O2N R4 O H O2N R4 NH2 H R3 O NH H R3 O NH2 R3 O N N O R1 N O R1 R1 N O N O O OH R2 OH R2 OH R2 (I), (II), (III), R5 R5 R6 OMe R6 OMe O2N R4 O2N R4 H H R3 O O R3 NH NH N N R1 N N NH2 R1 N N O O OH R2 (IV), OH R2 (V), R5 R6 OMe R6 OMe O2N R4 O2N R4 H R3 NH R3 O N N N O R1 N N NH2 R1 O O OH R2 (VI), or OH R2 (VII), wherein: R1 is triphosphate, α-thiotriphosphate or osphate; R2 is hydrogen or hydroxy; R3 is alkyl(C8) or substituted alkyl(C8); R4 is hydrogen; R5 is a −linker−reporter, wherein the reporter is based on a fluorophore; and the linker is a group of either of the following two formulas: N X H n , or H H N Y N X Y N O m n , wherein X is −O−, −S−, or −NH−; or alkanediyl(C12), alkenediyl(C12), alkynediyl(C12), arenediyl(C12), heteroarenediyl(C12), or a substituted version of any of these groups; Y is −O−, −NH−, alkanediyl(C12) or substituted alkanediyl(C12); n is an integer from 0–6; and m is an integer from 0–6; and R6 is hydrogen; to the sequencing apparatus, with the proviso that compounds of different formulas I-VII have ent fluorophores; (iii) adding a rase enzyme; and (iv) performing a polymerase reaction to incorporate at least one of the compounds of step (ii) into a growing nucleic acid , wherein steps (i)-(iii) can be med in any order.
101. The method according to claim 100, further comprising (v) analyzing the result of the polymerase chain reaction for incorporation of at least one compound from step (ii).
102. The method according to claim 100, wherein incorporation of at least one compound according to step (iv) is followed by termination of strand growth at an efficiency of from about 90% to about 100%.
103. The method according to claim 100, wherein the incorporation of at least one compound according to step (iv) occurs at about 70% to about 100% of the ency of incorporation of native substrate with the same base in the polymerase on a native substrate with the same base in the polymerase reaction.
104. A method of ming mini-sequencing or minisequencing-type sequencing comprising addition of a compound according to claim 1 to a mini-sequencing or minisequencing-type cing method.
105. The method according to any one of claims 95, 100, and 104, wherein the compound is further defined as a compound of formula (I).
106. The method according to any one of claims 95, 100, and 104, n the compound is further defined as a compound of formula (II).
107. The method according to any one of claims 95, 100, and 104, wherein the compound is further defined as a compound of a (III).
108. The method according to any one of claims 95, 100, and 104, wherein the compound is further defined as a compound of formula (IV).
109. The method according to any one of claims 95, 100, and 104, n the compound is further defined as a compound of formula (V).
110. The method according to any one of claims 95, 100, and 104, wherein the compound is further defined as a compound of formula (VI).
111. The method according to any one of claims 95, 100, and 104, wherein the nd is further defined as a compound of formula (VII).
112. A system comprising: a flowcell comprising a plurality of beads, wherein: each bead attached to a DNA molecule, wherein a compound ed from the group consisting of the following formulas and salts thereof: R5 R5 R6 OMe R6 OMe R6 OMe O2N R4 O2N R4 O H O2N R4 NH2 H NH H R3 O NH2 R3 O R3 O N N O R1 N O R1 R1 N O N O O OH R2 (I), OH R2 OH R2 (II), (III), R5 R5 R6 OMe R6 OMe O2N R4 O2N R4 H H R3 O O R3 NH NH N N R1 N N NH2 R1 N N O O OH R2 (IV), OH R2 (V), R5 R6 OMe R6 OMe O2N R4 O2N R4 H R3 NH R3 O N N N O R1 N N NH2 R1 O O OH R2 (VI), or OH R2 (VII), wherein: R1 is triphosphate, α-thiotriphosphate or polyphosphate; R2 is hydrogen or hydroxy; R3 is alkyl(C8) or substituted alkyl(C8); R4 is hydrogen; R5 is a −linker−reporter, wherein the reporter is based on a fluorophore; and the linker is a group of either of the following two formulas: N X H n , or H H N Y N X Y N O m n , X is −O−, −S−, or −NH−; or alkanediyl(C12), alkenediyl(C12), alkynediyl(C12), iyl(C12), heteroarenediyl(C12), or a substituted version of any of these groups; Y is −O−, −NH−, alkanediyl(C12) or substituted alkanediyl(C12); n is an integer from 0–6; and m is an integer from 0–6; and R6 is hydrogen; and has been incorporated into the DNA molecule using a polymerase; and the ll is at least partially transparent to visible and UV light; an imaging device ured to capture images of the flowcell; a filter wheel comprising at least four spectral filters, wherein the filter wheel is configured to cycle between each filter; a lamp configured to create a light path from the flowcell through a filter in the filter wheel to the imaging device; and an ultraviolet light source configured to provide ultraviolet light to the DNA molecules on the flowcell.
113. The system of claim 112, wherein the flowcell is a luidic flowcell.
114. The system of claim 112, further comprising an objective lens between the filter wheel and the flowcell.
115. The system of claim 112, further comprising a mirror ured to direct the light path to the imaging device.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161534347P | 2011-09-13 | 2011-09-13 | |
US61/534,347 | 2011-09-13 | ||
US201161627211P | 2011-10-07 | 2011-10-07 | |
US61/627,211 | 2011-10-07 | ||
PCT/US2012/055231 WO2013040257A1 (en) | 2011-09-13 | 2012-09-13 | 5-methoxy. 3'-oh unblocked, fast photocleavable terminating nucleotides and methods for nucleic acid sequencing |
Publications (2)
Publication Number | Publication Date |
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NZ622268A NZ622268A (en) | 2016-04-29 |
NZ622268B2 true NZ622268B2 (en) | 2016-08-02 |
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