DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claim(s) 1-9 and 16-18 is/are rejected under 35 U.S.C. 103 as being unpatentable over Blanchard et al. (WO 2013/109859, IDS) (Blanchard) in view of Baker et al. (US 2012/0259114) (Baker).
Regarding claim 1, Blanchard teaches a compound comprising a fluorescent dye moiety, a cyclooctatetraene (COT) moiety, covalent linkers, and a bioconjugate reactive moiety. Specifically, Blanchard teaches:
“cyanine fluorophore compositions in which a protective agent (e.g., triplet state quencher) and a reactive crosslinking group are attached to the cyanine moiety”
and further teaches that the compositions may include “a linking group of desired length (D) between the cyanine moiety and protective agent” (par [0009]).
Thus, Blanchard's cyanine moiety corresponds to the claimed R¹ fluorescent dye moiety, its linking groups correspond to the claimed L¹ and L² covalent linkers, and its reactive crosslinking group corresponds to the claimed R³ bioconjugate reactive moiety.
Blanchard further teaches the claimed eight-membered cyclooctatetraene ring optionally substituted with (R²)z2. Specifically, Blanchard teaches that the protective agent can be a cyclic polyene and states:
“Some examples of annulenes particularly considered herein include cyclooctatetraene (i.e., [8]annulene or COT)”
and further teaches that the annulene “can also be functionalized with any number of hydrocarbon groups, heteroatom-functionalized forms thereof, and heteroatom groups” (par [0065]).
Blanchard additionally teaches COT derivatives bearing substituents including carboxy, sulfonate, hydroxy, alkoxy, nitro, and halide groups, and specifically identifies “1,2-dicarboxycyclooctatetraene, 3-hydroxypropylcyclooctatetrane, sulfonatocyclooctetraene, and 3-sulfonatopropylcyclooctatetraene” (par [0069]). Alternatively, z2 may be 0, as expressly permitted by claim 1, such that Blanchard's unsubstituted COT directly corresponds to the claimed COT ring.
Blanchard further teaches the claimed covalent linker functionality. Blanchard teaches that hydrocarbon linkers may be straight-chain, branched, cyclic, or unsaturated and that the linkers may include oxygen, nitrogen, or sulfur heteroatoms, including ether, polyethyleneoxide, amine, amide, ester, and related functionalities (par [0041]-[0048]). Blanchard specifically demonstrates a three-carbon linker between COT and the fluorogenic center, stating that “Bromo-COT with a three-carbon linker 50 was then successfully linked to the fluorogenic center” (par [0236]).
Blanchard further teaches the claimed bioconjugate reactive moiety because its group M is “a reactive crosslinking group or a group that can be converted to a reactive crosslinking group” (par [0077]). Blanchard teaches reactive crosslinking groups including activated esters, aryl halides, and azide (N₃) (par [0070]). Blanchard further demonstrates a COT-Cy5 compound activated with an NHS ester and teaches that such compounds may be directly reacted with amine-containing biomolecules (par [0236]-[0237]).
Blanchard, however, does not expressly teach arranging the fluorescent dye moiety, COT moiety, and bioconjugate reactive moiety about a central 1,3,5-triazine scaffold as recited in Formula VI.
Baker teaches this missing feature. Baker teaches using:
“triazine scaffolds (e.g., trivalent reagent 2,4,6-trichloro-1,3,5-triazines as a core scaffold) to give access to multifunctional architectures” (par [0012]).
Baker further expressly identifies 1,3,5-triazine and teaches synthesis of the triazine scaffold from cyanuric chloride by consecutive aromatic nucleophilic substitution reactions (par [0013]).
More particularly, Baker teaches:
“trivalent triazine small molecules were used as a core scaffold on which two sites were used for binding (e.g., conjugation) to functional ligands and a third site was used for conjugation to an azide linker” (par [0014]).
Thus, Baker teaches the claimed arrangement of a trivalent 1,3,5-triazine central scaffold having two positions for attachment of functional moieties and a third position bearing a bioconjugate-reactive azide-containing moiety.
Baker further teaches attachment of substituents to the triazine through amino groups, corresponding to W¹ and W² being -NH-, which falls within the claimed -NR¹A- and -NR²A- wherein R¹A and R²A are hydrogen. For example, Baker teaches sequential substitution of cyanuric chloride with 3-azidopropan-1-amine, 2-aminoethanol, and an amino-containing ether linker (par [0194]).
It would have been obvious to one of ordinary skill in the art before the effective filing date to modify the fluorescent dye/COT composition of Blanchard by employing the trivalent 1,3,5-triazine core scaffold of Baker to connect the fluorescent dye-containing linker and COT-containing linker at two positions of the triazine while providing the bioconjugate reactive moiety at the third position, because Baker teaches that the triazine scaffold provides a multifunctional architecture capable of incorporating multiple functional components into a single molecule, and further teaches that “the sequential reactivity of the three chlorine atoms in the triazine backbone makes these molecules well-suited to get high variability, useful for combinatorial synthesis” (par [0074]). Such modification would have been motivated to improve the structural control and versatility of Blanchard's fluorescent dye/COT compositions while retaining Blanchard's photostabilizing COT and bioconjugation functionality. The resulting compound would have the structure recited in claim 1.
Regarding claim 2, Blanchard in view of Baker further teaches L¹ being L¹⁰¹-L¹⁰²-L¹⁰³, wherein the linker portions are selected from the recited linker groups. Baker teaches attaching to the 1,3,5-triazine an amino-containing polyether linker using tert-butyl (2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate, thereby providing a triazine-linked heteroalkylene/polyether chain having a terminal amine (par [0194]). Blanchard teaches NHS-activated fluorescent compounds that react with amino-containing groups and teaches that its linkers may contain alkylene, heteroalkylene, ether/polyethyleneoxide, amine, amide, and ester groups (par [0041]-[0048], [0227]-[0230]). Thus, coupling Baker's terminal amino-containing polyether linker to Blanchard's NHS-activated fluorescent dye provides an L¹ composed, for example, of a heteroalkylene portion, an -NHC(O)- portion, and a bond, each of which is expressly recited for L¹⁰¹, L¹⁰², and L¹⁰³.
Regarding claim 3, Blanchard in view of Baker further teaches the recited polyethylene glycol-containing L¹ linker wherein n1 is 1–10. Baker's amino-containing linker 2-(2-(2-aminoethoxy)ethoxy)ethyl contains repeating ethyleneoxy units and a terminal amino group suitable for subsequent conjugation (par [0194]). Blanchard expressly teaches varying linker length using “polyethyleneglycol building block[s]” and specifically prepares diglycol and tetraglycol linkers (par [0231]-[0232]). Thus, the references teach the recited PEG-containing L¹ linker with a repeat number falling within n1 = 1–10. It would have been obvious to select such a PEG linker because Blanchard expressly teaches varying PEG linker length to control the distance between functional components.
Regarding claim 4, Blanchard in view of Baker further teaches L² being L²⁰¹-L²⁰²-L²⁰³, wherein the linker portions are independently selected from the recited linker groups. Blanchard teaches COT coupled through covalent linker groups and expressly teaches linkers containing alkylene, heteroalkylene, ether/polyethyleneoxide, amine, amide, and ester functionality (par. [0041]-[0048]). Baker further teaches amino-containing polyether linker arms attached to the triazine scaffold (par [0194]). Accordingly, a linker between Baker's triazine and Blanchard's COT may comprise, for example, heteroalkylene, -NHC(O)-, and a bond, corresponding to L²⁰¹-L²⁰²-L²⁰³.
Regarding claim 5, Blanchard in view of Baker further teaches the recited PEG-containing L² linker wherein n2 is 1–10. Blanchard expressly teaches that the distance between the protective agent and fluorogenic center may affect performance and therefore prepares linkers having different lengths using polyethylene glycol building blocks, including diglycol and tetraglycol (par [0231]-[0232]). Baker likewise teaches a triazine-attached amino-containing polyether linker (par [0194]). Thus, the references teach the recited PEG-containing L² linker having a repeat number within n2 = 1–10. It would have been obvious to employ such a PEG-containing linker between Baker's triazine scaffold and Blanchard's COT moiety in order to provide the controlled linker spacing expressly sought by Blanchard.
Regarding claim 6, Baker further teaches W¹ being -NH-. Baker teaches preparing multifunctional 1,3,5-triazines by nucleophilic substitution of cyanuric chloride with amino-containing compounds. For example, Baker substitutes the triazine with 3-azidopropan-1-amine, 2-aminoethanol, and an amino-containing polyether linker (par [0194]), thereby forming substituent arms attached to the triazine through triazine-NH- bonds. Thus, Baker teaches W¹ = -NH-.
Regarding claim 7, Baker further teaches W² being -NH- for the same reasons discussed with respect to claim 6. Baker's multifunctional triazine has multiple positions functionalized by reaction with amino-containing compounds, thereby providing a second substituent arm attached through -NH- (par [0014], [0194]). Thus, Baker teaches W² = -NH-.
Regarding claim 8, Blanchard in view of Baker further teaches -W¹-L¹ being -NH-CH₂-CH₂-NH-C(O)-, which is one of the structures recited in claim 8. Blanchard teaches fluorescent compounds activated with an NHS ester to provide a chemical handle for reaction with a primary amine (par [0227]-[0230]). Blanchard further expressly teaches that “a diamino linker can be employed” in activated-ester coupling reactions (par [0106]). Reaction of an NHS-activated fluorescent compound with ethylenediamine provides fluorophore-C(O)-NH-CH₂-CH₂-NH₂. Baker teaches attachment of an amino group to its triazine through a triazine-NH bond. Thus, attachment of the remaining terminal amino group to Baker's triazine provides, when read outward from the triazine, triazine-NH-CH₂-CH₂-NH-C(O)-fluorophore, corresponding to the claimed -W¹-L¹ structure.
It would have been obvious to employ Blanchard's diamino linker for this attachment because Blanchard expressly teaches diamino linkers for activated-ester coupling, while Baker teaches amino substitution as the mechanism for functionalizing its triazine scaffold.
Regarding claim 9, Blanchard in view of Baker further teaches -W²-L² being -NH-CH₂-CH₂-NH-C(O)-, which is one of the structures recited in claim 9. Blanchard specifically prepares an ethylenediamine derivative of COT by reacting COT-NHS with ethylenediamine (par [0249]). This reaction provides a COT group attached through C(O)-NH-CH₂-CH₂-NH₂. Baker teaches attachment of amino-containing substituents to its triazine scaffold through triazine-NH bonds (par [0194]). Thus, attachment of the remaining amino group of Blanchard's COT-ethylenediamine derivative to Baker's triazine provides triazine-NH-CH₂-CH₂-NH-C(O)-COT, corresponding to the claimed -W²-L² structure.
Regarding claim 11, Blanchard in view of Baker further teaches R¹ being a cyanine moiety. Blanchard expressly teaches cyanine fluorescent dyes and states that when r is 1, 2, 3, and 4, the cyanine compounds correspond respectively to Cy3, Cy5, Cy7, and Cy9 derivatives (par [0082]). Thus, Blanchard teaches the claimed R¹ being a cyanine moiety, which is one of the alternatives recited in claim 11.
Regarding claim 16, Blanchard teaches a biomolecule covalently attached to a detectable fluorescent label. Blanchard teaches that its cyanine fluorophore compositions include a protective agent such as COT and a reactive crosslinking group for attaching the composition to a molecule of interest (par [0009]). Blanchard further teaches a dye-molecule composition wherein “Y is a biomolecule”, including peptides, proteins, nucleotides, oligonucleotides, and nucleic acids (par [0108]). Thus, Blanchard teaches a biomolecule covalently attached to a detectable label.
Blanchard further teaches the detectable label comprising a cyanine fluorescent dye moiety, a COT moiety, and covalent linkers. Blanchard specifically teaches COT-linked cyanine dyes and states that “Bromo-COT with a three-carbon linker 50 was then successfully linked to the fluorogenic center” (par [0236]). Blanchard also teaches that COT may be unsubstituted or functionalized with various groups, including carboxy, hydroxy, sulfonate, and other groups (par [0065], [0069]). This teaches the claimed R¹ fluorescent dye moiety, COT ring optionally substituted with (R²)z2, and L¹/L² covalent linker functionality.
Blanchard does not expressly teach arranging these functional portions about the 1,3,5-triazine central scaffold of Formula VII.
Baker teaches this limitation. Baker teaches 1,3,5-triazine as a trivalent central scaffold, wherein two sites are used for attachment of functional ligands and a third site is used for conjugation (par [0012]-[0014]). Baker further teaches functionalizing the triazine with amino-containing substituents, thereby forming -NH- connections encompassed by the claimed W¹ and W² groups (par [0194]).
It would have been obvious to one of ordinary skill in the art before the effective filing date to modify the biomolecule-attached fluorescent dye/COT composition of Blanchard by employing Baker's trivalent 1,3,5-triazine as a central scaffold for attachment of the fluorescent dye-containing linker and COT-containing linker, while using the remaining triazine position for covalent attachment to the biomolecule, because Baker teaches using the triazine scaffold to provide multifunctional architectures incorporating multiple functional components into a single molecule and teaches that its sequentially reactive triazine positions provide high structural variability. The resulting biomolecule and detectable label would have the structure recited in claim 16. Claim 16 recites this biomolecule-label architecture.
Regarding claim 17, Blanchard in view of Baker further teaches directing an excitation beam onto the biomolecule comprising the detectable moiety and detecting light emission from the detectable moiety, wherein the biomolecule is that of claim 16.
Blanchard specifically teaches Cy5-COT compounds covalently linked to DNA oligonucleotides and imaged using a total internal reflection microscope under continuous laser excitation at 641 nm (par [0032]). Blanchard further teaches that fluorescence from the molecules illuminated by the 641 nm laser was collected using an objective and imaged onto an EMCCD camera (par [0317]). Thus, Blanchard teaches directing an excitation beam onto the labeled biomolecule and detecting light emission from the detectable moiety, as required by claim 17.
Regarding claim 18, Blanchard in view of Baker teaches the compound of claim 1 as discussed above. Baker further expressly teaches providing functionalized triazine compositions in a kit. Specifically, Baker teaches:
“kits comprising one or more of the reagents and tools necessary to generate a dendrimer conjugated with one or more triazine compositions”
including “triazine compositions having one or more functional groups” (par [0160]).
It would have been obvious to one of ordinary skill in the art to provide the fluorescent triazine compound of Blanchard in view of Baker in a kit, because Baker expressly teaches providing functionalized triazine compositions and associated reagents in kit form for their subsequent conjugation and use. The resulting kit would comprise the compound of claim 1, as required by claim 18.
Claim(s) 10 is/are rejected under 35 U.S.C. 103 as being unpatentable over Blanchard in view of Baker as applied to claim 1 above, and further in view of Mori et al. (US 2013/0177770) (Mori).
Regarding claim 10, Blanchard in view of Baker teaches the compound of claim 1 as discussed above. Claim 10 further requires R³ to be -N₃, -NH₂, -CN, -COOH, the recited activated ester group, or an alkynyl group.
Blanchard teaches that the reactive crosslinking group used for bioconjugation may be an azide (“N3”). Specifically, Blanchard teaches that amino-reactive groups include “dichlorotriazines, aryl halides, and azide (‘N3’)” (par [0070]). Thus, Blanchard teaches -N₃ as a bioconjugate reactive moiety.
Baker teaches a multifunctional 1,3,5-triazine core scaffold having independently functionalizable positions. Baker teaches that “trivalent triazine small molecules were used as a core scaffold on which two sites were used for binding (e.g., conjugation) to functional ligands and a third site was used for conjugation to an azide linker” (par [0014]). However, Baker does not expressly teach R³ being -N₃ directly attached to the 1,3,5-triazine ring.
Mori teaches this limitation. Mori teaches 6-azide-2,4-bis(amino-substituted)-1,3,5-triazines, including 6-azide-2,4-bis(ethanolamino)-1,3,5-triazine, thereby teaching a 1,3,5-triazine having -N₃ directly attached to one position of the triazine while the other two positions are amino-substituted (par [0116]).
Mori further prepares 2-azide-4,6-dichloro-1,3,5-triazine and subsequently substitutes the two chlorine positions with amino-containing groups to form 2-azide-4,6-bis(3-triethoxysilylpropyl)amino-1,3,5-triazine, demonstrating that the directly attached azide may be retained while the other two triazine positions are functionalized (par [0193]-[0199]).
It would have been obvious to one of ordinary skill in the art before the effective filing date to modify the triazine scaffold of Blanchard in view of Baker by employing Mori's azido-substituted 1,3,5-triazine arrangement such that the third triazine position bears -N₃ directly, because Blanchard teaches azide as a suitable bioconjugate reactive moiety, Baker teaches independently functionalizing the positions of a multifunctional 1,3,5-triazine scaffold, and Mori teaches the known arrangement wherein an azide is directly attached to one triazine position while the remaining two positions are available for amino-linked functionalization. The resulting compound would therefore have R³ being -N₃, as required by claim 10.
Allowable Subject Matter
Claim 12-15 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
The following is a statement of reasons for the indication of allowable subject matter: The prior art of record does not disclose or fairly suggest the formulas as recited in claim 12-15.
Conclusion
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/XIAOYUN R XU, Ph.D./ Primary Examiner, Art Unit 1797
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