The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Figure 9 should be designated by a legend such as --Prior Art-- because only that which is old is illustrated. See MPEP § 608.02(g). Corrected drawings in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. The replacement sheet(s) should be labeled “Replacement Sheet” in the page header (as per 37 CFR 1.84(c)) so as not to obstruct any portion of the drawing figures. If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Figure 2 of the cited Frost reference (Analytical Chemistry 2015, NPL C79, IDS filed 5-13-24) is equivalent to what is shown in figure 9 so that the content of instant figure 9 is prior art.
Claim 1 is objected to because of the following informalities: “c) a conjugation moiety” was apparently intended. Appropriate correction is required.
Claims 1-10 and 13-22 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention. With respect to claims 1 and 13, it is not clear if the isobaric tag, the enrichment moiety and the conjugation moiety are bound together in any particular order or need to have a particular structural relationship in order to be usable. For example, can the isobaric tag be bonded between the enrichment moiety and the conjugation moiety in a linear type of configuration? Alternatively, can the enrichment moiety be bonded between the isobaric tag and the conjugation moiety in a linear type of configuration? Alternatively, can the conjugation moiety be bonded between the isobaric tag and the enrichment moiety in a linear type of configuration? Alternatively, are the three components linked to a central atom/moiety? All other claims in this rejection depend from the two independent claims and fail to correct the problem.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1, 4-6, 9, 13, 18-19 and 21 are rejected under 35 U.S.C. 102(a)(1) as being clearly anticipated by Li (Chemical Communications 2007, newly cited, applied and hereinafter called Li ‘07) or Hoffmann (US 2010/0190183, newly cited and applied).
In the paper Li ‘07 teaches the development of a new reagent, called CILAT (Cleavable Isobaric Labeled Affinity Tag), for quantitative proteomics, which represents an improvement over the ICAT (Isotope-Coded Affinity Tag) and iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) methods at that time. With respect to claim 1, the CILAT molecule shown in figure 1a constitutes mass spectrometry tagging reagent comprising a compound having: a) an isobaric tag comprising a reporter group having at least one atom that is optionally isotopically labeled and a balancing group also having at least one atom that is optionally isotopically labeled (see figure 1a and figure 1c with their associated discussion); b) an enrichment moiety (see the affinity tag and cleavable linker portions of the compound in figure 1a); and c) a conjugation moiety able to bind to a molecule of interest (the reactive group portion of the compound in figure 1a). With respect to claim 4, one or more carbon atoms in the reporter group are 13C, one or more oxygen atoms are 18O, one or more nitrogen atoms in the reporter group 15N, or combinations thereof (see figure 1a and figure 1c with their associated discussion). With respect to claims 5-6, the enrichment moiety comprises biotin or a biotin derivative having the formula required by claim 6 (see the affinity portion of figure 1a with its associated discussion). With respect to claim 9, there are one or more cleavable linkers positioned between the isobaric tag, enrichment moiety, conjugation moiety, or combinations thereof (see the cleavable linker portion of the compound in figure 1a). With respect to claim 13, Li teaches a method of analyzing a molecule, said method comprising the steps of: a) providing the molecule (see figures 1 and 2 with their associated discussion in the paragraph bridging pages 2181-2182 and the following paragraph on page 2182); b) labeling the molecule with a compound having: i) an isobaric tag comprising a reporter group having at least one atom that is optionally isotopically labeled and a balancing group also having at least one atom that is optionally isotopically labeled; ii) an enrichment moiety; and iii) a conjugation moiety able to bind to a molecule of interest (see the description related to claim 1 above); c) purifying or enriching the labeled molecule (see figures 1 and 2 with their associated discussion in the paragraph bridging pages 2181-2182 and the following paragraph on page 2182); d) fragmenting the purified molecule to generate an immonium ion from the reporter group of the purified molecule (see figures 1 and 2 with their associated discussion in the paragraph bridging pages 2181-2182 and the following paragraph on page 2182); and e) detecting and analyzing fragments of the purified molecule (see figures 1 and 2 with their associated discussion in the paragraph bridging pages 2181-2182 and the following paragraph on page 2182). With respect to claims 18-19, the enrichment moiety comprises biotin or a biotin derivative having the formula required by claim 19 (see the affinity portion of figure 1a with its associated discussion). With respect to claim 21, there are one or more cleavable linkers positioned between the isobaric tag, enrichment moiety, conjugation moiety, or combinations thereof (see the cleavable linker portion of the compound in figure 1a).
In the patent publication Hoffmann teaches new isotopic and isobaric tagged molecules, kits comprising them and methods of using them in mass spectrometry. With respect to claim 1, the molecules shown in figures 5-8 constitute mass spectrometry tagging reagent comprising a compound having: a) an isobaric tag comprising a reporter group having at least one atom that is optionally isotopically labeled and a balancing group also having at least one atom that is optionally isotopically labeled (see the isobaric part of the molecules in figures 5-8 in combination with figure 3 and their respective discussion); b) an enrichment moiety (see the acid cleavable biotin portion of figures 5-8); and c) a conjugation moiety able to bind to a molecule of interest (the maleimide Cys-reactive group portion of figures 5-8). With respect to claim 4, one or more carbon atoms in the reporter group are 13C, one or more oxygen atoms are 18O, one or more nitrogen atoms in the reporter group 15N, or combinations thereof (see figure 3 with its associated discussion). With respect to claims 5-6, the enrichment moiety comprises biotin or a biotin derivative having the formula required by claim 6 (see the acid cleavable biotin portion of figures 5-8). With respect to claim 9, there are one or more cleavable linkers positioned between the isobaric tag, enrichment moiety, conjugation moiety, or combinations thereof (see the acid cleavable biotin portion of figures 5-8). With respect to claim 13, Hoffmann teaches a method of analyzing a molecule, said method comprising the steps of: a) providing the molecule (see figures 1 and 3 with their associated discussion); b) labeling the molecule with a compound having: i) an isobaric tag comprising a reporter group having at least one atom that is optionally isotopically labeled and a balancing group also having at least one atom that is optionally isotopically labeled; ii) an enrichment moiety; and iii) a conjugation moiety able to bind to a molecule of interest (see the description related to claim 1 above in combination with figures 1 and 3 with their respective discussion); c) purifying or enriching the labeled molecule (see figure 1 with its associated discussion); d) fragmenting the purified molecule to generate an immonium ion from the reporter group of the purified molecule (see figures 1 and 3 with their associated discussion); and e) detecting and analyzing fragments of the purified molecule (see figures 1 and 3 with their associated discussion). With respect to claims 18-19, the enrichment moiety comprises biotin or a biotin derivative having the formula required by claim 19 (see the acid cleavable biotin portion of figures 5-8). With respect to claim 21, there are one or more cleavable linkers positioned between the isobaric tag, enrichment moiety, conjugation moiety, or combinations thereof (see the acid cleavable biotin portion of figures 5-8).
Claims 1, 4-6, and 9 are rejected under 35 U.S.C. 102(a)(1) as being clearly anticipated by Yuan (Chemical Communications 2013, newly cited and applied). global profiling of carbonyl metabolites with a photo-cleavable isobaric labeling affinity tag. A carbonyl-reactive photo-cleavable isobaric labeling affinity tag can provide a selective, high-throughput, and reproducible approach for the quantitative analysis of aldehyde and ketone metabolites in complex biological samples. As illustrated in figure 1A, this molecule consists of three parts, an aminooxy functional group for specific coupling with carbonyls (the conjugation moiety able to bind to a molecule of interest required by claim 1), an isobaric tag for quantification (the isobaric tag comprising a reporter group having at least one atom that is optionally isotopically labeled and a balancing group also having at least one atom that is optionally isotopically labeled required by claim 1), and a photo-cleavable biotin for affinity enrichment of labeled carbonyls (the enrichment moiety required by claim 1). Since each required tag/moiety is present in the Yuan compound, claim 1 is anticipated. Similar to other reagents for the labeling of aldehydes and ketones, carbonyl-CILAT introduces a tertiary amine to enhance the ionization characteristics of derivatized compounds for MS detection (see Table S1 and figure S4). The ability of purifying tagged carbonyls can further improve this sensitivity by removing interferences from other small molecule metabolites. More importantly, carbonyl-CILAT uses an isobaric, rather than a mass-shift tag, for the quantification of multiple analytes under different environmental conditions. This isobaric structure is adopted from a previously developed Deuterium isobaric Amine Reactive Tag (DiART) used for proteomic analysis. Thanks to isotopic nuclei on various positions, each isobaric tag has the same nominal mass, but yields a different low mass reporter ion upon MS/MS fragmentation. These reporter ions supply quantitative information and are used for relative quantitation. For example, CILAT114 has 13C on position 1 and 14N on position 2, while CILAT115 carries 12C on position 1 and 15N on position 2. Upon fragmentation, these two tags can generate strong signature peaks at m/z = 114 and 115 respectively. In addition, in the paragraph bridging pages 11080-11081, they emphasize that although they only synthesized CILAT114 and CILAT115 for the relative quantification of two samples, it can be easily expanded for concurrent quantification of up to six samples as in the DiART from which it was adopted. With respect to claim 4, one or more carbon atoms in the reporter group are 13C, one or more oxygen atoms are 18O, one or more nitrogen atoms in the reporter group 15N, or combinations thereof (see the paragraph bridging pages 11080-11081). With respect to claims 5-6, the enrichment moiety comprises biotin or a biotin derivative having the formula required by claim 6 (see the biotin portion of figure 1A with its associated discussion). With respect to claim 9, there are one or more cleavable linkers positioned between the isobaric tag, enrichment moiety, conjugation moiety, or combinations thereof (see the photo-cleavable linker portion of the compound in figure 1A).
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 7-8, 10, 20 and 22 are rejected under 35 U.S.C. 103 as being unpatentable over Li ‘07 as applied to claims 1, 9, 13 and 21 above, and further in view of Szychowski (Journal of the American Chemical Society 2010, newly cited and applied). The paragraph bridging the columns on page 2182 of Li ‘07 teaches that the module solid phase synthesis of the CILAT molecules would make it easy to exchange each component, such as the replacement of the biotin with a fluorous tag as a different route for affinity purification. With respect to claims 7-8 and 20, Li ‘07 does not teach that the conjugation moiety comprises an azide or protected azide group or is able to bind to a terminal alkyne or dibenzocyclooctyne (DBCO) group having the claimed formula. With respect to claims 10 and 22, Li ‘07 does not teach that the one or more linkers comprise a polyethylene glycol (PEG) linker, a Boc linker, a DADPS linker, a diol linker, an aminophenol linker, or combinations thereof.
In the paper Szychowski teaches the azide-alkyne cycloaddition provides a powerful tool for bio-orthogonal labeling of proteins, nucleic acids, glycans, and lipids. In some labeling experiments, e.g., in proteomic studies involving affinity purification and mass spectrometry, it is convenient to use cleavable probes that allow release of labeled biomolecules under mild conditions. Five cleavable biotin probes are described for use in labeling of proteins and other biomolecules via azide-alkyne cycloaddition. Subsequent to conjugation with metabolically labeled protein, these probes are subject to cleavage with either 50 mM Na2S2O4 (see schemes 2 and 6 with their associated discussion), 2% HOCH2CH2SH, 10% HCO2H, 95% CF3CO2H (see schemes 3-6 with their associated discussion), or irradiation at 365 nm (see schemes 1 and 6 with their associated discussion). Most strikingly, a probe constructed around a dialkoxydiphenylsilane (DADPS, see schemes 3 and 6 with their associated discussion) linker was found to be cleaved efficiently when treated with 10% HCO2H for 0.5 h. A model green fluorescent protein was used to demonstrate that the DADPS probe undergoes highly selective conjugation and leaves a small (143 Da) mass tag on the labeled protein after cleavage. These features make the DADPS probe especially attractive for use in biomolecular labeling and proteomic studies. The introduction on page 18351 teaches that the azide-alkyne cycloaddition is selective, efficient, and broad in scope: a paradigmatic example of a “click” reaction. The discovery that copper catalysis allows the cycloaddition to be effected under mild conditions has stimulated numerous studies of labeling of proteins, glycans, lipids, and RNA. For example, they recently reported the Bio-orthogonal Non-Canonical Amino Acid Tagging (BONCAT) method for selective identification of newly synthesized proteins in cells. In the BONCAT approach, newly synthesized proteins are distinguished from the preexisting pool of proteins by cotranslational incorporation of a reactive noncanonical amino acid (e.g., L-homopropargylglycine (Hpg, 1), Figure 1). Azide or alkyne functional groups on the side chains of such noncanonical amino acids can serve as bio-orthogonal handles for selective conjugation to affinity probes through the copper-catalyzed azide-alkyne cycloaddition reaction. Once conjugated, newly synthesized proteins can be selectively enriched through conventional affinity purification protocols that exploit the biotin-streptavidin interaction. Proteins are released from the resin and identified by mass spectrometry. Azide or alkyne functional groups incorporated into proteins can also be used for cell-selective labeling of newly synthesized proteins.
With respect to claims 7-8 and 20, it would have been obvious to one of ordinary skill in the art at the time the application was filed to modify the Li ‘07 conjugation moiety to the azide taught by Szychowski because of the ability to modify the different components of the Li ‘07 tag and the selective, efficient, and broad use of the azide-alkyne cycloaddition labeling of proteins, glycans, lipids, and RNA as taught by Szychowski. With respect to claims 10 and 22 it would have been obvious to one of ordinary skill in the art at the time the application was filed to modify the Li ‘07 tagging compound by replacing the cleavable linker in the Li ‘07 compound with any of the cleavable linkers taught by Szychowski because of the ability to modify the different components of the Li ‘07 tag and the use of the various cleavable linkers taught by Szychowski and in particular the benefits of the dialkoxydiphenylsilane (DADPS) linker in being able to remove a biotin moiety used for affinity purification prior to mass spectral analysis.
Claims 7-8, 10, 20 and 22 are rejected under 35 U.S.C. 103 as being unpatentable over Hoffmann as applied to claims 1, 9, 13 and 21 above, and further in view of Szychowski (as described above). Paragraphs [0069]-[0072] teach that the functional group, L, of the molecule may be an aliphatic hydrocarbon chain that contains a cleavable moiety, the latter may be selected from the group comprising an acid labile moiety, a base labile moiety, a moiety which is cleavable by UV irradiation, a moiety which is cleavable by microwave irradiation, a moiety which is cleavable by change of electric potential, a moiety which is cleavable by oxidation, a moiety which is cleavable by reduction, a moiety which can be altered by olefin metathesis, a moiety which can be altered by disulfide exchange and a moiety which is cleavable by a protease such as trypsin. The person skilled in the art will be aware that the specific molecular structure of the cleavable group L may differ depending on which cleavage mechanism is envisaged. If for example a site specific protease is used, the group L should comprise a corresponding peptide sequence. The advantage of using e.g. a protease for cleavage is that non-specific cleavage of the peptides to be analyzed is avoided. This, of course, presumes that no protease recognition sequences are found within the peptides to be analyzed. This can be assured by first digesting the polypeptides to be analyzed with the respective protease, to label the resulting peptides, to enrich the labelled peptides via the group A and to then cleave of the group A by using the same protease. In a preferred embodiment, L contains a cleavable moiety which is selected from the group comprising disulfide moieties and acid labile moieties. In a particularly preferred embodiment, L contains an acid labile moiety as a cleavable moiety. In another particularly preferred embodiment, L contains a disulfide moiety as a cleavable moiety. Paragraphs [0095]-[0103] teach that the functional group RG which is a reactive group for the selective binding of biomolecules such as, but not limited to proteins or peptides, can be selected from the group comprising amino-reactive chemical groups, thiol-reactive chemical groups, hydroxyl-reactive chemical groups and carboxyl-reactive chemical groups. In a preferred embodiment RG is a thiol-reactive chemical group. In a more preferred embodiment RG is iodoacetamide. In another more preferred embodiment RG is a maleimide. In another preferred embodiment RG amine-reactive chemical group. In a more preferred embodiment RG is an NHS-ester. In another more preferred embodiment RG is an imidoester. In terms of availability, stability and coupling efficiency maleimides and NHS-esters can be preferred as reactive groups. It is important to note at this point, that some combinations of cleavable linker groups L and reactive groups RG will lead to problems due to inter- or intramolecular reactivity of the molecules disclosed in the present invention with themselves. An example for this would be the combination of a disulfide-containing linker L and a thiol-reactive group RG. A person skilled in the art will have to take this fact into account upon designing molecules as disclosed in the present invention, and avoid such unfavorable combinations. Therefore, L and RG are chosen in a way that avoids inter- or intramolecular reactivity of the molecules with themselves. The before said also applies to the other groups of the inventive molecules. A person skilled in the art would consider this without being explicitly told. With respect to claims 7-8 and 20, Hoffmann does not teach that the conjugation moiety comprises an azide or protected azide group or is able to bind to a terminal alkyne or dibenzocyclooctyne (DBCO) group having the claimed formula. With respect to claims 10 and 22, Hoffmann does not teach that the one or more linkers comprise a polyethylene glycol (PEG) linker, a Boc linker, a DADPS linker, a diol linker, an aminophenol linker, or combinations thereof.
With respect to claims 7-8 and 20, it would have been obvious to one of ordinary skill in the art at the time the application was filed to modify the Hoffmann conjugation moiety to the azide taught by Szychowski because of the variability of the conjugation/reactive moiety of the Hoffmann tags and the selective, efficient, and broad use of the azide-alkyne cycloaddition labeling of proteins, glycans, lipids, and RNA as taught by Szychowski. With respect to claims 10 and 22 it would have been obvious to one of ordinary skill in the art at the time the application was filed to modify the Hoffmann tagging compounds by replacing the cleavable linker in the Hoffmann compound with any of the cleavable linkers taught by Szychowski because of the variability of the types of useable cleavable linkers taught by Hoffmann and the use of the various cleavable linkers taught by Szychowski and in particular the benefits of the dialkoxydiphenylsilane (DADPS) linker in being able to remove a biotin moiety used for affinity purification prior to mass spectral analysis.
Claims 2-3 and 17 are rejected under 35 U.S.C. 103 as being unpatentable over Hoffmann as applied to claims 1 and 13 above, and further in view of Li (US 2013/0078728, newly applied and hereinafter called Li ‘728). Paragraphs [0075]-[0086] of Hoffmann teach that the molecule which is subject-matter of the present invention the functional group IB (differently labelled isobaric tag groups IB) comprises an isotopically labelled reporter group and an isotopically labelled counterbalancing group (see figure 3A). The IB can be labelled with various atoms which occur in more than one stable isotopic form: hydrogen isotopes 1H and/or 2H; carbon isotopes 12C and/or 13C; nitrogen isotopes 14N and/or 15N and/or oxygen isotopes 16O and/or 18O. The functional group IB may comprise a heterocyclic moiety. Labelling with the carbon isotopes 12C and/or 13C may be particularly preferred as these can be incorporated into molecular structures which are less prone to non-intended reactions during fragmentation in MS analysis. The functional group IB of the molecule may comprise an isotopically labelled reporter group and an isotopically labelled counterbalancing group as mentioned above. More specifically, IB may comprise a predetermined breaking point between the isotopically labelled reporter group and the isotopically labelled counterbalancing group. In a preferred embodiment of the present invention, IB comprises a predetermined breaking point between the isotopically labelled reporter group and an isotopically labelled counterbalancing group, whereby the molecule breaks upon induction by collision induced fragmentation. In a more preferred embodiment of the present invention the functional group IB is an N-Alkyl-N-Acetyl-1,4-Piperazine derivative. In a particularly preferred embodiment the structure of the isobaric group IB is as shown in figure 3A). with respect to claims 2-3 and 17, Hoffmann does not teach the reporter group comprises one or more dimethylated, diethylated, acylated, or asymmetric alkylated amino acids, where the reporter group contains at least a portion of the one or more dimethylated, diethylated, acylated, or asymmetric alkylated amino acids comprising or derived from N,N - dimethyl leucine (DiLeu); N,N - dimethyl isoleucine (Di lle); N,N - dimethyl alanine (DiAla); N,N - dimethyl glycine (DiGly); N,N - dimethyl valine (DiVal); N,N - dimethyl histidine (DiHis); N,N - dimethyl phenylalanine (DiPhe); N,N - dimethyl tryptophan (DiTrp); N,N – dimethyl lysine (DiLys), or N,N - dimethyl tyrosine (DiTyr).
In the patent publication Li ‘728 teaches compositions and methods of tagging peptides and other molecules using novel isobaric tandem mass tagging reagents, including novel N,N-dimethylated amino acid 8-plex and 16-plex isobaric tandem mass tagging reagents. The tagging reagents comprise: a) a reporter group having at least one atom that is optionally isotopically labeled; b) a balancing group, also having at least one atom that is optionally isotopically labeled, and c) an amine reactive group. The tagging reagents disclosed herein serve as attractive alternatives for isobaric tag for relative and absolute quantitation (iTRAQ) and tandem mass tags (TMTs) due to their synthetic simplicity, labeling efficiency and improved fragmentation efficiency. Paragraph [0016] teaches that the tagging reagents of the present invention are derived from a dipeptide comprising two amino acids. Preferably the amino acids are natural amino acids, but the present invention contemplates the use of unnatural, non-standard and synthetic amino acids, such as b amino acids, as the amino acid which makes up the reporter group, the balancing group, or both. Further, the tagging reagents of the present invention are derived from a dipeptide where the amino group of one amino acid has been dimethylated. The free carboxyl group of the dipeptide can be attached to an amine reactive group or a target molecule, such as a peptide. During MS2 fragmentation, the dipeptide will fragment to form a reporter ion, preferably an immonium ion, which can be readily detected. The tagging reagents are derived from N,N-dimethyl leucine (DiLeu); N,N-dimethyl isoleucine (Di Ile); N,N-dimethyl alanine (DiAla); N,N-dimethyl glycine (DiGly); N,N-dimethyl valine (DiVal); N,N-dimethyl histidine (DiHis); N,N-dimethyl phenylalanine (DiPhe); N,N-dimethyl tryptophan (DiTrp); N,N-dimethyl lysine (DiLys) or N,N-dimethyl tyrosine (DiTyr). Paragraph [0183] describes previous work of the inventors related to a set of novel N,N-dimethylated leucine (DiLeu) 4-plex reagents as an attractive alternative to isobaric tag for relative and absolute quantitation (iTRAQ) reagent for protein and peptide quantitation. A notable feature of this labeling approach is the production of intense immonium al ions when a dimethylated amino acid (such as a dimethylated leucine) undergoes tandem mass spectroscopy (MS2) dissociation. The formation of the dimethylated al ion from the previous 4-plex reagent is shown in figure 1 along with the immonium ion formed by the iTRAQ reporter ion. Examiner notes that the iTRAQ tag is the same molecule shown in figure 3A) of Hoffmann. Paragraph [0184] teaches that the DiLeu 4-plex isobaric MS2 tagging reagents provided high quantitation efficacy and greatly reduced cost for peptidomics and proteomics. DiLeu reagents also serve as attractive alternatives for iTRAQ and tandem mass tags (TMTs) due to their synthetic simplicity, labeling efficiency and improved fragmentation efficiency. These reagents maintain the isobaric quantitation features present in the iTRAQ reagents, with comparable performance and greatly reduced cost. This makes the DiLeu tandem MS tags a more affordable tool for routine quantitation and methodology development as compared to commercially available 4-plex reagents. Paragraph [0185] teaches that while the previous 4-plex DiLeu reagents provide a cost effective alternative to previously existing isobaric labels (i.e. iTRAQ reagents) for peptide and protein quantitation, the demand for high throughput peptide/protein liquid chromatography MS2 makes 8-plex reagents and 16-plex reagents an even more attractive alternative. Building off of the previous work with 4-plex isobaric tagging reagents, the inventors of synthesized isobaric 8-plex tagging reagents which can also be used to provide 16-plex reagents. In the case of the 4-plex reagents, the number of reporter ions was limited by the balancing group, in that case a carbonyl group. Unfortunately, the carbonyl group could only be modified 4 different ways using 13C and 18O. To overcome this limitation, the present invention uses an amino acid to form the balancing group. Amino acids were chosen for the balancing group for several reasons: they can bear more isotopes than a carbonyl group, isotopic amino acids with various isotopic combinations are readily commercially available, and the methods of coupling two amino acids are well established. Figure 3 lists various immonium ions formed by different amino acids. Immonium ions able to give strong signals in MS2 are indicated in bold. Paragraph [0187] teaches that isotopic dimethylated leucines can readily be made from commercially available leucine compounds. Different possible reporter ions and their corresponding mass (m/z) from isotopic dimethylated leucines are shown in figure 2. Similar reporter ions are also available for other dimethylated amino acids. The incorporation of other dimethylated amino acids into the tagging reagents is also useful since several amino acids are able to form strong immonium ions during fragmentation (see figure 3). For example, figures 4-9 show synthesis steps used to form exemplary dimethylated amino acids and QTOF MS2 spectra of those dimethylated amino acids showing the presence of the immonium reporter ions: dimethylated leucine, dimethylated glycine, dimethylated alanine, dimethylated valine, dimethylated isoleucine and dimethylated phenylalanine.
With respect to claims 2-3 and 17, it would have been obvious to one of ordinary skill in the art at the time the application was filed to replace the iTRAQ isobaric reporter group/tags of Hoffmann with the demethylated amino acid isobaric reporter group/tags of Li ‘728 because of their cost and ease of preparation advantages compared to the iTRAQ isobaric reporter groups as taught by Li ‘728.
Claims 7-8, 15-16 and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Li ’07 or Hoffmann, both as described above and as applied to claims 1 or 13 above, and further in view of Chuh (Current Opinion in Chemical Biology 2015, newly cited and applied), Wright (Natural Product Reports 2016, newly cited and applied) or Wijetunge (Bioconjugate Chemistry 2021, newly cited and applied). With respect to claims 7-8, 15-16 and 20, Li ’07 and Hoffmann do not teach that the conjugation moiety comprises an azide or protected azide group or is able to bind to a terminal alkyne or dibenzocyclooctyne (DBCO) group having the claimed formula.
In the paper Chuh looks at chemical methods for the proteome-wide identification of post-translationally modified proteins. Thousands of proteins are subjected to posttranslational modifications that can have dramatic effects on their functions. Traditional biological methods have struggled to address some of the challenges inherit in the unbiased identification of certain posttranslational modifications. As with many areas of biological discovery, the development of chemoselective and bioorthogonal reactions and chemical probes has transformed our ability to selectively label and enrich a wide variety of posttranslational modifications. Collectively, these efforts are making significant contributions to the goal of mapping the protein modification landscape. Figure 1 presents chemical methods to tag and enrich post-translationally modified proteins. In portion (a) of the figure, some posttranslational modifications (PTMs) can be specifically reacted with enrichment tags using chemoselective reactions. In portion (b) of the figure, bioorthogonal reactions in combination with chemical reporters enable the installation of affinity tags. Chemical reporters can either be incorporated into PTMs using cellular metabolism or appended to existing PTMs using enzymes or selective chemical reactions. Figure 2 shows bioorthogonal reactions that occur selectively between two abiotic functional-groups. Of particular relevance to the instant claims is portion (b) showing the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) to give a stable triazole product from azides and alkynes. This common bioorthogonal reaction can be performed in both directions; however, azide-tags give reduced background signals. Figures 4-6 show methods of adding alkyne groups to report protein modifications.
In the paper Wright describes chemical proteomics approaches for identifying the cellular targets of natural products. Deconvoluting the mode of action of natural products and drugs remains one of the biggest challenges in chemistry and biology today. Chemical proteomics is a growing area of chemical biology that seeks to design small molecule probes to understand protein function. In the context of natural products, chemical proteomics can be used to identify the protein binding partners or targets of small molecules in live cells. Highlighted are recent examples of chemical probes based on natural products and their application for target identification. The review focuses on probes that can be covalently linked to their target proteins (either via intrinsic chemical reactivity or via the introduction of photocrosslinkers), and can be applied “in situ” – in living systems rather than cell lysates. They also focus on strategies that employ a click reaction, the copper-catalyzed azide–alkyne cycloaddition reaction (CuAAC), to allow minimal functionalization of natural product scaffolds with an alkyne or azide tag. They also discuss ‘competitive mode’ approaches that screen for natural products that compete with a well-characterized chemical probe for binding to a particular set of protein targets. Fueled by advances in mass spectrometry instrumentation and bioinformatics, many modern strategies are now embracing quantitative proteomics to help define the true interacting partners of probes, and they highlight the opportunities this rapidly evolving technology provides in chemical proteomics. Finally, some of the limitations and challenges of chemical proteomics approaches are discussed. Figure 1 shows a compound-centric chemical proteomics approach for identifying the targets of natural products (NPs). The probe is designed based on the structure of the NP, is added to live cells and binds to its target protein. It reacts covalently (via an electrophilic trap or a photocrosslinking group) with the target protein. Cells are lysed and samples subject to copper-catalyzed azide–alkyne cycloaddition (CuAAC) to attach a fluorophore or affinity label for downstream analysis. Figure 6 shows cleavable linkers for chemical proteomics. In portion (a) of the figure, proteins enriched on resin e.g. by biotin–streptavidin, can be specifically released and then analyzed by MS (bottom), or digested on bead and then the modified peptide selectively released (top). In portion (b) of the figure, the isotopically-encoded protease (TEV)-cleavable linker 1 for CuAAC and enrichment reported by Cravatt and co-workers is shown. Table 1 shows NP-inspired probes applied for target protein identification studies using gel-based proteomics. The probes are modified to introduce bioorthogonal alkyne tag. Figure 10 shows the alkylation of proteins by aspirin along with the terminal alkyne group containing aspirin-based probes 21 that have been reported and the azide containing cleavable linker 22 for the detection of aspirin probe-acylated peptide sites.
In the paper Wijetunge describes a copper-free Click enabled triazabutadiene for biorthogonal protein functionalization. Aryl diazonium ions have long been used in bioconjugation due to their reactivity toward electron-rich aryl residues, such as tyrosine. However, their utility in biological systems has been restricted due to the requirement of harsh conditions for their generation in situ, as well as limited hydrolytic stability. Previous work describing a scaffold known as triazabutadiene (TBD) has shown the ability to protect aryl diazonium ions allowing for increased synthetic utility, as well as triggered release under biologically relevant conditions. Herein, they describe the synthesis and application of a novel TBD, capable of installation of a cyclooctyne on protein surfaces for later use of copper-free click reactions involving functional azides. The probe shows efficient protein labeling across a wide pH range that can be accomplished in a convenient and timely manner. Orthogonality of the cyclooctyne modification was showcased by labeling a model protein in the presence of hen egg proteins, using an azide-containing fluorophore. They further confirmed that the azobenzene modification can be cleaved using sodium dithionite treatment. the first paragraph on page 254 teaches that tyrosine is recognized as an important amino acid in the study of protein−protein interactions as it is often targeted for post-translational modifications including phosphorylation, glycosylation, and oxidation. Tyrosine has also been identified as an attractive target in the realm of bioconjugate chemistry due to relatively low abundance on protein surfaces but high abundance at sites of protein−protein interactions. Additionally, tyrosine is uncharged at physiological pH, and therefore, tyrosine modifications do not alter the surface charge of proteins in contrast to those that modify charged residues like lysine. Figure 1 shows the steps in the workflow in general (a) and for the designed chemical probe. Figure 3 and its description in the second paragraph of the right column on page 256 teach the extent of protein labeling was quantified using a membrane scaffold protein (MSP1D1T2(-)), as a small model protein. MSP (deconvolved mass spectrum in purple, left/front side of the mass data) was first incubated with 1 (see scheme 1) at pH 7 to provide a cyclooctyne-modified MSP (deconvolved mass spectrum in red, middle portion of the mass data). At this point, part of the sample was split. One portion was treated with a fluorescent azide and the modification was observed by SDS-PAGE. The second portion was treated with a biotin-containing azide (deconvolved mass spectrum shown in orange, back/right side of the mass data). Masses corresponding with integer values of the appropriate modifications were observed and are noted with symbols (P = MSP protein, mod = chemical modification). Together, these data confirm that 1 is capable of robust labeling of several tyrosine residues on MSP, and importantly that the resulting modifications are capable of reacting with multiple azide substrates. This enables the same protein sample to be utilized in several workflows while retaining knowledge of the extent of modification. The final paragraph on page 257 teaches that overall, they have shown the ability to synthesize a versatile cyclooctyne containing TBD capable of efficient modification of proteins. Leveraging the physiological compatibility of the novel TBD, they were able to accomplish robust protein labeling across a range of pH, with relatively low reaction times. Importantly, they also displayed that this probe could be used to install a reactive cyclooctyne on the surface of proteins, allowing for quick and selective functionalization of proteins, even in the presence of complex protein mixtures. The triazabutadiene afforded the synthetic opportunity to show the cross-compatibility of cyclooctynes and aryl diazonium ions, two highly reactive and sought-after functional groups. Furthermore, the aryl diazonium ion chemistry described can be coupled with other strategies, such as chemoselective rapid azo-coupling reactions (CRACR) with 5-hydroxytryptophan to enhance selectivity. Taken together, they believe that this system poses great potential for a wide variety of bioconjugate applications including highly selective and bioorthogonal modification of proteins for purification, imaging, or drug conjugation.
With respect to claims 7-8, 14-15 and 20, it would have been obvious to one of ordinary skill in the art at the time the application was filed to modify the Hoffmann conjugation moiety to the azide taught by Chuh, Wright or Wijetunge because of the variability of the conjugation/reactive moiety of the Li ’07 or Hoffmann tags and the known use of reagents capable of reacting at/with a post-translational modification site of a peptide or protein to contain a terminal alkyne or DBCO group able to react with an azide group of the labeling compound as taught by at least Chu and Wijetunge and the utility and/or various advantages of such a process and/or the combination of a terminal alkyne or DBCO group with an azide conjugation moiety as taught by Chu, Wright and Wijetunge.
Claims 11-12 and 23-24 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 art of record fails to teach or fairly suggest the compounds being claimed in these claims. Examiner notes that the Hoffmann and Yuan references in particular teach the generic structure of claim 1 but would require a modification of each part of the generic structure to obtain the required isobaric tag, enrichment moiety and conjugation moiety of these claims. Compound/structure 12 of the Szychowski paper has the enrichment and conjugation moieties required by claims 11 and 23 but not the isobaric tag. As noted above, the applied anticipatory references teaching an isobaric tag in combination with enrichment and conjugation moieties do not teach the required isobaric tag while the Li ‘728 reference that teaches the required isobaric tag fails to teach it in combination with both an enrichment moiety and the required conjugation moiety. Thus modification of compound/structure 12 of the Szychowski paper by Hoffmann, Yuan or Li ‘728 would fail to reach the claimed compounds in these claims.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. The additionally cited art teaches various labels for proteins and other functional groups as well as cleavable linking structures.
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/ARLEN SODERQUIST/ Primary Examiner, Art Unit 1797